Quantitative Proteomics Strategy Research Articles

4D-FastDIA-based quantitative proteomics reveals protein biomarkers linked to flavor traits between triploid and diploid oysters (Crassostrea angulata)

In this study, the 4D-FastDIA-based quantitative proteomics strategy was employed to identify protein biomarkers linked to flavor traits in triploid and diploid oysters (Crassostrea angulata). A comprehensive analysis identified a total of 61,947 peptides and 9,703 protein groups across four sample groups, with 3,679 common proteins shared among all groups. Differentially expressed proteins (DEPs) were found to be significantly associated with flavor-related traits, revealing notable differences in amino acids, lipids, and nucleotides between triploid and diploid oysters. Enrichment analysis further elucidated significant pathways involved in metabolic processes, biosynthesis, and immune responses. Among the DEPs, 864 were correlated with flavor quality traits, highlighting key proteins such as S-adenosylmethionine synthase and pyruvate kinase as potential biomarkers for flavor traits. These findings provide valuable insights into the molecular mechanisms underlying flavor differences in oysters and offer potential biomarkers for evaluating and enhancing oyster quality.

DIA-based quantitative proteomic analysis on porcine meat quality at different chilling rates

The objective of this study was to evaluate the effects of chilling rate on porcine meat quality from the perspective of proteome using data independent acquisition (DIA)-based quantitative proteomic strategy. M. longissimus thoracis et lumborum (n = 9) was assigned randomly to the control group (3.72 °C/h), very fast chilling-I group (VFC-I, 9.31 °C/h) and VFC-II group (14.43 °C/h). The DIA was used to analyze the difference in proteins under different chilling rates. Results showed that tenderness was improved significantly in meat at the chilling rate of 14.43 °C/h. Seventy-nine differential abundant proteins (fold change > 1.5, P < 0.05), including 46 up-regulated and 33 down-regulated proteins, were identified and mainly involved in carbon metabolism, pyruvate metabolism and proteasome pathways. These pathways indicated that VFC delayed cell metabolism and glycolysis by down-regulating the expression of metabolic enzymes. The tenderness was improved by up-regulating the expression of proteasome and m-calpain.

Investigating the Neuroprotective Effect of Withaferin-A in a Parkinsonian Mouse Model

Parkinson's disease (PD) is an enduring and advancing neurodegenerative disorder that impacts millions worldwide. While options to manage its symptoms are available, a definitive cure remains elusive. To enhance the quality of life for PD patients and to alleviate symptoms, various classes of medications are employed including Levodopa (L-Dopa), Dopamine Agonists and Dopamine Agonists. Within this investigation, we investigated the neuroprotective potential of Withaferin-A within a mouse model of Parkinsonism, utilizing a proteomics methodology. Withaferin A, a natural compound extracted from the plant Withania somnifera, colloquially known as ashwagandha, has been explored for its therapeutic attributes across several conditions including PD. To assess the neuroprotective capacity of Withaferin A (WA), we employed a quantitative proteomics strategy to pinpoint proteins with distinct expression patterns in mouse models of Parkinson's disease (PD) treated with WA and 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). The experimental setup encompassed four groups of mice: Group I as control, group II administered MPTP, group III treated with Withaferin A and group IV receiving Withaferin A treatment. After humane termination and intracardial infusion, the substantia nigra (SN) region of the mice brains was collected. Employing tandem mass tags, quantitative proteomics was executed on the obtained brain samples through an Orbitrap Fusion Mass spectrometer (MS). The subsequent analysis of LC-MS/MS data was conducted via Proteome Discoverer and NCBI's mouse protein database using the MASCOT search engine. This comprehensive exploration unveiled a list of 6044 proteins among which alpha-synuclein was identified. Notably, 133 proteins exhibited differential expression. These differentially expressed proteins played pivotal roles in significant biological processes including cellular processes (signal transduction), metabolic processes (NADP metabolic process) and signaling (dopamine receptor signaling). Crucial pathways uncovered encompass Parkinson's disease and the dopamine receptor-mediated signaling pathway. It is our belief that the insights gleaned from this study provide a foundation for formulating targeted treatment strategies to combat Parkinson's disease, thus potentially enhancing the therapeutic landscape for individuals affected by this condition.

