Proteomic Insights into Edible Nut Seeds: Nutritional Value, Allergenicity, Stress Responses, and Processing Effects

Abstract

Nuts, including tree nuts such as almonds, walnuts, cashews, and macadamias, as well as peanuts, are widely consumed for their health benefits owing to their high-quality protein content. Globally, the nut industry represents a multi-billion-dollar sector, with increasing demand driven by consumer interest in nutrition, functional foods, and plant-based diets. Recent advances in proteomic technologies have enabled comprehensive analyses of nut seed proteins, shedding light on their roles in nutrition, allergenicity, stress responses, and food functionality. Seed storage proteins such as 2S albumins, 7S vicilins, and 11S legumins, are central to nutrition and allergenicity. Their behavior during processing has important implications for food safety. Proteomic studies have also identified proteins involved in lipid and carbohydrate metabolism, stress tolerance, and defense against pathogens. Despite technical challenges such as high lipid content and limited genomic resources for many nut species, progress in both extraction methods and mass spectrometry has expanded the scope of nut proteomics. This review underscores the central role of proteomics in improving nut quality, enhancing food safety, guiding allergen risk management, and supporting breeding strategies for sustainable crop improvement.

1. Introduction

Nuts, defined here as dry seeds with a lignified shell and lipid- and protein-rich kernels, are valued worldwide for their nutrient density, long shelf-life, and distinctive sensory qualities. They have been integral to Mediterranean, Middle Eastern, South Asian, and Indigenous American diets for millennia, serving as portable, energy-dense staples in hunter–gatherer societies and traditional cuisines [1,2,3]. Today, they remain central to global dietary patterns, appreciated for both flavour and health benefits.

According to the Nuts and Dried Fruits Statistical Yearbook [4], peanuts remain the most widely consumed legume grouped within the broader “nut” category, especially in middle-income countries, while among tree nuts, almonds accounted for the largest share of global consumption in 2023 (27%), followed by cashews (21%), walnuts (20%), pistachios (17%), and hazelnuts (10%). Pecans, macadamias, pine nuts, and Brazil nuts collectively represented the remaining 5%. Although peanuts are botanically legumes, their nutrient composition, culinary uses, and allergenic potential justify their inclusion in the broader “nut” category for nutritional and proteomic analyses [4]. In this review, “nut” refers to both botanical nuts and nutritionally equivalent seeds, reflecting their shared biological and dietary relevance.

Global consumption of nuts is increasing, driven by rising health awareness and promotion as nutrient-rich snacks by public health agencies and industry bodies [5,6]. Regular nut intake has been associated with reduced risks of cardiovascular disease, metabolic syndrome, type 2 diabetes, and Alzheimer’s disease [7,8,9,10,11]. These benefits are largely attributed to their favourable lipid profiles—rich in monounsaturated and polyunsaturated fatty acids—and high levels of antioxidant compounds [5,12,13,14,15,16,17].

Nuts are also important protein sources, although protein content varies widely between species—from ~6–11% in oil-rich nuts such as pecan to over 30% in pine nuts, with most tree nuts and peanuts containing ~15–25% protein [18,19,20]. These differences reflect species-specific seed storage strategies, where higher lipid allocation reduces relative protein content. Nut proteins are considered high-quality plant proteins: they supply all essential amino acids in proportions close to human nutritional requirements, have high digestibility (>70% versus ~46% for wheat gluten), and contain relatively low levels of anti-nutritional factors such as tannins and saponins [21]. The majority of amino acids are stored in the cotyledons as storage proteins from the cupin and prolamin superfamilies, with additional proteins participating in lipid metabolism and transfer, stress and defence responses, seed development, redox homeostasis, and other cellular functions. [22,23,24,25].

While the chemical composition and morphology of nuts are well described, their protein composition has not been comprehensively examined through a proteomic lens. Food proteomics—the large-scale identification and quantification of proteins—offers a powerful approach to link nut composition to nutritional value, allergenic potential, stress resilience, and processing behaviour. This review summarises recent advances in nut proteomics, highlighting applications in nutrition, allergen detection, and crop improvement, and addresses technical challenges such as high lipid interference and limited genomic resources. Understanding the nut proteome is crucial for enhancing nutritional quality, improving allergen detection and management, and guiding breeding strategies that support food security and sustainable agriculture.

2. Status and Utility of Proteomic Research in Nut Species

Despite the current research being limited, existing studies indicate that the applications of nut protein research are remarkably diverse, shedding light on the complex protein compositions of nuts and their significant roles in nutrition, health, and agriculture. These functional applications mainly involve nutritional profiling (proteomic identification of seed storage proteins and metabolic enzymes linked to protein quality and composition); food traceability and authentication (proteomic markers distinguishing nut species, cultivars, and geographic origins); allergen detection and risk assessment (detection of major and minor allergens, including heat-stable peptide markers in processed foods); processing effects and food functionality (impact of roasting, Maillard reactions, and cross-linking on protein solubility, allergenicity, and functionality); crop improvement and breeding markers (proteomic traits linked to seed quality, stress tolerance, and marker-assisted selection); stress and defense responses under abiotic and biotic conditions (proteins associated with drought tolerance, pathogen defense, and seed resilience); and bioactive peptide discovery (identification of peptides with health benefits) (Figure 1).

Research has reported on nuts including almonds (Prunus dulcis), cashews (Anacardium occidentale), peanuts (Arachis hypogaea), hazelnuts (Corylus avellana), walnuts (Juglans regia), pistachios (Pistacia vera), pecan (Carya illinoinensis), pine nut (Pinus koraiensis), and macadamia (Macadamia integrifolia); each presents unique challenges and opportunities for proteomic analysis with the aim to uncover their nutritional profiles, allergenic proteins, and responses to environmental stresses (Table 1).

One of the most intensely researched areas is allergen characterization and detection. Proteomics has identified and catalogued key storage protein allergens—2S albumins, 7S vicilins, and 11S legumins—across different nut species, and clarified patterns of IgE cross-reactivity, such as the strong homology between cashew and pistachio allergens [26,27,28,29]. These molecular insights are informing diagnostic tools and hypoallergenic breeding strategies. Advances in high-resolution LC–MS/MS, combined with targeted peptide selection, have enabled sub-mg/kg detection of multiple nut allergens in complex food matrices, and in many cases provide greater reliability than conventional immunoassays, which can be affected by heat-induced denaturation [30,31].

Table 1. Overview of Edible Nut Species Studied in Proteomics and Key Application Areas.

