Search Results (44)

Background: Type 2 diabetes mellitus (T2DM) is an epidemic chronic disease that affects millions of people worldwide. This study aims to explore the impact of T2DM on the tear proteome, specifically investigating whether alterations occur before the development of diabetic retinopathy. Methods: Flush tear samples were collected from healthy subjects and subjects with preDM and T2DM. Tear proteins were processed and analyzed by mass spectrometry-based shotgun proteomics using a data-independent acquisition parallel acquisition serial fragmentation (diaPASEF) approach. Machine learning algorithms, including random forest, lasso regression, and support vector machine, and statistical tools were used to identify potential biomarkers. Results: Machine learning models identified 17 proteins with high importance in classification. Among these, five proteins (cystatin-S, S100-A11, submaxillary gland androgen-regulated protein 3B, immunoglobulin lambda variable 3–25, and lambda constant 3) exhibited differential abundance across these three groups. No correlations were identified between proteins and clinical assessments of the ocular surface. Notably, the 17 important proteins showed superior prediction accuracy in distinguishing all three groups (healthy, preDM, and T2DM) compared to the five proteins that were statistically significant. Conclusions: Alterations in the tear proteome profile were observed in adults with preDM and T2DM before the clinical diagnosis of ocular abnormality, including retinopathy. Full article

Background and Objectives: In previous studies, we reported that the assessment of the cumulative thermal dose in the crystalline lens, conducted through computational modeling utilizing a supercomputer and the biothermal transport equation, exhibited a significant association with the incidence of nuclear cataracts. In this study, we have investigated the types of proteins that expressed underlying 35.0 °C (normal-temp) and 37.5 °C (warming-temp) by using the shotgun liquid chromatography (LC) with tandem mass spectrometry (MS/MS)-based global proteomic approach. Materials and Methods: We have discussed the changes in protein expression in warmed iHLEC-NY2 cells using Gene Ontology analysis and a label-free semiquantitative method based on spectral counting. Results: In iHLEC-NY2, 615 proteins were detected, including 307 (49.9%) present in both lenses cultured at normal-temp and warming-temp, 130 (21.1%) unique to the lens cultured at normal-temp, and 178 (29.0%) unique to the lens cultured at warming-temp. Furthermore, LC–MS/MS analysis showed that warming decreased the expression of actin, alpha cardiac muscle 1, actin-related protein 2, putative tubulin-like protein alpha-4B, ubiquitin carboxyl-terminal hydrolase 17-like protein 1, ubiquitin-ribosomal protein eL40 fusion protein, ribosome biogenesis protein BMS1 homolog, histone H2B type 1-M, and histone H2A.J. in iHLEC-NY2. Conclusions: The decreases in the specific protein levels of actin, tubulin, ubiquitin, ribosomes, and histones may be related to cataract development under warming conditions. This investigation could provide a critical framework for understanding the correlation between temperature dynamics and the development of nuclear cataracts. Full article

Background: Tear fluid, rich in proteins, is a promising source of novel biomarkers for ocular and systemic health. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) is the primary method for biomarker discovery. Still, factors such as limited sample volume, extracellular protein contamination, and reflex tearing can significantly impact results. Glass microcapillary tubes minimize these issues. Schirmer strips remain the most common collection method due to existing LC-MS/MS protocol optimization. Methods: In this study, we evaluated multiple digestion protocols for the shotgun quantitative LC-MS/MS analysis of small-volume tear fluid samples collected using glass capillary tubes. Protocol optimization was performed using pooled samples and then compared with the analysis of individual samples. Results: Using the optimized protocol, one μL samples were processed using a timsTOF Pro 2 mass spectrometer (Bruker) coupled online with an Evosep One liquid chromatography system (Evosep), leading to the identification of an average of 361 ± 63 proteins in pooled samples and 525 ± 123 proteins in individual small-volume tear fluid samples. Conclusions: This protocol highlights the practicality of using glass capillary tubes for comprehensive LC-MS/MS-based tear proteomics analysis, paving the way for detailed proteomics characterization of individual tear fluid samples rather than pooled samples. By shifting from pooled to individual samples, this approach greatly accelerates tear biomarker discovery, advancing precision and personalized medicine. Full article