6-Plex Tandem Phosphorus Tags (TPT) for Accurate Quantitative Proteomics.

Isobaric chemical labeling is a widely used strategy for high-throughput quantitative proteomics based on mass spectrometry. However, commercially available reagents have high costs in applications as well as the sensitivity limitations for detection of the trace protein samples. Previously, we developed a 2-plex isobaric labeling strategy based on phosphorus chemistry for ultrasensitive proteome quantification with high accuracy. In this work, 6-plex tandem phosphorus tags (TPT) were developed with 3-fold increase in the multiplexing quantitative capacity compared to the 2-plex isobaric phosphorus reagents introduced previously. High isotope enrichment of 18O labeling was incorporated into the phosphoryl group with three exchangeable oxygen atoms by using commercially available H218O. The combinational incorporations of 18O atom in reporter ions and balance group set up the low-cost foundation for development of multiplex TPT reagents. The novel 6-plex TPT reagents could produce phosphoramidate as unique reporter ions with approximately 1 Da mass difference and thus enable 6-plex quantitative analysis in high-resolution ESI-MS/MS analysis. Using HeLa cell tryptic peptides, we concluded that 6-plex TPT reagents could facilitate large-scale accurate quantitative proteomics with very high labeling efficiency.

Analysis of quality-related proteins in golden pompano (Trachinotus ovatus) fillets with modified atmosphere packaging under superchilling storage

Here, we aimed to study the changes in proteome of golden pompano fillets during post-mortem storage. Tandem mass tags (TMT)-labeled quantitative proteomic strategy was applied to investigate the relationships between protein changes and quality characteristics of modified atmosphere packaging (MAP) fillets during superchilling (-3 ℃) storage. Scanning electron microscopy was used to show that the muscle histology microstructure of fillets was damaged to varying degrees, and low-field nuclear magnetic resonance was used to find that the immobile water and free water in the muscle of fillets changed significantly. Total sulfhydryl content, TCA-soluble peptides and Ca2+-ATPase activity also showed that the fillet protein had a deterioration by oxidation and denaturation. The Fresh (FS), MAP, and air packaging (AP) groups were set. Total of 150 proteins were identified as differential abundant proteins (DAPs) in MAP/FS, while 209 DAPs were in AP/FS group. The KEGG pathway analysis indicated that most DAPs were involved in binding proteins and protein turnover. Correlation analysis found that 52 DAPs were correlated with quality traits. Among them, 8 highly correlated DAPs are expected to be used as potential quality markers for protein oxidation and water-holding capacity. These results provide a further understanding of the muscle deterioration mechanism of packaging golden pompano fillets during superchilling.

Proteomics analysis of histone deacetylase inhibitor-resistant solid tumors reveals resistant signatures and potential drug combinations.

Histone deacetylase inhibitors (HDACis) are important drugs for cancer therapy, but the indistinct resistant mechanisms of solid tumor therapy greatly limit their clinical application. In this study we conducted HDACi-perturbated proteomics and phosphoproteomics analyses in HDACi-sensitive and -resistant cell lines using a tandem mass tag (TMT)-based quantitative proteomic strategy. We found that the ribosome biogenesis proteins MRTO4, PES1, WDR74 and NOP16 vital to tumorigenesis might regulate the tumor sensitivity to HDACi. By integrating HDACi-perturbated protein signature with previously reported proteomics and drug sensitivity data, we predicted and validated a series of drug combination pairs potentially to enhance the sensitivity of HDACi in diverse solid tumor. Functional phosphoproteomic analysis further identified the kinase PDK1 and ROCK as potential HDACi-resistant signatures. Overall, this study reveals the potential HDACi-resistant signatures and may provide promising drug combination strategies to attenuate the resistance of solid tumor to HDACi.

Monitoring drug metabolic pathways through extracellular vesicles in mouse plasma.