Nut Species —Sample Tissue	Study Purpose	Protein Extraction Methods	Research Methods and Instruments	Search Database (If Applicable)	Key Findings	Application/Significance	Ref.
Macadamia (Macadamia integrifolia) - Nuts	Characterize proteomic diversity; assess parental genetic contributions to nut quality and allergenicity	Methanol homogenization + TCA precipitation; SDS-PAGE separation; in-gel digestion	SDS-PAGE densitometry (Bio-Rad); Nano-HPLC-MS/MS (Eksigent LC400 + TripleTOF 6600+); ProteinPilot; Scaffold; NSAF quantification	M. integrifolia genome database for ProteinPilot/Scaffold; functional annotation via BLASTP to UniProtKB/SwissProt Arabidopsis thaliana	431 proteins identified; ~50% were vicilin/legumin SSPs; paternal genotype strongly influenced SSP profiles; PTM and sequence coverage of SSPs varied across genotypes	Informs allergenicity risk assessment, highlights natural variation in SSP abundance, supports breeding strategies for hypoallergenic and nutritionally improved cultivars	[32]
Various nuts - Raw/baked nut powders; incurred bakery food samples	Develop HRMS-based method for simultaneous detection of multiple nut allergens in bakery foods	Buffers with urea/thiourea/CHAPS for raw nuts; Tris–HCl buffer for bakery food	LC-Q-TOF MS (Agilent 6545B) in EDR and auto MS/MS mode; HPLC-QQQ-MS (Sciex 6500) in MRM; PEAKS Xpro bioinformatics	NCBI Protein Database (taxon-specific entries for almond, cashew, peanut, walnut)	Identified 16 exclusive heat-stable marker peptides; LODs 0.10–0.31 mg/kg; recoveries 72.5–92.1%; detection of undeclared allergens in commercial bakery foods	Provides robust, sensitive, label-free quantification of nut allergens; supports allergen labeling regulation and risk management	[30]
Macadamia (Macadamia integrifolia) - Nuts	Identify new allergens and assess cross-reactivity with other tree nuts in Spanish cohort	Ground nuts in liquid N2; homogenized in PBS; centrifugation and dialysis; Bradford quantification	SDS-PAGE (reducing and non-reducing); Western blot with allergic patients’ sera; Inhibition assays (hazelnut, walnut extracts); MALDI-TOF/TOF MS for IgE-binding protein identification	Non-redundant protein sequence databases (MASCOT/NCBI search for peptide fingerprint)	Three new allergens identified: oleosin, pectin acetylesterase, aspartyl protease; confirmed Mac i 1 (vicilin) and antimicrobial peptide 1; Oleosin cross-reactive with hazelnut but not walnut	Expands macadamia allergen profile; reveals regional sensitization differences (Spanish vs. Australian/Japanese); supports improved allergy diagnosis and risk management	[27]
Walnut (Juglans regia) - Nuts (raw, boiled, roasted); oil body (oleosome) fraction	Characterize walnut oleosome proteome; assess processing effects and IgE recognition	Oil-body enrichment via sucrose/Tween/NaCl/urea buffers; methanol–chloroform–water precipitation	Off-gel label free shotgun proteomics (Orbitrap Q Exactive Plus + UHPLC); In-gel LDS-PAGE + MS/MS; Immunoblotting	UniProt J. regia database + mature protein sequences (signal peptide/propeptide removed forms)	Identified all 8 oleosins, 1 caleosin, 1 steroleosin detected; boiling enhanced protein solubility and recovery; phylogenetic alignment showed walnut oleosins similar to hazelnut Cor a 13 and peanut Ara h 15	First evidence of walnut oleosin allergenicity; shows thermal processing modulates allergen solubility and IgE reactivity; supports improved diagnosis and allergen risk assessment	[33]
Various nuts - (raw and roasted)	Develop a single LC-HRMS method to simultaneously detect/quantify nut allergens in processed bakery matrices	Tris-HCl buffer + urea; reduction (DTT), alkylation (IAA); SPE purification (Strata-X, SepPak C18); SEC cleanup for cookie extracts	LC-ESI-Q-Orbitrap MS (Q-Exactive Plus, Thermo Fisher); Full-MS/dd-MS2 & AIF modes; isotopically labelled AQUA peptides as internal standards; Proteome Discoverer	Customized database from UniProtKB entries: almond, hazelnut, peanut, walnut, cashew, pistachio	Identified reliable marker peptides common to raw and roasted nuts; 3 high-response peptides per nut selected; LOD 2.4–8.1 mg/kg, LOQ 8–27 mg/kg; roasting reduced peptide detection up to ~50%	Provides robust, multi-allergen detection at ppm levels in bakery foods; aligns with VITAL 3.0 thresholds; supports rational PAL use and consumer safety	[31]
Pecan (Carya illinoinensis) - Nuts from two cultivars at 6 developmental stages	Characterize proteomic changes during nut development; identify allergen accumulation timing; compare cultivars	Borate buffer; homogenization; centrifugation; SDS-PAGE quantification	SDS-PAGE + immunoblot with anti-pecan sera; LC-QTOF-MS/MS (Agilent 6520 Chip Cube); Mascot Distiller/Daemon; 2D-GE (isoelectric focusing pH 3–10, silver staining); image analysis	In-house protein libraries from Sumner (NCBI) and Pawnee transcriptomes; annotated with GO terms (InterProScan)	protein accumulation peaked at dough stage; allergens Car i 1 & Car i 2 first detected at dough stage; histones decreased as development progressed; cultivar differences	Provides molecular insight into timing of allergen accumulation and developmental regulation; informs breeding for reduced allergenicity, stress tolerance, improved seed quality	[28]
Almond (Prunus dulcis) - Commercial almond flour	Compare aqueous vs. protease-assisted aqueous extraction (AEP vs. EAEP) on protein profile, digestibility and antigenicity	AEP (alkaline, pH 9) vs. EAEP (AEP + neutral protease); centrifugation to separate protein/oil	SDS-PAGE; LC–MS/MS (Orbitrap); PEAKS Studio; simulated digestion and Degree of Hydrolysis (OPA); ELISA and Western blot (IgE/IgG sera)	UniProt P. dulcis (Swiss-Prot + TrEMBL)	EAEP mainly hydrolyzed prunin α-chains, improved digestibility (79→89%), increased peptide yield, and reduced immunoreactivity (~75%) with degradation of multiple allergens.	Demonstrates protease-assisted extraction improves almond protein digestibility and reduces allergenicity, supporting development of safer hypoallergenic almond-based foods	[34]
Chinese hickory (Carya cathayensis) - Embryos at 3 developmental stages	Characterize lipid droplet (LD) proteome during embryo development and identify key LD proteins linked to oil accumulation	Sucrose gradient centrifugation for LD isolation; protein precipitation (chloroform/acetone); lysis buffer (urea, Tris, NP40, NaDOC, protease inhibitors); reduction (DTT), alkylation (IAM)	LC–MS/MS (Q Exactive HF-X Orbitrap + Ultimate 3000 nano-UPLC); label-free quantification (LFQ, iBAQ, riBAQ); qRT-PCR validation; confocal microscopy (LD localization, GFP fusion); TEM ultrastructure	C. cathayensis genome searched with MaxQuant; Arabidopsis Plant Proteome Database for subcellular localization	Identified 6574 proteins, incl. 38 LD-associated; 62 DEPs linked to LD biogenesis; oleosins/CLO1/HSD5 increased during S2→S3, declined at S5; subcellular localization confirmed proteins at LDs	Provides first comprehensive LD proteome of Chinese hickory; reveals dynamic regulation of LD proteins during oil accumulation; supports breeding strategies for high-oil-yield nut cultivars	[35]
Hazelnut (Corylus avellana) - Nuts (raw and autoclaved variants)	Assess whether autoclaving (with/without hydration/drying) reduces allergenicity	Milling; protein extraction; Bradford assay for quantification; filtration	SDS-PAGE; In-gel digestion; LC–MS/MS (Q Exactive Orbitrap, Thermo) with UHPLC; Proteome Discoverer/SequestHT; Immunoblotting (IgE, IgG from 14 allergic patients)	UniProt C. avellana protein database	Autoclaving (especially with hydration/drying) reduced protein solubility (40–70%), degraded major allergens (Cor a 9, 11, 14), and markedly lowered patient skin test reactivity and IgE binding.	Demonstrates autoclaving, especially with hydration/drying, markedly reduces hazelnut allergenicity; provides proteomic and clinical evidence supporting hypoallergenic hazelnut product development	[36]
Pistachio (Pistacia vera) - Defatted flour from 4 cultivars	Characterize pistachio allergen proteome and compare cultivar-specific expression	Defatting with diethyl ether; extraction with urea + TBS buffer; Bradford assay; reduction (DTT), alkylation (IAA)	SDS-PAGE, 2-DE (IEF + SDS-PAGE); LC–MS/MS (Q Exactive Orbitrap + UltiMate 3000 RSLC nano-UPLC)	UniProtKB P. vera	Identified major allergens Pis v 1 (2S albumin), Pis v 2 & Pis v 5 (11S globulins), Pis v 3 (7S vicilin); multiple isoforms of 11S and 7S revealed; Pis v 5 differentially expressed in cultivars	First comprehensive pistachio allergen proteome; confirms dominance of cupin family allergens; highlights cultivar variation; supports allergen risk assessment and breeding for hypoallergenic cultivars	[26]
Macadamia (Macadamia integrifolia and hybrids with M. tetraphylla) - Nuts	Provide proteomic overview of macadamia nut; identify functional protein categories; assess potential allergens and cross-reactivity	Defatting (n-hexane/ethanol); protein extraction (NaCl buffer pH 8.4); acetone precipitation; SDS-PAGE; in-gel trypsin digestion	SDS-PAGE; LC–MS/MS (LTQ-FT Ultra Orbitrap + Ultimate 3000 nano-LC); Mascot and X!Tandem; Scaffold validation; spectral counting; GO/KEGG annotation	NCBI non-redundant Viridiplantae protein database; AllergenOnline (FARRP v16); IEDB-AR (linear epitopes)	Identified 1079 proteins; proteins classified into 46 categories; defense proteins most abundant; identified seed storage proteins; in silico matched macadamia proteins with allergens; cross-reactivity with peanut, walnut, lupin, sesame, birch pollen, fungi, dust mite indicated	First comprehensive macadamia nut proteome; highlights abundant defense and storage proteins; identifies potential allergenic proteins and cross-reactivity risks; provides resource for allergy assessment and breeding	[37]
Peanut (Arachis hypogaea) - Developing seeds at 7 underground stages (R1–R7)	Investigate peanut proteins at different development stages; analyze allergen accumulation and protein interaction networks	TCA/acetone precipitation; phenol extraction; MeOH/NH4OAc precipitation; guanidine-HCl solubilization; reduction (TBP); alkylation (2-VP); urea/thiourea/CHAPS lysis	2-DE (IEF pH 3–10, SDS-PAGE); colloidal CBB staining; densitometry (GS-800); MALDI-TOF/TOF-MS (Sciex 5800); Mascot search; Mfuzz clustering; qRT-PCR validation	Peanut A-genome (A. duranensis) and B-genome (A. ipaensis); EggNOG for functional annotation; STRING v9.1 for PPI network	Identified 264 proteins; clustered into 8 groups; 5 major functional categories; 14 allergen-like proteins identified, expression low at R1–R4, increased sharply from R5–R7; storage proteins mainly in cluster 7	Provides first proteome map of peanut underground seed development; reveals allergen accumulation timing; suggests harvesting immature seeds may lower allergenicity risk	[23]
Cashew (Anacardium occidentale) - Blanched raw nuts	Develop customized protein database; analyze effects of oil-roasting on allergen stability; improve MS peptide targets	Tris-HCl buffer + urea; reduction (DTT), alkylation (IAA); in-solution trypsin/chymotrypsin digestion; C18 cleanup	SDS-PAGE; LC–MS/MS (Q Exactive HF and Plus Orbitrap + UltiMate 3000 UHPLC); DDA and PRM targeted analysis	Customized cashew protein database from UniProt, Phytozome v12.1 genome (primary + selected alternative transcripts), and AUGUSTUS predictions	Identified 6595 peptides; revealed 17 new sequences for major allergens; Ana o 3 (2S) most heat-stable; roasting reduced solubility and abundance of Ana o 1; Maillard adducts identified on 11S peptides;	Provides optimized cashew protein database; reveals isoforms and processing impact on allergenicity; enables high-quality MS-based allergen detection; supports food safety regulation and risk assessment	[38]
Peanut (Arachis hypogaea) - Seeds from high and low-oleate cultivar at 6 developmental stages	Identify proteins linked to oleic acid accumulation during seed development	TCA/acetone precipitation; methanol/ammonium acetate wash; urea/Tris lysis; reduction (DTT), alkylation (IAM); iTRAQ labeling	iTRAQ-based LC–ESI–MS/MS (TripleTOF 5600, SCIEX); Mascot and ProteinPilot search; GO and KEGG annotation; STRING PPI analysis; qRT-PCR validation	UniprotKB peanut protein database; functional annotation with Arabidopsis TAIR and GO annotation; KEGG pathways	7666 proteins identified; 389 DEPs associated with FA metabolism; FAB2 upregulated early in high-oleate cultivar, downregulated later; KAS I/II upregulated early; multiple lipid oxidation enzymes identified; OA biosynthesis enriched at stage 3	Provides molecular insight into oleic acid accumulation dynamics; identifies FAB2 as key regulator; supports breeding strategies for high-oleate peanut varieties with improved oil stability and nutritional quality	[39]
Cashew (Anacardium occidentale) - Cashew nut protein fractions and protein isolate	Characterize molecular and functional properties of cashew protein fractions vs. isolate	Osborne fractionation (water, NaCl, ethanol, NaOH) for fractions; alkali extraction + isoelectric precipitation for protein isolate; petroleum ether defatting	SDS-PAGE (reducing/non-reducing), Circular dichroism spectroscopy, SEM, solubility tests, water/Oil holding capacity, foaming and emulsifying assays, amino acid analyzer (Hitachi L-8800)	N/A	Cashew proteins showed varied functional properties	Demonstrates functional specialization of fractions: albumin/globulin for beverages (solubility), glutelin for bakery/meat (foaming and water/oil binding), protein isolate for emulsified foods; supports cashew as valuable protein source for food industry	[40]
Hazelnut (Corylus avellana) - Nuts	Characterize storage, allergenic, and defense proteins; improve allergen detection	PBS extraction, defatting (hexane), TCA/acetone precipitation, buffer optimization (temp, ratio)	SDS-PAGE, 2-DE, MALDI-TOF, LC–ESI–MS/MS; RP–HPLC; Western blot; ELISA; MS-based targeted detection (SRM/PRM)	NCBI and UniProtKB databases (SwissProt entries for Cor a proteins); Mascot search; AllergenOnline cross-references	Identified major allergens; multiple isoforms and proteoforms; MS marker peptides developed for food allergen detection; roasting altered extractability but not IgE binding significantly	First comprehensive hazelnut proteome; provides allergen catalog and biomarker peptides; supports allergy diagnosis, risk assessment, and food authentication	[41]
Almond (Prunus dulcis) - Nuts	Characterize almond nut proteome; compare with American almonds; identify allergens and stress-related proteins	Phenol extraction with sucrose/KCl/EDTA Tris buffer; methanol–ammonium acetate precipitation; acetone wash; SDS-PAGE and in-gel trypsin digestion	2-DE + SDS-PAGE; LC–MS/MS (Q Exactive Orbitrap); Mascot search; GO annotation (Blast2GO); ImageMaster 2D Elite analysis	NCBI protein database (taxonomy: P. dulcis); UniProt for GO annotation; SwissProt cross-references	Identified 434 proteins (259 novel/hypothetical); 2104 protein spots in 2-DE; allergens Pru du 1, 2, 3, 4, 6 detected; 22 hypothetical proteins with glycosylation motifs; abundant metabolic/catalytic proteins; stress-related proteins	Provides reference almond proteome map; improves understanding of allergenicity, stress response proteins, and nutritional value; supports development of almond protein-based foods	[29]
Walnut (Juglans regia) - Defatted walnut flour (raw and roasted)	Assess effects of roasting on solubility and detectability of walnut allergens	Defatting (hexane); sequential vs. simultaneous extraction/trypsin digestion; DTT, IAA	SDS-PAGE; LC–MS/MS (Orbitrap Elite, UPLC C18); Label-free quantification (Progenesis LC-MS); Mascot search; Maillard adduct screening	UniProt Juglandaceae + UniProt A. hypogaea + AllergenOnline v12 + contaminants	2S albumin (Jug r 1), nsLTP (Jug r 3), 7S N-terminal showed minor roasting effects; Mature 7S (Jug r 2) and 11S (Jug r 4) increased detectability after roasting, likely due to improved digestibility; Maillard adducts mainly on 7S and 11S globulins	Reveals allergen-specific responses to roasting; highlights MS as powerful tool for monitoring allergen modifications; informs allergen risk assessment and MS-based detection in processed foods	[42]
Hazelnut (Corylus avellana) - Defatted flour; water-soluble protein extracts	Evaluate effect of high hydrostatic pressure (HHP) on protein solubility and immunoreactivity	Defatting (n-hexane); soaking in distilled water; extraction with borate saline buffer + PVP; centrifugation; dialysis; freeze-drying	SDS-PAGE; Western blot (IgE sera from 15 allergic patients); 2D-LC fractionation (ProteomeLab PF-2D: chromatofocusing + RP-HPLC); MALDI-TOF/TOF-MS; LC–ESI–MS/MS	SwissProt/NCBI protein databases (taxonomy: C. avellana); Mascot for peptide mass fingerprinting	HHP reduced solubility of major allergens Cor a 9 (11S globulin) and Cor a 11 (7S vicilin); SDS-PAGE and immunoblot showed similar IgE reactivity between control and HHP samples; PF-2D revealed fewer protein peaks in HHP extracts; Cor a 9 and Cor a 11 remained identifiable but less soluble	Demonstrates HHP alters solubility but not IgE-binding of hazelnut allergens; provides proteomic evidence for limited effect of HHP alone on allergenicity; suggests combining HHP with protease digestion for hypoallergenic food development	[43]
Hazelnut (Corylus avellana) - Nuts	Identify and characterize novel IgE-binding proteins in hazelnut	Defatting (diethyl ether); PBS extraction; reduction (DTT), alkylation (IAA); dialysis + lyophilization	SDS-PAGE (reducing and non-reducing), 2-DE; MALDI-TOF-MS; nano-LC–ESI-Q-TOF MS/MS (Mascot, Batch-Tag, Protein Prospector); de novo sequencing; Immunoblotting and inhibition assays; RP-HPLC purification	NCBI and SwissProt databases (green plants); homology search via BLAST/MS-Pattern; incomplete hazelnut genome noted	Identified a novel alkaline subunit (homologous to 11S globulin Cor a 9) as predominant IgE-binding protein; minor reactivity also to Cor a 11, Cor a 9, Cor a 8; de novo peptides showed homology with 11S isoforms from pistachio, cashew, soybean	First evidence of new 11S globulin isoallergen in hazelnut; explains complex IgE-reactivity patterns; provides platform for component-resolved diagnostics and new therapeutic strategies	[44]
Peanut (Arachis hypogaea) - Nuts	Compare proteomes of allergenic vs. hypoallergenic peanut cultivars	Defatting (hexane); extraction with Tris-HCl + urea + thiourea + CHAPS; reduction (DTT), alkylation (IAA); trypsin digestion	2-DE; MALDI-TOF/TOF-MS (AB Sciex 5800); LC–MS/MS; Progenesis PG240 software; Mascot and X!Tandem search; Scaffold validation	NCBI A. hypogaea database + expressed sequence tags	162 protein spots identified; 40 differential spots between allergenic and hypoallergenic lines; allergens Ara h 1, 2, 3, 6 more abundant in allergenic varieties; hypoallergenic types showed lower SSPs and higher stress-response proteins (LEA, HSPs, PR proteins)	Provides proteomic evidence for reduced allergenicity in selected peanut cultivars; supports breeding of hypoallergenic peanuts	[45]
Brazil nut (Bertholletia excelsa) - Nuts	Purify and characterize major 2S albumin isoforms; assess heterogeneity and structural features	Defatting (petroleum ether); water extraction; dialysis; gel filtration (Superdex 75); chromatofocusing	SDS-PAGE, IEF, MALDI-TOF-MS (Bruker Reflex III), RP-HPLC–ESI-MS (Micromass Quattro II), Circular dichroism spectroscopy, Fourier transform-infrared spectroscopy	NCBI protein database (Viridiplantae) searched with Mascot peptide mass fingerprinting	Identified ~10 2S albumin isoforms; heterogeneity due to post-translational processing; all predominantly α-helical; no glycosylation detected	Provides first detailed proteomic and structural characterization of Brazil nut 2S albumin isoforms; explains allergen heterogeneity (Ber e 1); supports allergen risk assessment and structure–function studies	[46]
Pili nut (Canarium ovatum) - Nuts	Characterize storage protein composition and physicochemical properties	Modified Osborne fractionation: water (albumins), NaCl (globulins), ethanol (prolamins), NaOH (glutelins); defatting with hexane	SDS-PAGE (reducing and non-reducing); Native-PAGE; Amino acid analysis (Beckman Analyzer); DSC (MC-2D calorimeter); proximate and mineral analysis	N/A	Protein content ~11.9% of nuts; Globulins major fraction (60.3% of total protein), mainly 11S globulin with 22.6 and 31.6 kDa subunits; Albumins minor (2.9%); No prolamins; Glutelins ~36.8%	First detailed characterization of pili nut proteins; shows similarity to other oilseed 11S globulins (soy, peanut, hemp); supports potential use of pili nut proteins as isolates in food applications	[47]
Peanut (Arachis hypogaea) - Nuts (raw and roasted, aqueous extracts)	Assess roasting effects on major allergens and PTM profiles	Defatting (n-hexane, tetrachloroethylene); aqueous PBS extraction under mild conditions	1D/2D SDS-PAGE; In-gel digestion; nLC–MS/MS (LTQ Orbitrap XL); PTM profiling via PEAKS XPro; Western blot with PTM-specific Abs; ELISA (IgE binding, patient sera)	UniProtKB A. hypogaea + contaminants; PTM search included 313 mods from Unimod	Roasting reduced protein solubility (~4 × lower); Ara h 3 enriched in raw, Ara h 6 in roasted; >40 PTMs; oxidation (Met), deamidation (Asn/Gln), hydroxylation (Pro), carbamoylation (Lys/Arg), and methylation more frequent in roasted; Ara h 1 most extensively modified; PTMs mapped to IgE-binding epitopes.	Provides first semi-quantitative PTM profiling of peanut allergens; shows roasting alters PTM patterns without markedly changing IgE binding; supports understanding of allergen proteoforms in food processing and allergy risk assessment	[48]