Background: Although estrogenic compounds promise therapeutic potential in treating various conditions, concerns regarding their endocrine-disrupting effects have been raised. Current methodologies for screening estrogenicity in rodent models are limited to the female-specific uterotrophic bioassay. Studies have reported enlargement of the seminal vesicles in orchiectomized males treated with estrogens. However, identifying estrogenicity strictly through changes in wet weights is uninformative regarding the molecular mechanisms of these agents. Therefore, protein-based biomarkers can complement and improve the sensitivity of weight-based assessments. To this end, we present a discovery-driven proteomic analysis of 17β-estradiol’s effects on the seminal vesicles. Methods: We treated orchidectomized mice with the hormone for five days and used the vehicle-treated group as a control. Seminal vesicles were analyzed by shotgun approach using data-dependent nanoflow liquid chromatography–tandem mass spectrometry and label-free quantification. Proteins found to be differentially expressed between the two groups were processed through a bioinformatics pipeline focusing on pathway analyses and assembly of protein interaction networks. Results: Out of 668 identified proteins that passed rigorous validation criteria, 133 were regulated significantly by 17β-estradiol. Ingenuity Pathway Analysis^® linked them to several hormone-affected pathways, including those associated with immune function such as neutrophil degranulation. The altered protein interaction networks were also related to functions including endocrine disruption, abnormal metabolism, and therapeutic effects. Conclusions: We identified several potential biomarkers for estrogenicity in mouse seminal vesicles, many of them not previously linked with exogenous 17β-estradiol exposure. Full article

Cow uterine infections pose a challenge in dairy farming, resulting in reproductive disorders. Uterine fluid extracellular vesicles (UF-EVs) play a key role in cell-to-cell communication in the uterus, potentially holding the signs of aetiology for endometritis. We used mass spectrometry-based quantitative shotgun proteomics to compare UF-EV proteomic profiles in healthy cows (H), cows with subclinical (SE) or clinical endometritis (CLE) sampled at 28–35 days postpartum. Functional analysis was performed on embryo cultures with the exposure to different EV types. A total of 248 UF-EV proteins exhibited differential enrichment between the groups. Interestingly, in SE, EV protein signature suggests a slight suppression of inflammatory response compared to CLE-UF-EVs, clustering closer with healthy cows’ profile. Furthermore, CLE-UF-EVs proteomic profile highlighted pathways associated with cell apoptosis and active inflammation aimed at pathogen elimination. In SE-UF-EVs, the regulation of normal physiological status was aberrant, showing cell damage and endometrial repair at the same time. Serine peptidase HtrA1 (HTRA1) emerged as a potential biomarker for SE. Supplementation of CLE- and SE-derived UF-EVs reduced the embryo developmental rates and quality. Therefore, further research is warranted to elucidate the precise aetiology of SE in cattle, and HTRA1 should be further explored as a potential diagnostic biomarker. Full article

The mechanism underlying podocyte dysfunction in minimal change disease (MCD) remains unknown. This study aimed to shed light on the potential pathophysiology of MCD using glomerular proteomic analysis. Shotgun proteomics using label-free quantitative mass spectrometry was performed on formalin-fixed, paraffin-embedded (FFPE) renal biopsies from two groups of samples: control (CTR) and MCD. Glomeruli were excised from FFPE renal biopsies using laser capture microdissection (LCM), and a single-pot solid-phase-enhanced sample preparation (SP3) digestion method was used to improve yield and protein identifications. Principal component analysis (PCA) revealed a distinct separation between the CTR and MCD groups. Forty-eight proteins with different abundance between the two groups (p-value ≤ 0.05 and |FC| ≥ 1.5) were identified. These may represent differences in podocyte structure, as well as changes in endothelial or mesangial cells and extracellular matrix, and some were indeed found in several of these structures. However, most differentially expressed proteins were linked to the podocyte cytoskeleton and its dynamics. Some of these proteins are known to be involved in focal adhesion (NID1 and ITGA3) or slit diaphragm signaling (ANXA2, TJP1 and MYO1C), while others are structural components of the actin and microtubule cytoskeleton of podocytes (ACTR3 and NES). This study suggests the potential of mass spectrometry-based shotgun proteomic analysis with LCM glomeruli to yield valuable insights into the pathogenesis of podocytopathies like MCD. The most significantly dysregulated proteins in MCD could be attributable to cytoskeleton dysfunction or may be a compensatory response to cytoskeleton malfunction caused by various triggers. Full article

Streck tubes are commonly used to collect blood samples to preserve cell-free circulating DNA. They contain imidazolidinyl urea as a formaldehyde-releasing agent to stabilize cells. We investigated whether the released formaldehyde leads to crosslinking of intracellular proteins. Therefore, we employed a shotgun proteomics experiment on human peripheral blood mononuclear cells (PBMCs) that were isolated from blood collected in Streck tubes, EDTA tubes, EDTA tubes containing formaldehyde, or EDTA tubes containing allantoin. The identified crosslinks were validated in parallel reaction monitoring LC/MS experiments. In total, we identified and validated 45 formaldehyde crosslinks in PBMCs from Streck tubes, which were also found in PBMCs from formaldehyde-treated blood, but not in EDTA- or allantoin-treated samples. Most were derived from cytoskeletal proteins and histones, indicating the ability of Streck tubes to fix cells. In addition, we confirm a previous observation that formaldehyde crosslinking of proteins induces a +24 Da mass shift more frequently than a +12 Da shift. The crosslinking capacity of Streck tubes needs to be considered when selecting blood-collection tubes for mass-spectrometry-based proteomics or metabolomic experiments. Full article