The ability to monitor the response of metabolic enzymes to drug exposure in individuals is highly appealing and critical to personalized medicine. Although pharmacogenomics assesses genotypic differences, it does not report changes in metabolic enzyme activities due to environmental factors such as drug interactions. Here, we report a quantitative proteomics strategy to monitor drug metabolic pathways by profiling metabolic enzymes in circulating extracellular vesicles (EVs) upon drug exposure. Mass spectrometry (MS)-based measurement revealed that changes in metabolic enzyme abundance in EVs paralleled those in hepatic cells isolated from liver tissue. Coupling with multiplexed isotopic labeling, we temporally quantified 34 proteins involved in drug absorption, distribution, metabolism, and excretion (ADME) pathways. Out of 44 known ADME proteins in plasma EVs, previously annotated mouse cytochrome P450 3A11 (Cyp3a11), homolog to human CYP3A4, and uridine 5'-diphospho (UDP) glucuronosyltransferase 2A3 (Ugt2a3), increased upon daily rifampicin dosage. Dasatinib, a tyrosine kinase inhibitor to treat leukemia, also elevated Cyp3a11 levels in plasma EVs, but to a lesser extent. Altogether, this study demonstrates that measuring drug enzymes in circulating EVs as an effective surrogate is highly feasible and may transform today's drug discovery and development for personalized medicine.

FGF-23 is a biomarker of RV dysfunction and congestion in patients with HFrEF

There is no biomarker reflecting right ventricular dysfunction in HFrEF patients used in clinical practice. We have aimed to look for a circulating marker of RV dysfunction employing a quantitative proteomic strategy. The Olink Proteomics Multiplex panels (Cardiovascular Disease II, III, Cardiometabolic, and Inflammation Target Panels) identified FGF-23 to be the most differentially abundant (more than 2.5-fold) in blood plasma of HF patients with severe RV dysfunction (n = 30) compared to those with preserved RV function (n = 31). A subsequent ELISA-based confirmatory analysis of circulating FGF-23 in a large cohort of patients (n = 344, 72.7% NYHA III/IV, LVEF 22.5%, 54.1% with moderate/severe RV dysfunction), followed by multivariable regression analysis, revealed that the plasma FGF-23 level was most significantly associated with RV dysfunction grade (p = 0.0004) and congestion in the systemic circulation (p = 0.03), but not with LV-ejection fraction (p = 0.69) or estimated glomerular filtration rate (eGFR, p = 0.08). FGF-23 was associated with the degree of RV dysfunction in both sub-cohorts (i.e. in patients with and without congestion, p < 0.0001). The association between FGF-23 and RV-dysfunction remained significant after the adjustment for BNP (p = 0.01). In contrast, when adjusted for BNP, FGF-23 was no longer associated with LV dysfunction (p = 0.59). The Cox proportional hazard model revealed that circulating FGF-23 was significantly associated with adverse outcomes even after adjusting for BNP, LVEF, RV dysfunction grade and eGFR. Circulating FGF-23 is thus a biomarker of right ventricular dysfunction in HFrEF patients regardless of congestion status.

Loss of ACTL7A causes small head sperm by defective acrosome-acroplaxome-manchette complex

BackgroundActin-like 7 A (ACTL7A) is essential for acrosome formation, fertilization and early embryo development. ACTL7A variants cause acrosome detachment responsible for male infertility and early embryonic arrest. In this study, we aim to explore the additional functions of ACTL7A beyond the process of acrosome biogenesis and investigate the possible underlying mechanisms.MethodsNuclear morphology analysis was used to observe the sperm head shape of ACTL7A-mutated patients. Actl7a knock-out (KO) mouse model was generated. Immunofluorescence and transmission electron microscopy (TEM) were performed to analyze the structure of spermatids during spermiogenesis. Tandem mass tags labeling quantitative proteomics strategy was employed to explore the underlying molecular mechanisms. The expression levels of key proteins in the pathway were analyzed by western blotting. Intracytoplasmic sperm injection (ICSI)-artificial oocyte activation (AOA) technology was utilized to overcome fertilization failure in male mice with a complete knockout of Actl7a.ResultsThe new phenotype of small head sperm associated with loss of ACTL7A in patients was discovered, and further confirmed in Actl7a-KO mice. Immunofluorescence and TEM analyses revealed that the deletion of ACTL7A damaged the formation of acrosome-acroplaxome-manchette complex, leading to abnormalities in the shaping of sperm heads. Moreover, a proteomic analysis of testes from WT and Actl7a-KO mice revealed that differentially expressed genes were notably enriched in PI3K/AKT/mTOR signaling pathway which is strongly associated with autophagy. Inhibition of autophagy via PI3K/AKT/mTOR signaling pathway activation leading to PDLIM1 accumulation might elucidate the hindered development of manchette in Actl7a-KO mice. Remarkably, AOA successfully overcame fertilization failure and allowed for the successful production of healthy offspring from the Actl7a complete knockout male mice.ConclusionsLoss of ACTL7A causes small head sperm as a result of defective acrosome-acroplaxome-manchette complex via autophagy inhibition. ICSI-AOA is an effective technique to rescue male infertility resulting from ACTL7A deletion. These findings provide essential evidence for the diagnosis and treatment of patients suffering from infertility.