Abbreviations: TCA: Trichloroacetic acid; SDS-PAGE: Sodium dodecyl sulfate–polyacrylamide gel electrophoresis; HPLC: High-performance liquid chromatography; MS/MS: Tandem mass spectrometry; NSAF: Normalized spectral abundance factor; SSP: Seed storage protein; PTM: Post-translational modification; HRMS: High-resolution mass spectrometry; CHAPS: 3-[(3-cholamidopropyl) dimethylammonio]-1-propanesulfonate; Q-TOF: Quadrupole time-of-flight; QQQ-MS: Triple quadrupole mass spectrometry; MRM: Multiple reaction monitoring; IgE: Immunoglobulin E; MALDI: Matrix-assisted laser desorption/ionization; LDS-PAGE: Lithium dodecyl sulfate–polyacrylamide gel electrophoresis; DTT: Dithiothreitol; IAA: Iodoacetamide; SPE: Solid-phase extraction; SEC: Size exclusion chromatography; Q-Orbitrap: Quadrupole-Orbitrap high-resolution MS; AIF: All-ion fragmentation; PAL: Precautionary allergen labeling; VITAL: Voluntary Incidental Trace Allergen Labelling; GO: Gene Ontology; GST: Glutathione S-transferase; DAP: Days after pollination; IAM: Iodoacetamide; NaDOC: Sodium deoxycholate; iBAQ/riBAQ: Intensity-based absolute quantification/relative iBAQ; TEM: Transmission electron microscopy; TBP: Tributylphosphane; 2-VP: 2-vinylpyridine; PPI: Protein–protein interaction; DDA: Data-dependent acquisition; PRM: Parallel reaction monitoring; UHPLC: Ultra-high performance liquid chromatography; KEGG: Kyoto Encyclopedia of Genes and Genomes; FAB2: Stearoyl-acyl carrier protein desaturase; KAS: 3-ketoacyl-ACP synthase; SEM: Scanning electron microscopy; nsLTP: Non-specific lipid transfer protein; LEA: Late embryogenesis abundant proteins; HSPs: Heat shock proteins; PR proteins: Pathogenesis-related proteins; PBS: Phosphate-buffered saline; OPA: o-Phthaldialdehyde method.

Table 1. Overview of Edible Nut Species Studied in Proteomics and Key Application Areas.

