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Keywords = nano liquid chromatography tandem mass spectrometry (nanoLC–nanoESI MS/MS)

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21 pages, 2506 KiB  
Article
Integrated Gel Electrophoresis and Mass Spectrometry Approach for Detecting and Quantifying Extraneous Milk in Protected Designation of Origin Buffalo Mozzarella Cheese
by Sabrina De Pascale, Giuseppina Garro, Silvia Ines Pellicano, Andrea Scaloni, Stefania Carpino, Simonetta Caira and Francesco Addeo
Foods 2025, 14(7), 1193; https://doi.org/10.3390/foods14071193 - 28 Mar 2025
Cited by 1 | Viewed by 531
Abstract
Ensuring the authenticity of Mozzarella di Bufala Campana (MdBC), a Protected Designation of Origin (PDO) cheese, is essential for regulatory enforcement and consumer protection. This study evaluates a multi-technology analytical platform developed to detect adulteration due to the addition of non-buffalo milk or [...] Read more.
Ensuring the authenticity of Mozzarella di Bufala Campana (MdBC), a Protected Designation of Origin (PDO) cheese, is essential for regulatory enforcement and consumer protection. This study evaluates a multi-technology analytical platform developed to detect adulteration due to the addition of non-buffalo milk or non-PDO buffalo milk in PDO dairy buffalo products. Peripheral laboratories use gel electrophoresis combined with polyclonal antipeptide antibodies for initial screening, enabling the detection of foreign caseins, including those originating outside the PDO-designated regions. For more precise identification, Matrix-Assisted Laser Desorption Ionization Time of Flight Mass Spectrometry (MALDI-TOF-MS) differentiates species by detecting proteotypic peptides. In cases requiring confirmation, nano-liquid chromatography coupled to electrospray tandem mass spectrometry (nano-LC-ESI-MS/MS) is used in central state laboratories for the highly sensitive detection of extraneous milk proteins in PDO buffalo MdBC cheese. On the other hand, analysis of the pH 4.6 soluble fraction from buffalo blue cheese identified 2828 buffalo-derived peptides and several bovine specific peptides, confirming milk adulteration. Despite a lower detection extent in the pH 4.6 insoluble fraction following tryptic hydrolysis, the presence of bovine peptides was still sufficient to verify fraud. This integrated proteomic approach, which combines electrophoresis and mass spectrometry technologies, significantly improves milk adulteration detection, providing a robust tool to face increasingly sophisticated fraudulent practices. Full article
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16 pages, 2684 KiB  
Article
Identification of the Hepatic Metabolites of Flumazenil and their Kinetic Application in Neuroimaging
by Wei-Hsi Chen, Chuang-Hsin Chiu, Shiou-Shiow Farn, Kai-Hung Cheng, Yuan-Ruei Huang, Shih-Ying Lee, Yao-Ching Fang, Yu-Hua Lin and Kang-Wei Chang
Pharmaceuticals 2023, 16(5), 764; https://doi.org/10.3390/ph16050764 - 18 May 2023
Cited by 2 | Viewed by 2524
Abstract
Studies of the neurobiological causes of anxiety disorders have suggested that the γ-aminobutyric acid (GABA) system increases synaptic concentrations and enhances the affinity of GABAA (type A) receptors for benzodiazepine ligands. Flumazenil antagonizes the benzodiazepine-binding site of the GABA/benzodiazepine receptor (BZR) complex [...] Read more.