Use of Tandem Mass Spectrometry Quantitative Proteomics to Identify Potential Biomarkers to Follow the Effects of Cold and Frozen Storage of Muscle Tissue of Litopenaeus vannamei.

L. vannamei has become one of the most productive species. However, it is susceptible to microbial contamination during fishing, transportation, and storage, which can lead to spoilage and quality deterioration. This study investigates the relationship between changes in the proteome of Litopenaeus vannamei (L. vannamei) muscle and quality characteristics during low-temperature storage using the tandem mass spectrometry technology of quantitative proteomics strategy. The differential expression of proteins under cold storage (4 °C, CS), partial slight freezing (-3 °C, PFS), and frozen storage (-18 °C, FS) conditions was compared with the fresh group (CK), resulting in 1572 proteins identified as differentially expressed. The purpose of this research is to identify potential biochemical markers by analyzing quality changes and relative differential proteins through searches in the UniProt database, Gene Ontology database, and Genome Encyclopedia. Correlation analysis revealed that seven DEPs were significantly related to physical and chemical indicators. Bioinformatics analysis demonstrated that most DEPs are involved in binding proteins, metabolic enzymes, and protein turnover. Additionally, some DEPs were identified as potential biomarkers for muscle decline. These findings contribute to understanding the mechanism of freshness decline in L. vannamei under low-temperature storage and the changes in muscle proteome.

STRAP binds to and promotes the repair of N1-methyldeoxyadenosine in DNA

N1-methyladenosine (m1A) is an important RNA modification that functions in various biological processes by interacting with cellular proteins. However, the binding proteins of N1-methyldeoxyadenosine (1mdA) in DNA remain largely unknown. Herein, we employed a quantitative proteomics strategy to identify the potential binding proteins of 1mdA in human cells. Our results revealed that serine‑threonine kinase receptor-associated protein (STRAP) can bind to 1mdA-carrying DNA. We further demonstrated that STRAP participates in alkylating agent-induced DNA damage response and can promote the repair of 1mdA embedded in DNA. Moreover, we investigated the effects of STRAP on 1mdA-induced perturbation in transcription using a shuttle vector- and next-generation sequencing-based assay, and found that STRAP is involved in the transcriptional bypass of 1mdA in human cells. Together, our study revealed STRAP as a novel 1mdA-binding protein in human cells and provided new insight into the biological implications of STRAP and 1mdA modification in human diseases.

Development of analytical methods fort he quantitative analysis of the enzyme abundance and activity in the arachidonic acid cascade