Nut Species —Sample Tissue	Study Purpose	Protein Extraction Methods	Research Methods and Instruments	Search Database (If Applicable)	Key Findings	Application/Significance	Ref.
Macadamia (Macadamia integrifolia) - Nuts	Characterize proteomic diversity; assess parental genetic contributions to nut quality and allergenicity	Methanol homogenization + TCA precipitation; SDS-PAGE separation; in-gel digestion	SDS-PAGE densitometry (Bio-Rad); Nano-HPLC-MS/MS (Eksigent LC400 + TripleTOF 6600+); ProteinPilot; Scaffold; NSAF quantification	M. integrifolia genome database for ProteinPilot/Scaffold; functional annotation via BLASTP to UniProtKB/SwissProt Arabidopsis thaliana	431 proteins identified; ~50% were vicilin/legumin SSPs; paternal genotype strongly influenced SSP profiles; PTM and sequence coverage of SSPs varied across genotypes	Informs allergenicity risk assessment, highlights natural variation in SSP abundance, supports breeding strategies for hypoallergenic and nutritionally improved cultivars	[32]
Various nuts - Raw/baked nut powders; incurred bakery food samples	Develop HRMS-based method for simultaneous detection of multiple nut allergens in bakery foods	Buffers with urea/thiourea/CHAPS for raw nuts; Tris–HCl buffer for bakery food	LC-Q-TOF MS (Agilent 6545B) in EDR and auto MS/MS mode; HPLC-QQQ-MS (Sciex 6500) in MRM; PEAKS Xpro bioinformatics	NCBI Protein Database (taxon-specific entries for almond, cashew, peanut, walnut)	Identified 16 exclusive heat-stable marker peptides; LODs 0.10–0.31 mg/kg; recoveries 72.5–92.1%; detection of undeclared allergens in commercial bakery foods	Provides robust, sensitive, label-free quantification of nut allergens; supports allergen labeling regulation and risk management	[30]
Macadamia (Macadamia integrifolia) - Nuts	Identify new allergens and assess cross-reactivity with other tree nuts in Spanish cohort	Ground nuts in liquid N2; homogenized in PBS; centrifugation and dialysis; Bradford quantification	SDS-PAGE (reducing and non-reducing); Western blot with allergic patients’ sera; Inhibition assays (hazelnut, walnut extracts); MALDI-TOF/TOF MS for IgE-binding protein identification	Non-redundant protein sequence databases (MASCOT/NCBI search for peptide fingerprint)	Three new allergens identified: oleosin, pectin acetylesterase, aspartyl protease; confirmed Mac i 1 (vicilin) and antimicrobial peptide 1; Oleosin cross-reactive with hazelnut but not walnut	Expands macadamia allergen profile; reveals regional sensitization differences (Spanish vs. Australian/Japanese); supports improved allergy diagnosis and risk management	[27]
Walnut (Juglans regia) - Nuts (raw, boiled, roasted); oil body (oleosome) fraction	Characterize walnut oleosome proteome; assess processing effects and IgE recognition	Oil-body enrichment via sucrose/Tween/NaCl/urea buffers; methanol–chloroform–water precipitation	Off-gel label free shotgun proteomics (Orbitrap Q Exactive Plus + UHPLC); In-gel LDS-PAGE + MS/MS; Immunoblotting	UniProt J. regia database + mature protein sequences (signal peptide/propeptide removed forms)	Identified all 8 oleosins, 1 caleosin, 1 steroleosin detected; boiling enhanced protein solubility and recovery; phylogenetic alignment showed walnut oleosins similar to hazelnut Cor a 13 and peanut Ara h 15	First evidence of walnut oleosin allergenicity; shows thermal processing modulates allergen solubility and IgE reactivity; supports improved diagnosis and allergen risk assessment	[33]
Various nuts - (raw and roasted)	Develop a single LC-HRMS method to simultaneously detect/quantify nut allergens in processed bakery matrices	Tris-HCl buffer + urea; reduction (DTT), alkylation (IAA); SPE purification (Strata-X, SepPak C18); SEC cleanup for cookie extracts	LC-ESI-Q-Orbitrap MS (Q-Exactive Plus, Thermo Fisher); Full-MS/dd-MS2 & AIF modes; isotopically labelled AQUA peptides as internal standards; Proteome Discoverer	Customized database from UniProtKB entries: almond, hazelnut, peanut, walnut, cashew, pistachio	Identified reliable marker peptides common to raw and roasted nuts; 3 high-response peptides per nut selected; LOD 2.4–8.1 mg/kg, LOQ 8–27 mg/kg; roasting reduced peptide detection up to ~50%	Provides robust, multi-allergen detection at ppm levels in bakery foods; aligns with VITAL 3.0 thresholds; supports rational PAL use and consumer safety	[31]
Pecan (Carya illinoinensis) - Nuts from two cultivars at 6 developmental stages	Characterize proteomic changes during nut development; identify allergen accumulation timing; compare cultivars	Borate buffer; homogenization; centrifugation; SDS-PAGE quantification	SDS-PAGE + immunoblot with anti-pecan sera; LC-QTOF-MS/MS (Agilent 6520 Chip Cube); Mascot Distiller/Daemon; 2D-GE (isoelectric focusing pH 3–10, silver staining); image analysis	In-house protein libraries from Sumner (NCBI) and Pawnee transcriptomes; annotated with GO terms (InterProScan)	protein accumulation peaked at dough stage; allergens Car i 1 & Car i 2 first detected at dough stage; histones decreased as development progressed; cultivar differences	Provides molecular insight into timing of allergen accumulation and developmental regulation; informs breeding for reduced allergenicity, stress tolerance, improved seed quality	[28]
Almond (Prunus dulcis) - Commercial almond flour	Compare aqueous vs. protease-assisted aqueous extraction (AEP vs. EAEP) on protein profile, digestibility and antigenicity	AEP (alkaline, pH 9) vs. EAEP (AEP + neutral protease); centrifugation to separate protein/oil	SDS-PAGE; LC–MS/MS (Orbitrap); PEAKS Studio; simulated digestion and Degree of Hydrolysis (OPA); ELISA and Western blot (IgE/IgG sera)	UniProt P. dulcis (Swiss-Prot + TrEMBL)	EAEP mainly hydrolyzed prunin α-chains, improved digestibility (79→89%), increased peptide yield, and reduced immunoreactivity (~75%) with degradation of multiple allergens.	Demonstrates protease-assisted extraction improves almond protein digestibility and reduces allergenicity, supporting development of safer hypoallergenic almond-based foods	[34]
Chinese hickory (Carya cathayensis) - Embryos at 3 developmental stages	Characterize lipid droplet (LD) proteome during embryo development and identify key LD proteins linked to oil accumulation	Sucrose gradient centrifugation for LD isolation; protein precipitation (chloroform/acetone); lysis buffer (urea, Tris, NP40, NaDOC, protease inhibitors); reduction (DTT), alkylation (IAM)	LC–MS/MS (Q Exactive HF-X Orbitrap + Ultimate 3000 nano-UPLC); label-free quantification (LFQ, iBAQ, riBAQ); qRT-PCR validation; confocal microscopy (LD localization, GFP fusion); TEM ultrastructure	C. cathayensis genome searched with MaxQuant; Arabidopsis Plant Proteome Database for subcellular localization	Identified 6574 proteins, incl. 38 LD-associated; 62 DEPs linked to LD biogenesis; oleosins/CLO1/HSD5 increased during S2→S3, declined at S5; subcellular localization confirmed proteins at LDs	Provides first comprehensive LD proteome of Chinese hickory; reveals dynamic regulation of LD proteins during oil accumulation; supports breeding strategies for high-oil-yield nut cultivars	[35]
Hazelnut (Corylus avellana) - Nuts (raw and autoclaved variants)	Assess whether autoclaving (with/without hydration/drying) reduces allergenicity	Milling; protein extraction; Bradford assay for quantification; filtration	SDS-PAGE; In-gel digestion; LC–MS/MS (Q Exactive Orbitrap, Thermo) with UHPLC; Proteome Discoverer/SequestHT; Immunoblotting (IgE, IgG from 14 allergic patients)	UniProt C. avellana protein database	Autoclaving (especially with hydration/drying) reduced protein solubility (40–70%), degraded major allergens (Cor a 9, 11, 14), and markedly lowered patient skin test reactivity and IgE binding.	Demonstrates autoclaving, especially with hydration/drying, markedly reduces hazelnut allergenicity; provides proteomic and clinical evidence supporting hypoallergenic hazelnut product development	[36]
Pistachio (Pistacia vera) - Defatted flour from 4 cultivars	Characterize pistachio allergen proteome and compare cultivar-specific expression	Defatting with diethyl ether; extraction with urea + TBS buffer; Bradford assay; reduction (DTT), alkylation (IAA)	SDS-PAGE, 2-DE (IEF + SDS-PAGE); LC–MS/MS (Q Exactive Orbitrap + UltiMate 3000 RSLC nano-UPLC)	UniProtKB P. vera	Identified major allergens Pis v 1 (2S albumin), Pis v 2 & Pis v 5 (11S globulins), Pis v 3 (7S vicilin); multiple isoforms of 11S and 7S revealed; Pis v 5 differentially expressed in cultivars	First comprehensive pistachio allergen proteome; confirms dominance of cupin family allergens; highlights cultivar variation; supports allergen risk assessment and breeding for hypoallergenic cultivars	[26]
Macadamia (Macadamia integrifolia and hybrids with M. tetraphylla) - Nuts	Provide proteomic overview of macadamia nut; identify functional protein categories; assess potential allergens and cross-reactivity	Defatting (n-hexane/ethanol); protein extraction (NaCl buffer pH 8.4); acetone precipitation; SDS-PAGE; in-gel trypsin digestion	SDS-PAGE; LC–MS/MS (LTQ-FT Ultra Orbitrap + Ultimate 3000 nano-LC); Mascot and X!Tandem; Scaffold validation; spectral counting; GO/KEGG annotation	NCBI non-redundant Viridiplantae protein database; AllergenOnline (FARRP v16); IEDB-AR (linear epitopes)	Identified 1079 proteins; proteins classified into 46 categories; defense proteins most abundant; identified seed storage proteins; in silico matched macadamia proteins with allergens; cross-reactivity with peanut, walnut, lupin, sesame, birch pollen, fungi, dust mite indicated	First comprehensive macadamia nut proteome; highlights abundant defense and storage proteins; identifies potential allergenic proteins and cross-reactivity risks; provides resource for allergy assessment and breeding	[37]
Peanut (Arachis hypogaea) - Developing seeds at 7 underground stages (R1–R7)	Investigate peanut proteins at different development stages; analyze allergen accumulation and protein interaction networks	TCA/acetone precipitation; phenol extraction; MeOH/NH4OAc precipitation; guanidine-HCl solubilization; reduction (TBP); alkylation (2-VP); urea/thiourea/CHAPS lysis	2-DE (IEF pH 3–10, SDS-PAGE); colloidal CBB staining; densitometry (GS-800); MALDI-TOF/TOF-MS (Sciex 5800); Mascot search; Mfuzz clustering; qRT-PCR validation	Peanut A-genome (A. duranensis) and B-genome (A. ipaensis); EggNOG for functional annotation; STRING v9.1 for PPI network	Identified 264 proteins; clustered into 8 groups; 5 major functional categories; 14 allergen-like proteins identified, expression low at R1–R4, increased sharply from R5–R7; storage proteins mainly in cluster 7	Provides first proteome map of peanut underground seed development; reveals allergen accumulation timing; suggests harvesting immature seeds may lower allergenicity risk	[23]
Cashew (Anacardium occidentale) - Blanched raw nuts	Develop customized protein database; analyze effects of oil-roasting on allergen stability; improve MS peptide targets	Tris-HCl buffer + urea; reduction (DTT), alkylation (IAA); in-solution trypsin/chymotrypsin digestion; C18 cleanup	SDS-PAGE; LC–MS/MS (Q Exactive HF and Plus Orbitrap + UltiMate 3000 UHPLC); DDA and PRM targeted analysis	Customized cashew protein database from UniProt, Phytozome v12.1 genome (primary + selected alternative transcripts), and AUGUSTUS predictions	Identified 6595 peptides; revealed 17 new sequences for major allergens; Ana o 3 (2S) most heat-stable; roasting reduced solubility and abundance of Ana o 1; Maillard adducts identified on 11S peptides;	Provides optimized cashew protein database; reveals isoforms and processing impact on allergenicity; enables high-quality MS-based allergen detection; supports food safety regulation and risk assessment	[38]
Peanut (Arachis hypogaea) - Seeds from high and low-oleate cultivar at 6 developmental stages	Identify proteins linked to oleic acid accumulation during seed development	TCA/acetone precipitation; methanol/ammonium acetate wash; urea/Tris lysis; reduction (DTT), alkylation (IAM); iTRAQ labeling	iTRAQ-based LC–ESI–MS/MS (TripleTOF 5600, SCIEX); Mascot and ProteinPilot search; GO and KEGG annotation; STRING PPI analysis; qRT-PCR validation	UniprotKB peanut protein database; functional annotation with Arabidopsis TAIR and GO annotation; KEGG pathways	7666 proteins identified; 389 DEPs associated with FA metabolism; FAB2 upregulated early in high-oleate cultivar, downregulated later; KAS I/II upregulated early; multiple lipid oxidation enzymes identified; OA biosynthesis enriched at stage 3	Provides molecular insight into oleic acid accumulation dynamics; identifies FAB2 as key regulator; supports breeding strategies for high-oleate peanut varieties with improved oil stability and nutritional quality	[39]
Cashew (Anacardium occidentale) - Cashew nut protein fractions and protein isolate	Characterize molecular and functional properties of cashew protein fractions vs. isolate	Osborne fractionation (water, NaCl, ethanol, NaOH) for fractions; alkali extraction + isoelectric precipitation for protein isolate; petroleum ether defatting	SDS-PAGE (reducing/non-reducing), Circular dichroism spectroscopy, SEM, solubility tests, water/Oil holding capacity, foaming and emulsifying assays, amino acid analyzer (Hitachi L-8800)	N/A	Cashew proteins showed varied functional properties	Demonstrates functional specialization of fractions: albumin/globulin for beverages (solubility), glutelin for bakery/meat (foaming and water/oil binding), protein isolate for emulsified foods; supports cashew as valuable protein source for food industry	[40]
Hazelnut (Corylus avellana) - Nuts	Characterize storage, allergenic, and defense proteins; improve allergen detection	PBS extraction, defatting (hexane), TCA/acetone precipitation, buffer optimization (temp, ratio)	SDS-PAGE, 2-DE, MALDI-TOF, LC–ESI–MS/MS; RP–HPLC; Western blot; ELISA; MS-based targeted detection (SRM/PRM)	NCBI and UniProtKB databases (SwissProt entries for Cor a proteins); Mascot search; AllergenOnline cross-references	Identified major allergens; multiple isoforms and proteoforms; MS marker peptides developed for food allergen detection; roasting altered extractability but not IgE binding significantly	First comprehensive hazelnut proteome; provides allergen catalog and biomarker peptides; supports allergy diagnosis, risk assessment, and food authentication	[41]
Almond (Prunus dulcis) - Nuts	Characterize almond nut proteome; compare with American almonds; identify allergens and stress-related proteins	Phenol extraction with sucrose/KCl/EDTA Tris buffer; methanol–ammonium acetate precipitation; acetone wash; SDS-PAGE and in-gel trypsin digestion	2-DE + SDS-PAGE; LC–MS/MS (Q Exactive Orbitrap); Mascot search; GO annotation (Blast2GO); ImageMaster 2D Elite analysis	NCBI protein database (taxonomy: P. dulcis); UniProt for GO annotation; SwissProt cross-references	Identified 434 proteins (259 novel/hypothetical); 2104 protein spots in 2-DE; allergens Pru du 1, 2, 3, 4, 6 detected; 22 hypothetical proteins with glycosylation motifs; abundant metabolic/catalytic proteins; stress-related proteins	Provides reference almond proteome map; improves understanding of allergenicity, stress response proteins, and nutritional value; supports development of almond protein-based foods	[29]
Walnut (Juglans regia) - Defatted walnut flour (raw and roasted)	Assess effects of roasting on solubility and detectability of walnut allergens	Defatting (hexane); sequential vs. simultaneous extraction/trypsin digestion; DTT, IAA	SDS-PAGE; LC–MS/MS (Orbitrap Elite, UPLC C18); Label-free quantification (Progenesis LC-MS); Mascot search; Maillard adduct screening	UniProt Juglandaceae + UniProt A. hypogaea + AllergenOnline v12 + contaminants	2S albumin (Jug r 1), nsLTP (Jug r 3), 7S N-terminal showed minor roasting effects; Mature 7S (Jug r 2) and 11S (Jug r 4) increased detectability after roasting, likely due to improved digestibility; Maillard adducts mainly on 7S and 11S globulins	Reveals allergen-specific responses to roasting; highlights MS as powerful tool for monitoring allergen modifications; informs allergen risk assessment and MS-based detection in processed foods	[42]
Hazelnut (Corylus avellana) - Defatted flour; water-soluble protein extracts	Evaluate effect of high hydrostatic pressure (HHP) on protein solubility and immunoreactivity	Defatting (n-hexane); soaking in distilled water; extraction with borate saline buffer + PVP; centrifugation; dialysis; freeze-drying	SDS-PAGE; Western blot (IgE sera from 15 allergic patients); 2D-LC fractionation (ProteomeLab PF-2D: chromatofocusing + RP-HPLC); MALDI-TOF/TOF-MS; LC–ESI–MS/MS	SwissProt/NCBI protein databases (taxonomy: C. avellana); Mascot for peptide mass fingerprinting	HHP reduced solubility of major allergens Cor a 9 (11S globulin) and Cor a 11 (7S vicilin); SDS-PAGE and immunoblot showed similar IgE reactivity between control and HHP samples; PF-2D revealed fewer protein peaks in HHP extracts; Cor a 9 and Cor a 11 remained identifiable but less soluble	Demonstrates HHP alters solubility but not IgE-binding of hazelnut allergens; provides proteomic evidence for limited effect of HHP alone on allergenicity; suggests combining HHP with protease digestion for hypoallergenic food development	[43]
Hazelnut (Corylus avellana) - Nuts	Identify and characterize novel IgE-binding proteins in hazelnut	Defatting (diethyl ether); PBS extraction; reduction (DTT), alkylation (IAA); dialysis + lyophilization	SDS-PAGE (reducing and non-reducing), 2-DE; MALDI-TOF-MS; nano-LC–ESI-Q-TOF MS/MS (Mascot, Batch-Tag, Protein Prospector); de novo sequencing; Immunoblotting and inhibition assays; RP-HPLC purification	NCBI and SwissProt databases (green plants); homology search via BLAST/MS-Pattern; incomplete hazelnut genome noted	Identified a novel alkaline subunit (homologous to 11S globulin Cor a 9) as predominant IgE-binding protein; minor reactivity also to Cor a 11, Cor a 9, Cor a 8; de novo peptides showed homology with 11S isoforms from pistachio, cashew, soybean	First evidence of new 11S globulin isoallergen in hazelnut; explains complex IgE-reactivity patterns; provides platform for component-resolved diagnostics and new therapeutic strategies	[44]
Peanut (Arachis hypogaea) - Nuts	Compare proteomes of allergenic vs. hypoallergenic peanut cultivars	Defatting (hexane); extraction with Tris-HCl + urea + thiourea + CHAPS; reduction (DTT), alkylation (IAA); trypsin digestion	2-DE; MALDI-TOF/TOF-MS (AB Sciex 5800); LC–MS/MS; Progenesis PG240 software; Mascot and X!Tandem search; Scaffold validation	NCBI A. hypogaea database + expressed sequence tags	162 protein spots identified; 40 differential spots between allergenic and hypoallergenic lines; allergens Ara h 1, 2, 3, 6 more abundant in allergenic varieties; hypoallergenic types showed lower SSPs and higher stress-response proteins (LEA, HSPs, PR proteins)	Provides proteomic evidence for reduced allergenicity in selected peanut cultivars; supports breeding of hypoallergenic peanuts	[45]
Brazil nut (Bertholletia excelsa) - Nuts	Purify and characterize major 2S albumin isoforms; assess heterogeneity and structural features	Defatting (petroleum ether); water extraction; dialysis; gel filtration (Superdex 75); chromatofocusing	SDS-PAGE, IEF, MALDI-TOF-MS (Bruker Reflex III), RP-HPLC–ESI-MS (Micromass Quattro II), Circular dichroism spectroscopy, Fourier transform-infrared spectroscopy	NCBI protein database (Viridiplantae) searched with Mascot peptide mass fingerprinting	Identified ~10 2S albumin isoforms; heterogeneity due to post-translational processing; all predominantly α-helical; no glycosylation detected	Provides first detailed proteomic and structural characterization of Brazil nut 2S albumin isoforms; explains allergen heterogeneity (Ber e 1); supports allergen risk assessment and structure–function studies	[46]
Pili nut (Canarium ovatum) - Nuts	Characterize storage protein composition and physicochemical properties	Modified Osborne fractionation: water (albumins), NaCl (globulins), ethanol (prolamins), NaOH (glutelins); defatting with hexane	SDS-PAGE (reducing and non-reducing); Native-PAGE; Amino acid analysis (Beckman Analyzer); DSC (MC-2D calorimeter); proximate and mineral analysis	N/A	Protein content ~11.9% of nuts; Globulins major fraction (60.3% of total protein), mainly 11S globulin with 22.6 and 31.6 kDa subunits; Albumins minor (2.9%); No prolamins; Glutelins ~36.8%	First detailed characterization of pili nut proteins; shows similarity to other oilseed 11S globulins (soy, peanut, hemp); supports potential use of pili nut proteins as isolates in food applications	[47]
Peanut (Arachis hypogaea) - Nuts (raw and roasted, aqueous extracts)	Assess roasting effects on major allergens and PTM profiles	Defatting (n-hexane, tetrachloroethylene); aqueous PBS extraction under mild conditions	1D/2D SDS-PAGE; In-gel digestion; nLC–MS/MS (LTQ Orbitrap XL); PTM profiling via PEAKS XPro; Western blot with PTM-specific Abs; ELISA (IgE binding, patient sera)	UniProtKB A. hypogaea + contaminants; PTM search included 313 mods from Unimod	Roasting reduced protein solubility (~4 × lower); Ara h 3 enriched in raw, Ara h 6 in roasted; >40 PTMs; oxidation (Met), deamidation (Asn/Gln), hydroxylation (Pro), carbamoylation (Lys/Arg), and methylation more frequent in roasted; Ara h 1 most extensively modified; PTMs mapped to IgE-binding epitopes.	Provides first semi-quantitative PTM profiling of peanut allergens; shows roasting alters PTM patterns without markedly changing IgE binding; supports understanding of allergen proteoforms in food processing and allergy risk assessment	[48]