Studies of the neurobiological causes of anxiety disorders have suggested that the γ-aminobutyric acid (GABA) system increases synaptic concentrations and enhances the affinity of GABAA (type A) receptors for benzodiazepine ligands. Flumazenil antagonizes the benzodiazepine-binding site of the GABA/benzodiazepine receptor (BZR) complex in the central nervous system (CNS). The investigation of flumazenil metabolites using liquid chromatography (LC)-tandem mass spectrometry will provide a complete understanding of the in vivo metabolism of flumazenil and accelerate radiopharmaceutical inspection and registration. The main goal of this study was to investigate the use of reversed-phase high performance liquid chromatography (PR-HPLC), coupled with electrospray ionization triple-quadrupole tandem mass spectrometry (ESI-QqQ MS), to identify flumazenil and its metabolites in the hepatic matrix. Carrier-free nucleophilic fluorination with an automatic synthesizer for [18F]flumazenil, combined with nano-positron emission tomography (NanoPET)/computed tomography (CT) imaging, was used to predict the biodistribution in normal rats. The study showed that 50% of the flumazenil was biotransformed by the rat liver homogenate in 60 min, whereas one metabolite (M1) was a methyl transesterification product of flumazenil. In the rat liver microsomal system, two metabolites were identified (M2 and M3), as their carboxylic acid and hydroxylated ethyl ester forms between 10 and 120 min, respectively. A total of 10–30 min post-injection of [18F]flumazenil showed an immediate decreased in the distribution ratio observed in the plasma. Nevertheless, a higher ratio of the complete [18F]flumazenil compound could be used for subsequent animal studies. [18F] According to in vivo nanoPET/CT imaging and ex vivo biodistribution assays, flumazenil also showed significant effects on GABAA receptor availability in the amygdala, prefrontal cortex, cortex, and hippocampus in the rat brain, indicating the formation of metabolites. We reported the completion of the biotransformation of flumazenil by the hepatic system, as well as [18F]flumazenil’s potential as an ideal ligand and PET agent for the determination of the GABAA/BZR complex for multiplex neurological syndromes at the clinical stage. Full article
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17 pages, 1694 KiB  
Article
In-Depth Analysis of the N-Glycome of Colorectal Cancer Cell Lines
by Di Wang, Valeriia Kuzyk, Katarina Madunić, Tao Zhang, Oleg A. Mayboroda, Manfred Wuhrer and Guinevere S. M. Lageveen-Kammeijer
Int. J. Mol. Sci. 2023, 24(5), 4842; https://doi.org/10.3390/ijms24054842 - 2 Mar 2023
Cited by 5 | Viewed by 3886
Abstract
Colorectal cancer (CRC) is the third most commonly diagnosed cancer and the second leading cause of cancer deaths worldwide. A well-known hallmark of cancer is altered glycosylation. Analyzing the N-glycosylation of CRC cell lines may provide potential therapeutic or diagnostic targets. In [...] Read more.
Colorectal cancer (CRC) is the third most commonly diagnosed cancer and the second leading cause of cancer deaths worldwide. A well-known hallmark of cancer is altered glycosylation. Analyzing the N-glycosylation of CRC cell lines may provide potential therapeutic or diagnostic targets. In this study, an in-depth N-glycomic analysis of 25 CRC cell lines was conducted using porous graphitized carbon nano-liquid chromatography coupled to electrospray ionization mass spectrometry. This method allows for the separation of isomers and performs structural characterization, revealing profound N-glycomic diversity among the studied CRC cell lines with the elucidation of a number of 139 N-glycans. A high degree of similarity between the two N-glycan datasets measured on the two different platforms (porous graphitized carbon nano-liquid chromatography electrospray ionization tandem mass spectrometry (PGC-nano-LC-ESI-MS) and matrix-assisted laser desorption/ionization time of flight-mass spectrometry (MALDI-TOF-MS)) was discovered. Furthermore, we studied the associations between glycosylation features, glycosyltransferases (GTs), and transcription factors (TFs). While no significant correlations between the glycosylation features and GTs were found, the association between TF CDX1 and (s)Le antigen expression and relevant GTs FUT3/6 suggests that CDX1 contributes to the expression of the (s)Le antigen through the regulation of FUT3/6. Our study provides a comprehensive characterization of the N-glycome of CRC cell lines, which may contribute to the future discovery of novel glyco-biomarkers of CRC. Full article
(This article belongs to the Special Issue Glyco-Dynamics and Cell Signaling)
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26 pages, 854 KiB  
Review
Detergent-Assisted Protein Digestion—On the Way to Avoid the Key Bottleneck of Shotgun Bottom-Up Proteomics
by Katerina Danko, Elena Lukasheva, Vladimir A. Zhukov, Viktor Zgoda and Andrej Frolov
Int. J. Mol. Sci. 2022, 23(22), 13903; https://doi.org/10.3390/ijms232213903 - 11 Nov 2022
Cited by 24 | Viewed by 6910
Abstract
Gel-free bottom-up shotgun proteomics is the principal methodological platform for the state-of-the-art proteome research. This methodology assumes quantitative isolation of the total protein fraction from a complex biological sample, its limited proteolysis with site-specific proteases, analysis of the resulted peptides with nanoscaled reversed-phase [...] Read more.