Elevated intraocular pressure is considered a major cause of glaucomatous retinal neurodegeneration. To facilitate a better understanding of the underlying molecular processes and mechanisms, we report a study focusing on alterations of the retina proteome by induced ocular hypertension in a rat model of the disease. Glaucomatous processes were modeled through sclerosing the aqueous outflow routes of the eyes by hypertonic saline injections into an episcleral vein. Mass spectrometry-based quantitative retina proteomics using a label-free shotgun methodology identified over 200 proteins significantly affected by ocular hypertension. Various facets of glaucomatous pathophysiology were revealed through the organization of the findings into protein interaction networks and by pathway analyses. Concentrating on retinal neurodegeneration as a characteristic process of the disease, elevated intraocular pressure-induced alterations in the expression of selected proteins were verified by targeted proteomics based on nanoflow liquid chromatography coupled with nano-electrospray ionization tandem mass spectrometry using the parallel reaction monitoring method of data acquisition. Acquired raw data are shared through deposition to the ProteomeXchange Consortium (PXD042729), making a retina proteomics dataset on the selected animal model of glaucoma available for the first time. Full article

The ABC transporter ABCA7 has been found to be aberrantly expressed in a variety of cancer types, including breast cancer. We searched for specific epigenetic and genetic alterations and alternative splicing variants of ABCA7 in breast cancer and investigated whether these alterations are associated with ABCA7 expression. By analyzing tumor tissues from breast cancer patients, we found CpGs at the exon 5–intron 5 boundary aberrantly methylated in a molecular subtype-specific manner. The detection of altered DNA methylation in tumor-adjacent tissues suggests epigenetic field cancerization. In breast cancer cell lines, DNA methylation levels of CpGs in promoter-exon 1, intron 1, and at the exon 5–intron 5 boundary were not correlated with ABCA7 mRNA levels. By qPCR involving intron-specific and intron-flanking primers, we identified intron-containing ABCA7 mRNA transcripts. The occurrence of intron-containing transcripts was neither molecular subtype-specific nor directly correlated with DNA methylation at the respective exon–intron boundaries. Treatment of breast cancer cell lines MCF-7, BT-474, SK-BR3, and MDA-MB-231 with doxorubicin or paclitaxel for 72 h resulted in altered ABCA7 intron levels. Shotgun proteomics revealed that an increase in intron-containing transcripts was associated with significant dysregulation of splicing factors linked to alternative splicing. Full article

Shotgun proteomics has proven to be an attractive alternative for identifying a pathogen and characterizing the antimicrobial resistance genes it produces. Because of its performance, proteotyping of microorganisms by tandem mass spectrometry is expected to become an essential tool in modern healthcare. Proteotyping microorganisms that have been isolated from the environment by culturomics is also a cornerstone for the development of new biotechnological applications. Phylopeptidomics is a new strategy that estimates the phylogenetic distances between the organisms present in the sample and calculates the ratio of their shared peptides, thus improving the quantification of their contributions to the biomass. Here, we established the limit of detection of tandem mass spectrometry proteotyping based on MS/MS data recorded for several bacteria. The limit of detection for Salmonella bongori with our experimental set-up is 4 × 10⁴ colony-forming units from a sample volume of 1 mL. This limit of detection is directly related to the amount of protein per cell and therefore depends on the shape and size of the microorganism. We have demonstrated that identification of bacteria by phylopeptidomics is independent of their growth stage and that the limit of detection of the method is not degraded in presence of additional bacteria in the same proportion. Full article

The common octopus (Octopus vulgaris) is nowadays the most demanded cephalopod species for human consumption. This species was also postulated for aquaculture diversification to supply its increasing demand in the market worldwide, which only relies on continuously declining field captures. In addition, they serve as model species for biomedical and behavioral studies. Body parts of marine species are usually removed before reaching the final consumer as by-products in order to improve preservation, reduce shipping weight, and increase product quality. These by-products have recently attracted increasing attention due to the discovery of several relevant bioactive compounds. Particularly, the common octopus ink has been described as having antimicrobial and antioxidant properties, among others. In this study, the advanced proteomics discipline was applied to generate a common octopus reference proteome to screen potential bioactive peptides from fishing discards and by-products such as ink. A shotgun proteomics approach by liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) using an Orbitrap Elite instrument was used to create a reference dataset from octopus ink. A total of 1432 different peptides belonging to 361 non-redundant annotated proteins were identified. The final proteome compilation was investigated by integrated in silico studies, including gene ontology (GO) term enrichment, pathways, and network studies. Different immune functioning proteins involved in the innate immune system, such as ferritin, catalase, proteasome, Cu/Zn superoxide dismutase, calreticulin, disulfide isomerase, heat shock protein, etc., were found in ink protein networks. Additionally, the potential of bioactive peptides from octopus ink was addressed. These bioactive peptides can exert beneficial health properties such as antimicrobial, antioxidant, antihypertensive, and antitumoral properties and are therefore considered lead compounds for developing pharmacological, functional foods or nutraceuticals. Full article