Abbreviations: TCA: Trichloroacetic acid; SDS-PAGE: Sodium dodecyl sulfate–polyacrylamide gel electrophoresis; HPLC: High-performance liquid chromatography; MS/MS: Tandem mass spectrometry; NSAF: Normalized spectral abundance factor; SSP: Seed storage protein; PTM: Post-translational modification; HRMS: High-resolution mass spectrometry; CHAPS: 3-[(3-cholamidopropyl) dimethylammonio]-1-propanesulfonate; Q-TOF: Quadrupole time-of-flight; QQQ-MS: Triple quadrupole mass spectrometry; MRM: Multiple reaction monitoring; IgE: Immunoglobulin E; MALDI: Matrix-assisted laser desorption/ionization; LDS-PAGE: Lithium dodecyl sulfate–polyacrylamide gel electrophoresis; DTT: Dithiothreitol; IAA: Iodoacetamide; SPE: Solid-phase extraction; SEC: Size exclusion chromatography; Q-Orbitrap: Quadrupole-Orbitrap high-resolution MS; AIF: All-ion fragmentation; PAL: Precautionary allergen labeling; VITAL: Voluntary Incidental Trace Allergen Labelling; GO: Gene Ontology; GST: Glutathione S-transferase; DAP: Days after pollination; IAM: Iodoacetamide; NaDOC: Sodium deoxycholate; iBAQ/riBAQ: Intensity-based absolute quantification/relative iBAQ; TEM: Transmission electron microscopy; TBP: Tributylphosphane; 2-VP: 2-vinylpyridine; PPI: Protein–protein interaction; DDA: Data-dependent acquisition; PRM: Parallel reaction monitoring; UHPLC: Ultra-high performance liquid chromatography; KEGG: Kyoto Encyclopedia of Genes and Genomes; FAB2: Stearoyl-acyl carrier protein desaturase; KAS: 3-ketoacyl-ACP synthase; SEM: Scanning electron microscopy; nsLTP: Non-specific lipid transfer protein; LEA: Late embryogenesis abundant proteins; HSPs: Heat shock proteins; PR proteins: Pathogenesis-related proteins; PBS: Phosphate-buffered saline; OPA: o-Phthaldialdehyde method.

Manufacturer, city, and country of instruments/reagents (where applicable) are available in the original cited sources.

Proteomics is also being applied to evaluate processing effects on allergenicity and functionality. Comparative analyses have shown that thermal, high-pressure processing, and enzymatic treatments can alter solubility, digestibility, and IgE-binding capacity of major allergens in a protein- and process-dependent manner [33,38,41,43,44]. These findings are guiding the development of processing strategies aimed at reducing allergenic potential while maintaining nutritional and sensory quality.

Beyond allergen research, proteomics has also advanced understanding of nut developmental biology and quality traits. For example, studies in Chinese hickory (Carya cathayensis) have linked lipid droplet structural proteins (oleosins, caleosins, steroleosins, lipid droplet–associated proteins) to oil stability and seed viability [35], while work in peanut has shown that stearoyl-ACP desaturase (FAB2) abundance in early development correlates with oleic acid content and oil oxidative stability [39]. Similar proteomic profiling in other nuts has mapped fatty acid biosynthesis enzymes (e.g., FAD2, FAD3) and their roles in oil composition [49,50,51]. In parallel, in silico digestion of storage proteins has revealed peptides with antihypertensive, antioxidant, and antidiabetic potential [52,53,54]. In macadamia, proteomic profiling has revealed that parental genotype significantly shapes storage protein abundance and post-translational modifications, linking proteomic diversity to nutritional quality, allergenicity, and breeding potential [32].

Furthermore, proteomics is increasingly used to study stress responses and disease resistance, identifying stress-responsive proteins, antioxidant enzymes, and pathogenesis-related proteins in nuts and related tissues [55,56,57,58,59,60]. These findings provide molecular markers for breeding programs targeting resilience and quality improvement.

3. Functional Proteomic Landscape of Nut Seeds

Edible nuts possess a diverse protein profile that supports storage, metabolism, defence, and processing-related activities. In this section, we review the seed proteomes of major commercial nuts—including peanut, almond, walnut, pistachio, cashew, hazelnut, Brazil nut, pecan, macadamia, and pine nut—organized by functional categories. These encompass the principal storage protein families (2S albumins, 7S vicilins, and 11S legumins) and their allergenic counterparts, as well as proteins involved in primary metabolism, abiotic and biotic stress responses, and food-quality traits. Examples are drawn from proteomic studies across multiple nut species.

3.1. Storage Proteins and Allergens in Nuts

The predominant seed storage proteins in edible nuts belong to three conserved families—2S albumins, 7S vicilin-type globulins, and 11S legumin-type globulins [61]. From a proteomic perspective, these proteins are typically among the most abundant features in 2D gel maps or the highest-intensity identifications in LC–MS datasets. Their accumulation patterns during seed maturation have been characterised in several nut species, most notably in peanut, where both 7S vicilins and 11S legumins increase sharply during late maturation, marking the transition from active metabolism to storage deposition [23].