Gel-free bottom-up shotgun proteomics is the principal methodological platform for the state-of-the-art proteome research. This methodology assumes quantitative isolation of the total protein fraction from a complex biological sample, its limited proteolysis with site-specific proteases, analysis of the resulted peptides with nanoscaled reversed-phase high-performance liquid chromatography-(tandem) mass spectrometry (nanoRP-HPLC-MS and MS/MS), protein identification by sequence database search and peptide-based quantitative analysis. The most critical steps of this workflow are protein reconstitution and digestion; therefore, detergents and chaotropic agents are strongly mandatory to ensure complete solubilization of complex protein isolates and to achieve accessibility of all protease cleavage sites. However, detergents are incompatible with both RP separation and electrospray ionization (ESI). Therefore, to make LC-MS analysis possible, several strategies were implemented in the shotgun proteomics workflow. These techniques rely either on enzymatic digestion in centrifugal filters with subsequent evacuation of the detergent, or employment of MS-compatible surfactants, which can be degraded upon the digestion. In this review we comprehensively address all currently available strategies for the detergent-assisted proteolysis in respect of their relative efficiency when applied to different biological matrices. We critically discuss the current progress and the further perspectives of these technologies in the context of its advances and gaps. Full article
(This article belongs to the Section Biochemistry)
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20 pages, 3367 KiB  
Article
Aggregation and Molecular Properties of β-Glucosidase Isoform II in Chayote (Sechium edule)
by Alberto Cruz Rodríguez, Fabiola Anaid Sánchez Esperanza, Eduardo Pérez-Campos, María Teresa Hernández-Huerta, Laura Pérez-Campos Mayoral, Carlos Alberto Matias-Cervantes, Alexis Martínez Barras, Gabriel Mayoral-Andrade, Luis Ángel Santos Pineda, Aymara Judith Díaz Barrita, Edgar Zenteno, Carlos Romero Díaz, Ruth Martínez Cruz, Eduardo Pérez-Campos Mayoral, Edith Alhelí Bernabé Pérez, Alma Dolores Pérez Santiago, María del Socorro Pina-Canseco and Margarito Martínez Cruz
Molecules 2020, 25(7), 1699; https://doi.org/10.3390/molecules25071699 - 8 Apr 2020
Cited by 2 | Viewed by 3979
Abstract
The presence of isoforms of β-glucosidase has been reported in some grasses such as sorghum, rice and maize. This work aims to extract and characterize isoform II in β-glucosidase from S. edule. A crude extract was prepared without buffer solution and adjusted [...] Read more.