(1) Background: Untargeted mass spectrometry (MS)-based proteomic analysis is highly amenable to automation. Software algorithms translate raw spectral data into protein information obtained by a comparison to sequence databases. However, the technology has limitations, especially for analytes measured at the limit of detection. In a protein expression study of human gastric biopsies, the question arose whether or not it is possible, as well as sensible, to search for viral proteins in addition to those from the human host. (2) Methods: Experimental data-independent MS data were analyzed using protein sequences for oncoviruses, and BLAST analyses were performed to elucidate the level of sequence homology to host proteins. (3) Results: About one hundred viral proteins were assigned, but there was also up to 43% sequence homology to human proteins. (4) Conclusions: There are at least two reasons why the matches to viral proteins should be used with care. First, it is not plausible that large amounts of viral proteins should be present in human gastric biopsies, so the spectral quality of the peptides derived from viral proteins is likely low. As a consequence, the number of false assignments is high. Second, homologous peptides found both in human and virus proteomes contribute to matching errors. Thus, though shotgun proteomics raw data can technically be analyzed using any database, meaningful results cannot be always expected and a sanity check must be performed. Both instrumentation and bioinformatic processing in MS-based proteomics are continuously improving at lowering the limit of detection even further. Nevertheless, data output should always be controlled in order to avoid the over-interpretation of results. Full article

Motivated by the clinical success of gold(I) metallotherapeutic Auranofin in the effective treatment of both inflammatory and cancer diseases, we decided to prepare, characterize, and further study the [Au(kin)(PPh₃)] complex (1), where Hkin = kinetin, 6-furfuryladenine, for its in vitro anti-cancer and anti-inflammatory activities. The results revealed that the complex (1) had significant in vitro cytotoxicity against human cancer cell lines (A2780, A2780R, PC-3, 22Rv1, and THP-1), with IC₅₀ ≈ 1–5 μM, which was even significantly better than that for the conventional platinum-based drug Cisplatin while comparable with Auranofin. Although its ability to inhibit transcription factor NF-κB activity did not exceed the comparative drug Auranofin, it has been found that it is able to positively influence peroxisome-proliferator-activated receptor-gamma (PPARγ), and as a consequence of this to have the impact of moderating/reducing inflammation. The cellular effects of the complex (1) in A2780 cancer cells were also investigated by cell cycle analysis, induction of apoptosis, intracellular ROS production, activation of caspases 3/7 and disruption of mitochondrial membrane potential, and shotgun proteomic analysis. Proteomic analysis of R2780 cells treated with complex (1) and starting compounds revealed possible different places of the effect of the studied compounds. Moreover, the time-dependent cellular accumulation of copper was studied by means of the mass spectrometry study with the aim of exploring the possible mechanisms responsible for its biological effects. Full article

Quinoa proteins are attracting global interest for their wide amino acid profile and as a promising source for the development of biomedical treatments, including those against immune-mediated diseases. However, information about the bioactivity of quinoa proteins is scarce. In this study, a quinoa grain proteome map obtained by label-free mass spectrometry-based shotgun proteomics was investigated for the identification of quinoa grain proteins with potential immunonutritional bioactivities, including those related to cancer. After carefully examining the sequence similarities of the 1211 identified quinoa grain proteins against already described bioactive proteins from other plant organisms, 71, 48, and 3 of them were classified as antimicrobial peptides (AMPs), oxidative stress induced peptides (OSIPs), and serine-type protease inhibitors (STPIs), respectively, suggesting their potential as immunomodulatory, anti-inflammatory, and anticancer agents. In addition, data interpretation using Venn diagrams, heat maps, and scatterplots revealed proteome similarities and differences with respect to the AMPs, OSIPs, and STPIs, and the most relevant bioactive proteins in the predominant commercial quinoa grains (i.e., black, red, white (from Peru), and royal (white from Bolivia)). The presented proteomics data mining strategy allows easy screening for potentially relevant quinoa grain proteins and commercial classes for immunonutrition, as a basis for future bioactivity testing. Full article

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