Structurally, 7S vicilins are generally trimeric (~150–200 kDa) and 11S legumins are hexameric (~300–400 kDa), with each legumin subunit post-translationally cleaved into an acidic polypeptide (~30–40 kDa) and a basic polypeptide (~20 kDa) linked by disulfide bonds. The smaller 2S albumins (~10–15 kDa) are rich in intramolecular disulfide bridges, conferring high thermal and proteolytic stability [62]. In addition to these classical reserves, oleosins and caleosins—structural proteins of oil bodies—are also part of the storage apparatus in high-oil nuts such as pecan and macadamia, stabilising lipid droplets and contributing indirectly to seed energy supply. Table 2 lists the major storage protein families and allergenic counterparts identified in common edible nuts.

These proteins represent the main nitrogen and carbon reserves for germination. For example, the 11S legumin-type protein amandin constitutes ~65–70% of total protein in almond nuts [63], while peanut storage proteins (arachins and conarachins) account for ~87–95% of total seed protein [64]. Amino acid profiles vary by species: peanut and almond seed storage proteins are rich in arginine (e.g., ~3.5 g per 100 g protein in peanut), a precursor of nitric oxide beneficial for cardiovascular health [65], whereas Brazil nut storage proteins—particularly Ber e 2—are exceptionally high in sulfur-containing amino acids (~18% methionine and ~8% cysteine) [66].

Seed storage proteins are also the primary allergens in most nuts (Table 2). In peanut, Ara h 1 (7S vicilin), Ara h 2 and Ara h 6 (2S albumins), and Ara h 3 (11S legumin) are the major clinically relevant allergens, with Ara h 2 and Ara h 6 showing pronounced resistance to gastrointestinal proteolysis [67]. In walnut, major allergens include Jug r 1 (2S), Jug r 2 (7S), and Jug r 4 (11S), with Jug r 1 considered a reliable diagnostic marker [68]. Almond similarly contains Pru du 6 (amandin, an 11S legumin) as a major allergen, alongside Pru du 1, a 2S albumin-like protein with some classification uncertainty [69]. Cashew and pistachio (Pistacia vera), both from the Anacardiaceae family, exhibit highly homologous storage protein allergens: Ana o 1/2/3 in cashew (7S/11S/2S) show ~70–80% sequence identity with Pis v 3/2/1 in pistachio, explaining the high rate of cross-reactivity between the two [70]. In hazelnut (Corylus avellana), the storage proteins Cor a 9 (11S globulin) and Cor a 14 (2S albumin) are major allergens linked to true hazelnut food allergy. They serve as biomarkers to differentiate genuine nut allergy from mild oral symptoms caused by birch pollen cross-reactivity [71].

Cross-reactivity is largely driven by the high structural conservation of homologous storage proteins across species. For example, walnut Jug r 1 and Brazil nut Ber e 1 (both 2S albumins) share ~46% sequence identity, leading to IgE co-recognition [72]. This means a person allergic to walnut often reacts to the homologous protein in Brazil nut. Despite their allergenicity to susceptible individuals, these storage proteins are nutritionally valuable and pose no harm to the general population. This creates a challenge for allergy management, as patients allergic to one nut are frequently advised to avoid all nuts. Proteomic tools, however, are helping to dissect such cross-reactivities at the molecular level. For example, epitope mapping of pistachio and cashew allergens has pinpointed shared peptide regions responsible for IgE binding, improving our understanding of cross-allergy mechanisms [73]. Furthermore, the growing catalogue of nut allergens informs the development of component-resolved diagnostics to more accurately identify which specific nuts a patient must avoid.

Table 2. Major Allergenic Protein Families Identified in Edible Nut Species.

Nut Species	2S Albumin (Family)	7S Vicilin (Family)	11S Legumin (Family)	Other Allergenic Proteins (Family)	Note	Ref.
Almond (Prunus dulcis)	Pru du 1 (AMP or 2S albumin)	Pru du 2 (7S conglutin), Pru du 5 (amposin; 7S vicilin), Pru du 8 (antimicrobial protein, related to 7S storage globulin)	Pru du 6 (amandin; 11S legumin)	Pru du 3 (nsLTP), Pru du 4 (profilin), Pru du 7 (Bet v 1-like PR-10 protein), Pru du 10 (mandelonitrile lyase)	Amandin (Pru du 6) is the major almond allergen and can account for ~65% of soluble protein in almonds.	[74]
Brazil Nut (Bertholletia excelsa)	Ber e 1 (2S albumin)	(7S vicilin not well-characterized)	Ber e 2 (11S globulin)	-	Ber e 1 heat-/digestion-stable, key diagnostic marker for true Brazil nut allergy. Ber e 2 also IgE-reactive. Cross-reactivity with other tree nut and seed 2S/11S proteins.	[75]
Cashew (Anacardium occidentale)	Ana o 3 (2S albumin)	Ana o 1 (7S vicilin)	Ana o 2 (11S legumin)	-	2S/7S/11S allergens are all IgE-reactive; contribute to severe reactions and cross-reactivity with pistachio, hazelnut, etc.	[76]
Hazelnut (Corylus avellana)	Cor a 14 (2S albumin)	Cor a 11 (7S vicilin)	Cor a 9 (11S legumin)	Cor a 8 (nsLTP), Cor a 1 (PR-10, Bet v 1 homolog), Cor a 12, 13, 15 (Oleosins)	Cor a 9, 11, 14 are major allergens. Oleosins (Cor a 12, 13, 15) are minor allergens.	[71]
Peanut (Arachis hypogaea)	Ara h 2,6, 7 (2S albumins)	Ara h 1 (7S vicilin)	Ara h 3 (11S legumin; Ara h 4 isoform)	Ara h 8 (Bet v 1–like PR-10 protein), Ara h 9 (nsLTP), Ara h 5 (profilin), Ara h 10/11 (oleosins), Ara h 12/13 (defensins)	Ara h 2 and Ara h 6 are the most potent peanut allergens. Major allergens include Ara h 1 (7S), Ara h 3/4 (11S), Ara h 2/6/7 (2S). At least 17 allergens identified	[77]
Pecan (Carya illinoinensis)	Car i 1 (2S albumin)	Car i 2 (7S vicilin)	Car i 4 (11S legumin)	-	Car i 1, 2 are major allergens, accumulate in late seed filling; show IgE cross-reactivity with walnut, hazelnut.	[78]
Pine Nut (Pinus pinea)	Pin p 1 (2S albumin)	(Globulins present, not allergenic)	(Globulins present, not allergenic)	-	Pin p 1 is the major pine nut allergen. Though less common as an allergen, pine nut allergy can be severe and may cross-react with peanut or tree nut allergens.	[79]
Pistachio (Pistacia vera)	Pis v 1 (2S albumin)	Pis v 3 (7S vicilin)	Pis v 2, 5 (11S globulin)	Pis v 4 (superoxide dismutase)	Pistachio shares major allergens (2S, 7S, 11S) with cashew, causing frequent cross-reactivity. Interestingly, a defense enzyme (Pis v 4) is also an allergen in pistachio.	[80]
Walnut (Juglans regia)	Jug r 1 (2S albumin)	Jug r 2 (7S vicilin)	Jug r 4 (11S legumin-like)	Jug r 3 (nsLTP), Jug r 5 (profilin)	Jug r 4 (11S globulin) is the major walnut allergen and shares homology with 11S proteins from hazelnut and cashew, explaining cross-reactivity.	[81]
Macadamia (Macadamia integrifolia)	(Not yet identified)	Mac i 1 (7S vicilin)	Mac i 2 (11S globulin)	Putative: oleosin, pectin acetylesterase, aspartyl protease	Putative allergens identified by IgE-reactive proteomics; oleosin cross-reacts with hazelnut IgE. Allergy rare but may be severe.	[27]

Table 2. Major Allergenic Protein Families Identified in Edible Nut Species.

Nut Species	2S Albumin (Family)	7S Vicilin (Family)	11S Legumin (Family)	Other Allergenic Proteins (Family)	Note	Ref.
Almond (Prunus dulcis)	Pru du 1 (AMP or 2S albumin)	Pru du 2 (7S conglutin), Pru du 5 (amposin; 7S vicilin), Pru du 8 (antimicrobial protein, related to 7S storage globulin)	Pru du 6 (amandin; 11S legumin)	Pru du 3 (nsLTP), Pru du 4 (profilin), Pru du 7 (Bet v 1-like PR-10 protein), Pru du 10 (mandelonitrile lyase)	Amandin (Pru du 6) is the major almond allergen and can account for ~65% of soluble protein in almonds.	[74]
Brazil Nut (Bertholletia excelsa)	Ber e 1 (2S albumin)	(7S vicilin not well-characterized)	Ber e 2 (11S globulin)	-	Ber e 1 heat-/digestion-stable, key diagnostic marker for true Brazil nut allergy. Ber e 2 also IgE-reactive. Cross-reactivity with other tree nut and seed 2S/11S proteins.	[75]
Cashew (Anacardium occidentale)	Ana o 3 (2S albumin)	Ana o 1 (7S vicilin)	Ana o 2 (11S legumin)	-	2S/7S/11S allergens are all IgE-reactive; contribute to severe reactions and cross-reactivity with pistachio, hazelnut, etc.	[76]
Hazelnut (Corylus avellana)	Cor a 14 (2S albumin)	Cor a 11 (7S vicilin)	Cor a 9 (11S legumin)	Cor a 8 (nsLTP), Cor a 1 (PR-10, Bet v 1 homolog), Cor a 12, 13, 15 (Oleosins)	Cor a 9, 11, 14 are major allergens. Oleosins (Cor a 12, 13, 15) are minor allergens.	[71]
Peanut (Arachis hypogaea)	Ara h 2,6, 7 (2S albumins)	Ara h 1 (7S vicilin)	Ara h 3 (11S legumin; Ara h 4 isoform)	Ara h 8 (Bet v 1–like PR-10 protein), Ara h 9 (nsLTP), Ara h 5 (profilin), Ara h 10/11 (oleosins), Ara h 12/13 (defensins)	Ara h 2 and Ara h 6 are the most potent peanut allergens. Major allergens include Ara h 1 (7S), Ara h 3/4 (11S), Ara h 2/6/7 (2S). At least 17 allergens identified	[77]
Pecan (Carya illinoinensis)	Car i 1 (2S albumin)	Car i 2 (7S vicilin)	Car i 4 (11S legumin)	-	Car i 1, 2 are major allergens, accumulate in late seed filling; show IgE cross-reactivity with walnut, hazelnut.	[78]
Pine Nut (Pinus pinea)	Pin p 1 (2S albumin)	(Globulins present, not allergenic)	(Globulins present, not allergenic)	-	Pin p 1 is the major pine nut allergen. Though less common as an allergen, pine nut allergy can be severe and may cross-react with peanut or tree nut allergens.	[79]
Pistachio (Pistacia vera)	Pis v 1 (2S albumin)	Pis v 3 (7S vicilin)	Pis v 2, 5 (11S globulin)	Pis v 4 (superoxide dismutase)	Pistachio shares major allergens (2S, 7S, 11S) with cashew, causing frequent cross-reactivity. Interestingly, a defense enzyme (Pis v 4) is also an allergen in pistachio.	[80]
Walnut (Juglans regia)	Jug r 1 (2S albumin)	Jug r 2 (7S vicilin)	Jug r 4 (11S legumin-like)	Jug r 3 (nsLTP), Jug r 5 (profilin)	Jug r 4 (11S globulin) is the major walnut allergen and shares homology with 11S proteins from hazelnut and cashew, explaining cross-reactivity.	[81]
Macadamia (Macadamia integrifolia)	(Not yet identified)	Mac i 1 (7S vicilin)	Mac i 2 (11S globulin)	Putative: oleosin, pectin acetylesterase, aspartyl protease	Putative allergens identified by IgE-reactive proteomics; oleosin cross-reacts with hazelnut IgE. Allergy rare but may be severe.	[27]

3.2. Metabolism-Related Proteins in Nut Tissues

Beyond their roles as storage reserves, nuts also express a wide array of enzymes involved in lipid, carbohydrate, and nitrogen metabolism, particularly during seed development. Given the high oil and carbohydrate content of nuts, these metabolic enzymes feature prominently in proteomic profiles. In almond nuts, 434 proteins have been identified, with approximately 67.5% linked to primary metabolic processes [29]. In macadamia, shotgun proteomics revealed enzymes across 26 functional categories, with 11% involved in lipid metabolism, 9% in carbohydrate metabolism, 5% in protein metabolism, and 3% in amino acid metabolism [32]. Similarly, in peanut seeds, 70.9% of metabolic proteins are associated with carbohydrate, amino acid, or lipid pathways [23]. Collectively, these findings underscore the metabolic activity in nut tissues and the central role of energy conversion and biosynthesis during seed filling.