The presence of isoforms of β-glucosidase has been reported in some grasses such as sorghum, rice and maize. This work aims to extract and characterize isoform II in β-glucosidase from S. edule. A crude extract was prepared without buffer solution and adjusted to pH 4.6. Contaminating proteins were precipitated at 4 °C for 24 h. The supernatant was purified by chromatography on carboxymethyl cellulose (CMC) column, molecular exclusion on Sephacryl S-200HR, and exchange anionic on QFF column. Electrophoretic analyzes revealed a purified enzyme with aggregating molecular complex on SDS-PAGE, Native-PAGE, and AU-PAGE. Twelve peptides fragments were identified by nano liquid chromatography-tandem mass spectrometry (nano LC-ESI-MS/MS), which presented as 61% identical to Cucurbita moschata β-glucosidase and 55.74% identical to β-glucosidase from Cucumis sativus, another Cucurbitaceous member. The relative masses which contained 39% hydrophobic amino acids ranged from 982.49 to 2,781.26. The enzyme showed a specificity to β-d-glucose with a Km of 4.59 mM, a Vmax value of 104.3 μM∙min−1 and a kcat of 10,087 μM∙min−1 using p-nitrophenyl-β-D-glucopyranoside. The presence of molecular aggregates can be attributed to non-polar amino acids. This property is not mediated by a β-glucosidase aggregating factor (BGAF) as in grasses (maize and sorghum). The role of these aggregates is discussed. Full article
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19 pages, 4621 KiB  
Article
Novel Peptidomic Approach for Identification of Low and High Molecular Weight Tauopathy Peptides Following Calpain Digestion, and Primary Culture Neurotoxic Challenges
by Hamad Yadikar, Connor Johnson, Niko Pafundi, Edwin Mouhawasse, Lynn Nguyen, Isabel Torres, Milin Kurup, Zhihui Yang, Firas Kobeissy, Richard Yost and Kevin K. Wang
Int. J. Mol. Sci. 2019, 20(20), 5213; https://doi.org/10.3390/ijms20205213 - 21 Oct 2019
Cited by 8 | Viewed by 4707
Abstract
Tauopathy is a class of a neurodegenerative disorder linked with tau hyperphosphorylation, proteolysis, and aggregation. Tau can be subjected to proteolysis upon calpain activation in Alzheimer disease (AD), and traumatic brain injury (TBI). We and others have extensively researched calpain-mediated tau breakdown products [...] Read more.
Tauopathy is a class of a neurodegenerative disorder linked with tau hyperphosphorylation, proteolysis, and aggregation. Tau can be subjected to proteolysis upon calpain activation in Alzheimer disease (AD), and traumatic brain injury (TBI). We and others have extensively researched calpain-mediated tau breakdown products (Tau-BDP; 45K, 35K, and 17K). Tau proteolysis might also generate low molecular weight (LMW ≤10K) proteolytic peptides after neurodegenerative damage. In this study, we have subjected purified tau protein (phospho and non-phospho) and mouse brain lysate to calpain-1 digestion to characterize the LMW generated by nano-liquid chromatography coupled to electrospray ionization to tandem mass spectrometry (nano-LC-ESI-MS/MS). We have also challenged differentiated primary cerebrocortical neuronal cultures (CTX) with neurotoxic agents (calcium ionophore calcimycin (A23187), staurosporine (STS), N-methyl-D-aspartate (NMDA), and Maitotoxin (MTX)) that mimic neurodegeneration to investigate the peptidome released into the conditioned cell media. We used a simple workflow in which we fractionate LMW calpain-mediated tau peptides by ultrafiltration (molecular weight cut-off value (MWCO) of 10K) and subject filtrate fractions to nano-LC-MS/MS analysis. The high molecular weight (HMW) peptides and intact proteins retained on the filter were analyzed separately by western blotting using total and phospho-specific tau antibodies. We have identified several novel proteolytic tau peptides (phosphorylated and non-phosphorylated) that are only present in samples treated with calpain or cell-based calpain activation model (particularly N- and C-terminal peptides). Our findings can help in developing future research strategies emphasizing on the suppression of tau proteolysis as a target. Full article
(This article belongs to the Special Issue Biological Systems at the Protein Level)
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16 pages, 2323 KiB  
Article
Prediction of Bioactive Peptides from Chlorella sorokiniana Proteins Using Proteomic Techniques in Combination with Bioinformatics Analyses
by Lhumen A. Tejano, Jose P. Peralta, Encarnacion Emilia S. Yap, Fenny Crista A. Panjaitan and Yu-Wei Chang
Int. J. Mol. Sci. 2019, 20(7), 1786; https://doi.org/10.3390/ijms20071786 - 11 Apr 2019
Cited by 64 | Viewed by 8076
Abstract
Chlorella is one of the most nutritionally important microalgae with high protein content and can be a good source of potential bioactive peptides. In the current study, isolated proteins from Chlorella sorokiniana were subjected to in silico analysis to predict potential peptides with [...] Read more.
Chlorella is one of the most nutritionally important microalgae with high protein content and can be a good source of potential bioactive peptides. In the current study, isolated proteins from Chlorella sorokiniana were subjected to in silico analysis to predict potential peptides with biological activities. Molecular characteristics of proteins were analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and proteomics techniques. A total of eight proteins were identified by proteomics techniques from 10 protein bands of the SDS-PAGE. The predictive result by BIOPEP’s profile of bioactive peptides tools suggested that proteins of C. sorokiniana have the highest number of dipeptidyl peptidase-IV (DPP IV) inhibitors, with high occurrence of other bioactive peptides such as angiotensin-I converting enzyme (ACE) inhibitor, glucose uptake stimulant, antioxidant, regulating, anti-amnestic and antithrombotic peptides. In silico analysis of enzymatic hydrolysis revealed that pepsin (pH > 2), bromelain and papain were proteases that can release relatively larger quantity of bioactive peptides. In addition, combinations of different enzymes in hydrolysis were observed to dispense higher numbers of bioactive peptides from proteins compared to using individual proteases. Results suggest the potential of protein isolated from C. sorokiniana could be a source of high value products with pharmaceutical and nutraceutical application potential. Full article
(This article belongs to the Special Issue Peptides for Health Benefits 2019)
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12 pages, 9064 KiB  
Article
Rapid Identification of Dipeptidyl Peptidase-IV (DPP-IV) Inhibitory Peptides from Ruditapes philippinarum Hydrolysate
by Rui Liu, Lei Zhou, Yan Zhang, Nai-Juan Sheng, Zhi-Kang Wang, Ti-Zhi Wu, Xin-Zhi Wang and Hao Wu
Molecules 2017, 22(10), 1714; https://doi.org/10.3390/molecules22101714 - 13 Oct 2017
Cited by 62 | Viewed by 5590
Abstract
Dipeptidyl peptidase-IV (DPP-IV) inhibitory peptides were rapidly identified from Ruditapes philippinarum hydrolysate. The hydrolysate was fractionated by ethanol precipitation and preparative reverse phase high-performance liquid chromatography (RP-HPLC). The fraction which showed the highest DPP-IV inhibitory activity was then analyzed by a high-throughput nano-liquid [...] Read more.
Dipeptidyl peptidase-IV (DPP-IV) inhibitory peptides were rapidly identified from Ruditapes philippinarum hydrolysate. The hydrolysate was fractionated by ethanol precipitation and preparative reverse phase high-performance liquid chromatography (RP-HPLC). The fraction which showed the highest DPP-IV inhibitory activity was then analyzed by a high-throughput nano-liquid chromatography electrospray ionization tandem mass spectrometry (nano-LC ESI-MS/MS) method, and the sequences of peptides were identified based on the MS/MS spectra against the Mollusca protein data from the UniProt database. In total, 50 peptides were identified. Furthermore, molecular docking was used to identify potential DPP-IV inhibitors from the identified peptides. Docking results suggested that four peptides: FAGDDAPR, LAPSTM, FAGDDAPRA, and FLMESH, could bind pockets of DPP-IV through hydrogen bonds, π-π bonds, and charge interactions. The four peptides were chemically synthesized and tested for DPP-IV inhibitory activity. The results showed that they possessed DPP-IV inhibitory activity with IC50 values of 168.72 μM, 140.82 μM, 393.30 μM, and >500 μM, respectively. These results indicate that R. philippinarum-derived peptides may have potential as functional food ingredients for the prevention of diabetes. Full article
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