Proteome profiling of developing peanut and walnut illustrates the timed expression of fatty acid biosynthetic enzymes. In peanut, a comprehensive iTRAQ proteomic analysis, using an isobaric tag-based quantification approach, detected the onset of fatty acid synthesis soon after fertilization [82], with acetyl-CoA carboxylase and ketoacyl-ACP synthases upregulated early to channel photosynthate into fatty acids. In the high-oleic cultivar, stearoyl-ACP desaturase (FAB2/SAD, which converts stearic to oleic acid in plastids) abundance rises sharply during early seed development and declines toward maturity, whereas in the low-oleic cultivar, FAB2 abundance decreases in early stages but increases again at late development [39]. This contrasting pattern suggests that early, high FAB2 activity in high-oleic cultivars drives rapid oleic acid accumulation, while late upregulation in low-oleic cultivars may represent a compensatory response to limited oleic acid levels.

The degree of fatty acid unsaturation in nut oils varies by species, and proteomic data highlight corresponding differences in desaturase enzyme activity. Walnut, for instance, is rich in polyunsaturated fatty acids (e.g., linoleate and linolenate) and shows strong upregulation of endoplasmic reticulum desaturases fatty acid desaturase 2 (FAD2) and FAD3 during seed filling, especially during early and mid-developmental stages [51]. In contrast, oleic-acid–rich nuts such as pecan and hickory show lower FAD2 transcript expression during seed development [49,50], a pattern that reduces FAD2 protein accumulation and oleate-to-linoleate desaturation, thereby favoring monounsaturated (oleate-rich) oil profiles. In peanut, a well-characterized FAD2 mutation that limits the conversion of oleic to linoleic acid leads to a dramatic shift in seed oil composition—raising oleic acid content while reducing polyunsaturates. This change is accompanied by widespread alterations in protein expression, particularly during the stearic-to-oleic acid transition stage. Differential abundance of proteins related to carbohydrate metabolism, energy generation, and lipid biosynthesis suggests that the FAD2 mutation reshapes not only lipid profiles but also broader metabolic networks that support oil accumulation [83].

Once synthesized and desaturated, fatty acids are incorporated into triacylglycerols (TAGs) via the Kennedy pathway, with coordinated increases in TAG assembly enzymes during oil deposition. In walnut, the “lipogenesis” stage (~60–88 days post pollination) coincides with a sharp rise in total oil content and lipid metabolic proteins [51]. In peanut, these pathways peak by mid-maturation, after which synthesis slows as accumulation plateaus. Mature seeds also begin storing lipid catabolic enzymes such as lipoxygenases (LOX), which remain dormant until germination, when they initiate TAG breakdown via LOX-dependent pathways [82].

To safely store large amounts of oil in the cytosol, nuts form lipid droplets (oil bodies) stabilized by structural proteins, primarily oleosins (except for chestnut)—small hydrophobic proteins (~15–24 kDa) that prevent droplet coalescence [84]. Proteomic studies confirm the presence of oleosins across multiple nut species. In hazelnut, the oleosins, Cor a 12, 13, and 15 were identified [85], while moderate levels of the allergenic oleosins Ara h 10 and Ara h 11 were detected in the peanut proteome [86]. In walnut, eight oleosin isoforms were detected during seed development, with expression peaking during oil-filling stages [51], paralleling lipid droplet accumulation. Importantly, walnut proteomics revealed that oleosins are not merely structural placeholders but increase sharply in concentration during the key oil-filling phase, reflecting their role in oil droplet proliferation; Caleosins, calcium-binding oil body proteins, are also present [51]. The accumulation of oleosins and presence of caleosins only became evident once a comprehensive walnut protein database was available, highlighting previous under-detection.

Lipid biosynthesis is tightly coupled with carbohydrate metabolism. During seed filling, nuts convert imported sucrose into glycolytic intermediates and fatty acid precursors. For example, peanut seeds import sucrose (~90% of seed sugars), and sucrose synthase and fructokinase genes are expressed to feed glycolysis and downstream lipid synthesis [87]. Notably, during the stearic-to-oleic acid conversion stage, most differentially abundant proteins between high- and low-oleic peanut cultivars were linked to carbohydrate metabolism, underscoring its critical role in lipid accumulation [39]. Although starch is not a major storage form in nuts (except in chestnut), glycolytic enzymes such as fructose-bisphosphate aldolase, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), pyruvate dehydrogenase, and phosphoglucomutase (catalyzing sugar interconversion), peak in early development to supply energy and precursors, such as direct carbon flux toward lipid accumulation [23]. In macadamia, 74 carbohydrate-metabolism-related proteins, including GAPDH, enolase, and pyruvate kinase, have been detected alongside enzymes of galactose, mannose, sucrose, and glyoxylate pathways [37].

In addition, nitrogen assimilation and amino acid biosynthesis are also critical. For example, during early seed filling of peanuts, proteomic analysis revealed key enzymes such as glutamine synthetase (ammonia assimilation), S-adenosylmethionine synthetase (methionine metabolism), argininosuccinate synthase (arginine biosynthesis), and 2-isopropylmalate synthase (leucine synthesis), along with enriched proteasome subunits involved in protein turnover [23]. The active presence of proteolytic machinery suggests that seeds not only synthesize proteins but also recycle them to optimize amino acid availability [23]. This dynamic amino acid metabolism supports the coordinated synthesis of storage proteins and oils, effectively linking nitrogen and carbon metabolism during seed development. At later stages of maturation, nut seeds channel the amassed amino acids mainly into assembling storage proteins, which serve as long-term nitrogen reservoirs for germination.

3.3. Abiotic Stress Response Proteins

Although most abiotic-stress studies in nut crops target leaves or roots, proteomic data show that the nut also plays a role in stress resilience. This is especially relevant in Mediterranean and arid production zones where seed maturation overlaps seasonal drought and heat extremes [88,89].

In peanut, mid-maturation drought triggers metabolic reprogramming, downregulating glycolysis, starch biosynthesis, and fatty-acid metabolism, while increasing storage proteins [90]. These adjustments reflect a shift in carbon and nitrogen allocation under limited water availability. More notably, drought-exposed nut seeds exhibit pronounced induction of late embryogenesis abundant (LEA) proteins, particularly dehydrins (Group 2 LEA), which function as hydrophilic molecular chaperones that stabilize proteins and membranes during water loss [90,91]. These proteins accumulate during the drying (curing) phase of seed maturation, forming a core component of desiccation tolerance machinery [92]. Similar mechanisms are evident in tree nuts. In pistachio seeds, multiple dehydrin isoforms (23–48 kDa) show isoform-specific responses to desiccation: some decline after drying, suggesting storage/regulatory roles, whereas others persist or increase, indicating classical protection [93]. Dehydrins have also been proposed to serve dual roles in pistachio—accumulating in buds under cold/drought and later being remobilised during seed development [94].

In addition to drought, nut seeds also encounter temperature extremes during development and storage. High temperature stress induces a distinct set of molecular safeguards, notably the accumulation of small heat shock proteins (sHSPs). In walnut, several class II sHSPs are enriched in maturing seed coats, likely acting as molecular chaperones that buffer both desiccation and thermal stress during late seed drying [59]. Heat can compromise seed storage protein stability through denaturation and aggregation, a phenomenon well-documented in legumes [95]. Proteomic profiles from thermotolerant genotypes further suggest upregulation of sHSPs, chaperonins, and proteases to maintain protein integrity and embryo viability [59].

Cold stress, particularly during imbibition, also triggers protein-level responses in nuts. A quantitative proteomic analysis of peanut seeds exposed to chilling temperatures during early germination identified over 100 differentially abundant proteins, many associated with hormonal signaling and stress adaptation [96]. Notably, cold-stressed seeds showed reduced cytokinin oxidase levels and corresponding increases in endogenous cytokinins; exogenous cytokinin application further alleviated chilling injury, underscoring the importance of hormone-regulated protein networks in cold tolerance. These proteomic shifts were accompanied by enhanced expression of structural protectants, such as dehydrins and other LEA proteins [96].

In plants, abiotic stresses such as drought, salinity, and chilling often trigger the accumulation of reactive oxygen species (ROS), leading to oxidative damage. Similar mechanisms are evident in nut seeds; for instance, PEG-induced dehydration in walnut significantly elevates ROS levels, posing risks to lipids, proteins, and DNA [97]. To mitigate oxidative damage, drought-tolerant genotypes activate enzymatic defense systems—comprising catalase (CAT), superoxide dismutase (SOD), peroxidase (POD), ascorbate peroxidase (APX), and glutathione S-transferases (GST)—alongside synthesising osmo-protectants like proline and soluble sugars, which work synergistically to preserve redox balance and cellular integrity under water-deficit conditions. In situ proteomic profiling of raw walnut has corroborated the presence and relative abundance of these enzymatic defenses. Specifically, SOD and CAT were identified at approximately 0.32% and 0.07% of total protein, respectively—among the highest-abundance antioxidant enzymes detected in the walnut seed proteome [98]. Comparable findings in developing pecan nuts revealed Cu/Zn-SOD, APX, glutathione reductase, and multiple GST isoforms, forming a coordinated redox buffering network [28]. GSTs detoxify lipid peroxides and electrophilic compounds, while glyoxalase I—also detected in pecan—plays a complementary role in redox regulation by detoxifying methylglyoxal, a cytotoxic byproduct whose accumulation is exacerbated under oxidative stress. Importantly, many of these antioxidant enzymes were first identified in nuts (e.g., almond) via LC-MS/MS [29], demonstrating the capacity of proteomics to uncover low-abundance yet critical defense proteins. This inherent antioxidant machinery likely contributes not only to seed abiotic tolerance but also to postharvest protection against lipid oxidation and rancidity.

3.4. Disease and Pest Defense Proteins (Pathogenesis-Related)

Nuts possess a well-developed innate immune system that relies heavily on a repertoire of pathogenesis-related (PR) proteins for defence against microbial pathogens and insect pests. Unlike vegetative tissues that can rapidly recruit active immune responses, nuts depend primarily on constitutive defense proteins pre-accumulated during development to protect their nutrient-rich tissues throughout dormancy [99]. The full spectrum of PR1 to PR14 proteins has been well characterized in various plants, β-1,3-glucanases (PR-2), chitinases (PR-3/4), thaumatin-like proteins (PR-5), protease inhibitors (PR-6), Bet v 1-like proteins (PR-10), defensins (PR-12), and non-specific lipid transfer proteins (PR-14)—have so far been identified in seeds [100]. Although all major PR classes are known from seeds in general, their occurrence and roles in tree nuts and peanuts remain incompletely characterised.

Peanut is currently the most intensively studied model. In Aspergillus flavus–infected nuts, resistant genotypes accumulate high levels of PR proteins, particularly PR-2 β-1,3-glucanase, whose enzymatic activity rises sharply post-inoculation, whereas susceptible genotypes show little change [101]. Isoform-resolved electrophoretic profiles further indicate that resistant seeds produce multiple β-1,3-glucanase variants, reinforcing the role of this protein in fungal defence. In a separate proteomic study combining chromatographic purification, 2D electrophoresis, and mass spectrometry, Aspergillus parasiticus infection differentially induced protease inhibitors: Bowman–Birk inhibitors (a small, protease inhibitor protein) appeared early in susceptible cultivars, while resistant cultivars mounted a delayed but stronger response, characterised by higher Bowman–Birk levels and specific induction of a Kunitz-type inhibitor (another class of serine protease inhibitor)—both targeting fungal proteases [102].

In tree nuts, studies on PR proteins remain limited, but emerging evidence supports their involvement in pathogen responses. In walnut, for example, a quantitative proteomic analysis of fruit hulls infected with Xanthomonas arboricola pv. juglandis, the causative agent of walnut blight, identified 2046 differentially accumulated proteins, including significant upregulation (log₂ fold-change > 1.5) of several PR-2 and PR-5 isoforms [103]. To support this, transcriptomic analysis during Colletotrichum gloeosporioides infection in walnut revealed strong (up to 60-fold) induction of seven PR-10/Bet v 1-like genes, many of which were co-expressed with chitinases, suggesting a coordinated Bet v1–chitinase defense module [104]. In pistachio, integrated genomic and transcriptomic analyses also pointed to chitinases as potential players in abiotic and biotic stress adaptation, including salinity and fungal resistance [105]. This strategic deployment of multiple PR families ensures that the nutritive tissues of nuts are “armed but not idle,” capable of mounting an effective defense even in the absence of inducible immune signaling. These findings underscore the biological relevance of a core set of PR protein families in the innate immune system of nut seeds and highlight the potential of proteomics-driven approaches to further elucidate their diversity, abundance, and stress-responsive dynamics across nut species.

4. Proteomic Insights into Processing-Induced Changes in Nut Proteins

Nut proteins not only serve structural and defence roles but also undergo marked alterations during processing, influencing allergen stability, digestibility, and functional performance. Heat treatment—whether dry (roasting) or moist (boiling, steaming)—induces protein unfolding, aggregation, solubility changes, and chemical modifications, all of which can now be tracked with LC-MS/MS–based proteomics. In cashew, comparative profiling showed that compact, disulfide-rich 2S albumins persisted after oil roasting at 166 °C for 10 min, whereas multimeric 7S vicilins were almost entirely lost under identical conditions [38]. In walnut, roasting at 180 °C increased the digestibility of 7S and 11S globulins, while 2S albumins and LTPs showed only minor abundance changes [42]. These differences likely reflect structural properties: 2S albumins are compact and stabilized by dense disulfide bonding, whereas 7S and 11S globulins form larger, multimeric assemblies more prone to thermal dissociation.

Moist-heat treatments (boiling, steaming) further illustrate the interplay of denaturation and solubility. When applied after hydration, these treatments substantially reduce allergenicity by promoting the degradation of major allergens including Cor a 8, Cor a 9, Cor a 11, and Cor a 14, with proteomics detecting shifts toward low–molecular weight fragments and fewer intact allergens [36]. In peanuts, nanoLC-MS/MS analysis following blanching revealed a redistribution of allergenic proteins from nut to skin and cooking water. Post-treatment, peanut skins retained all major allergens and remained immunoreactive in IgE-binding assays [45]. Interestingly, phenolic compounds in peanut skins can bind to proteins, thereby masking their detectability—a reminder that plant matrices and polyphenols influence protein extractability during processing.

Thermal processing not only unfolds proteins but also induces specific chemical modifications, which can now be comprehensively profiled by advanced proteomics. Extending structural insights from stability analyses, recent work has revealed how post-translational modifications (PTMs) accumulate on nut allergens during roasting. In roasted peanuts, methionine oxidation was most frequent, while tryptophan hydroxylation, arginine/lysine formylation, and asparagine oxidation were unique to heat-treated samples [48]. These modifications, arising from Maillard and oxidative chemistry, were especially extensive on Ara h 1 (7S globulin), indicating both structural and chemical susceptibility. In cashew, Maillard adducts on peptides from roasted nuts confirmed direct glycation under typical roasting conditions [38]. Such modifications can either stabilize proteins—reducing proteolysis—or disrupt epitopes, lowering allergenicity, depending on severity.

Processing-induced structural and chemical changes directly influence functional properties. High-temperature roasting causes extensive aggregation and loss of solubility in walnut proteins [42] whereas protease-assisted hydrolysis enhanced solubility and emulsifying capacity in almond protein isolates [34]. Proteomic workflows integrating quantitative profiling, PTM mapping, and solubility fractionation can resolve native, aggregated, and hydrolyzed protein states; time-resolved analyses can track functional proteins through roasting or enzymatic treatment. Linking these protein signatures to measured functional outputs—such as interfacial tension, rheology, or textural metrics—could enable predictive models of processing performance. Cultivar-dependent proteomic differences also affect processing outcomes. In walnuts, nut pellicle proteomes from two cultivars at different maturity stages showed variation in phenolic- and heat stress–related proteins, correlating with differences in color stability during roasting [59]. Such protein-level markers may guide breeding for improved appearance, flavour stability, and shelf life.

5. Challenges and Technical Advances in Nut Proteomics

Nuts are botanically seeds and often have extremely high lipid contents (e.g., 50–75% fat in walnuts, pecans, macadamias), which can severely interfere with protein extraction. Many seed proteins are embedded in oil bodies or associated with membranes [106]. A range of extraction methods have been trialed across nut species as summarized in Table 1. Conventional organic defatting (e.g., hexane or petroleum ether) improves extraction but can deplete lipid-associated proteins such as oleosins, including allergenic isoforms. For instance, Johnson et al. [86] reported that peanut oleosin allergens became ~100-fold less abundant after defatting and roasting relative to the major storage proteins Ara h 1/3. Heat processing (roasting, baking) further complicates analysis by inducing Maillard reactions and protein cross-linking, altering solubility and detectability; in cashew, prolonged roasting markedly decreased soluble protein yield [107]. To recover proteins more effectively, optimized extraction protocols tailored to each nut matrix are essential. As shown by Sealey-Voyksner et al. [108] different solvent and precipitation combinations yield variable proteome profiles across nut types.

Another key challenge is the predominance of highly abundant storage proteins, such as 2S albumins, 7S vicillins and 11 S legumins, as we introduced above, which can obscure detection of low-abundance proteins such as enzymes and lesser-known allergens; overlapping peptides from the storage proteins can also cause misidentification of rare proteins. To address this, various depletion strategies have been developed in seed proteome studies, including extraction with aqueous isopropanol [109], protamine sulfate precipitation (compatible with both label-free and tag-based quantification) [110], and CaCl₂-based removal of seed globulins [111]. Moreover, combinatorial peptide ligand libraries and multi-step fractionation can improve access to minor protein components [112,113].

The lack of high-quality genomic and proteomic references for many tree nuts also remains a bottleneck. Nut proteomic studies typically rely on available genomic entries from NCBI or UniPort (Table 1); Unlike model crops, most edible nut species genomes remain unsequenced and incompletely annotated which restricts confident MS/MS spectrum matching. To address this, transcriptome-based protein libraries have been constructed. For example, Pirone-Davies et al. [114] demonstrated that optimizing RNA-Seq-based transcriptome assembly for walnut improved peptide identifications aligned to seed allergen proteins by approximately 20%. Nonetheless, for under-studied or regionally important species, researchers often rely on cross-species homology or de novo sequencing. Although recent genome sequencing efforts in hazelnut, pistachio, walnut, and macadamia are improving the situation [115,116,117,118,119], annotation gaps still remain a key barrier to achieving comprehensive profiling.

Analytical complexity further increases in food safety applications, particularly for allergen detection. Nut allergens may be “hidden” by complex food matrices and by protein modifications occurring during the heating process, high-pressure processing or Maillard chemistry (as introduced in the previous section). Such processing can induce protein denaturation, aggregation, or chemical modifications which in turn alter peptide masses or disrupt conformation or linear epitopes, reducing detectability by antibodies or MS. In theory one can target heat-stable peptide markers, but finding uniquely specific peptides is difficult because of high sequence homology of proteins among related nuts. High-resolution proteomics approaches must therefore focus on selecting heat-stable, species-specific peptide markers that remain detectable post-processing. This is especially important for closely related species such as cashew and pistachio, which share highly homologous allergens. Luparelli et al. [31] demonstrated an LC–MS/MS workflow capable of detecting trace allergens from multiple nuts in baked cookies, down to mg/kg levels, by targeting unique peptides for each species. However, such assays require extensive validation and are not yet widely standardized for industrial use [25].

The field is still underdeveloped compared to staple crops, partly due to the long generation times of tree nuts, which slow breeding and field trials. There is no comprehensive “nut proteome atlas,” and published studies vary widely in scope and methodology, complicating cross-study comparisons. In addition to these experimental advances, bioinformatics tools play an increasingly central role in nut proteomics. Accurate protein identification and quantification in nuts requires the use of well-curated reference databases, allergen repositories, and customized pipelines that can handle extensive sequence homology within seed storage protein families. Public resources such as UniProt, NCBI, and the World Health Organization/International Union of Immunological Societies (WHO/IUIS) Allergen Nomenclature database (www.allergen.org) provide a foundation, while transcriptome-derived libraries can bridge gaps for under-studied species. Modern software platforms (e.g., MaxQuant, Proteome Discoverer, DIA-NN) allow integration of spectral libraries, open modification searches, and label-free quantification, thereby improving the resolution of allergen detection and the identification of post-translational modifications induced by processing [120,121,122]. Targeted approaches such as Skyline or Spectronaut further enable validation of species-specific peptide markers for regulatory applications [123,124].

Alongside bioinformatics improvements, advances in mass spectrometry technology have transformed nut proteomics. High-resolution instruments such as Orbitrap and ZenoTOF systems offer greater sensitivity, dynamic range, and fragmentation accuracy, facilitating the detection of low-abundance proteins that were previously masked by dominant storage proteins [125]. Data-independent acquisition workflows now allow reproducible profiling of complex nut matrices, while parallel accumulation and serial fragmentation strategies on timsTOF platforms accelerate data collection without compromising depth [126]. The integration of improved MS instrumentation with advanced computational pipelines is therefore essential for comprehensive nut proteomics, enabling both discovery-driven analyses and targeted applications in allergen risk management and breeding.

6. Future Prospects in Nut Proteomics

Building on these recent experimental, bioinformatic, and mass spectrometry advances, the future of nut proteomics lies in translating these technical improvements into practical applications. Nut proteomics holds great promise across multiple domains: in nutrition, nut proteomics enables identification of bioactive peptides with cardiometabolic benefits (e.g., angiotensin-converting enzyme- or dipeptidyl peptidase IV-inhibitory activity) [127]; in food safety, it improves allergen detection and risk assessment, informs processing strategies to reduce allergenicity, and supports accurate regulatory labelling [31]; in breeding, it links protein profiles to stress tolerance and seed quality traits [128]. In addition, advances in protein structure prediction platforms such as AlphaFold will further expand future applications by enabling detailed modeling of nut storage proteins and allergens. This will improve our understanding of allergenicity and support the identification of previously unrecognized allergenic proteins [129]. In addition, emerging approaches such as single-cell proteomics, although not yet applied to nut seeds, may eventually provide spatial and developmental resolution of protein expression, offering new insights into allergen accumulation and seed quality traits. Translating these advances into practice will require standardized extraction and quantification protocols, genome–proteome integration, and collaborative initiatives such as the WHO/IUIS Allergen Nomenclature database (www.allergen.org), which provides harmonized allergen definitions. As these resources develop, comparative nut proteomics will become increasingly feasible, enabling innovations in health, safety, and sustainable agriculture.