The arachidonic acid (ARA) cascade is a key regulatory pathway where eicosanoids and other oxylipins are formed via the oxidation of polyunsaturated fatty acids. These oxylipins are involved in controlling physiological functions such as vascular tone, blood clotting and immune regulation. Changes in the oxylipin pattern can be caused by direct enzyme inhibition or modulated gene expression. In order to understand the effect mechanisms modulating the ARA cascade it is essential to not only investigate the metabolite, i.e. oxylipin, concentrations but also the abundances of the enzymes involved in their formation. For this reason, a quantitative targeted proteomics method for the ARA cascade was developed within this thesis.A detailed workflow for method development was established where enzyme/protein levels are measured via sequence‐specific, representative peptides chosen on the basis of several criteria. With liquid chromatography‐tandem mass spectrometry‐based analysis, the cyclooxygenase (COX) pathway of the ARA cascade was investigated in human colon carcinoma cell lines and primary macrophages where strong correlations were found between the oxylipins formed via the COX pathway and the COX‐2 abundance.The modulating effects of polyphenols on the 5‐lipoxygenase (LOX) pathway of the ARA cascade were investigated in cell‐free enzyme assays and human neutrophils. Resveratrol, its dimer ɛ‐viniferin and a resveratrol imine analogue directly inhibited the 5‐LOX with micromolar potencies and modulated the total oxylipin pattern in the neutrophils with remarkably different effects. Genistein did not act as a 5‐LOX inhibitor in the cell‐free assay but the modulated oxylipin pattern in the cells suggests a global cellular interference.The enzymes of the LOX pathway were included in the targeted proteomics method which together with an extended targeted oxylipin metabolomics platform enables a sensitive multi‐ omics analysis of the ARA cascade in a single sample. The comparison of the multiple reaction monitoring (MRM) detection and the MRM^‐based approach with an additional fragmentation step demonstrated that MRM is more favorable in targeted proteomics due to its higher sensitivity, greater linear range and higher multiplexing capacities. The analysis with the multi‐ omics approach showed that the 5 LOX protein is induced together with its product formation during the differentiation of human monocytic THP 1 cells to macrophages. M1‐like human primary macrophages were also characterized by the abundance of 5‐LOX and its activating protein (FLAP), while 15‐LOX and 15‐LOX‐2 dominated the protein pattern of the M2 like macrophages accompanied by high levels of the oxylipins formed via these enzymes. The developed methodology allows mechanistic investigation of a modulation of the ARA cascade as demonstrated by lipo‐poly‐saccharide stimulation as well as pharmaceutical treatment of the cells.Overall, with this thesis, a new quantitative proteomics strategy for the enzymes of the ARA cascade is established which can be easily extended to further proteins. The use of this technique in combination with targeted oxylipin metabolomics has provided new insights into the mechanisms involved in the modulation of the ARA cascade in human immune cells.

An integrated multi-level analysis reveals learning-memory deficits and synaptic dysfunction in the rat model exposure to austere environment

Austere environment existing in tank, submarine and vessel has many risk factors including high temperature and humidity, confinement, noise, hypoxia, and high level of carbon dioxide, which may cause depression and cognitive impairment. However, the underlying mechanism is not fully understood yet. We attempt to investigate the effects of austere environment (AE) on emotion and cognitive function in a rodent model. After 21 days of AE stress, the rats exhibit depressive-like behavior and cognitive impairment. Compared with control group, the glucose metabolic level of the hippocampus is significantly decreased using whole-brain positron emission tomography (PET) imaging, and the density of dendritic spines of the hippocampus is remarkably reduced in AE group. Then, we employ a label-free quantitative proteomics strategy to investigate the differentially abundant proteins in rats' hippocampus. It is striking that the differentially abundant proteins annotated by KEGG enrich in oxidative phosphorylation pathway, synaptic vesicle cycle pathway and glutamatergic synapses pathway. The synaptic vesicle transport related proteins (Syntaxin-1A, Synaptogyrin-1 and SV-2) are down-regulated, resulting in the accumulation of intracellular glutamate. Furthermore, the concentration of hydrogen peroxide and malondialdehyde is increased while the activity of superoxide dismutase and complex I and IV of mitochondria is decreased, indicating that oxidative damage to hippocampal synapses is associated with the cognitive decline. The results of this study offer direct evidence, for the first time, that austere environment can substantially cause learning and memory deficits and synaptic dysfunction in a rodent model via behavioral assessments, PET imaging, label-free proteomics, and oxidative stress tests. SignificanceThe incidence of depression and cognitive decline in military occupations (for example, tanker and submariner) is significantly higher than that of global population. In the present study, we first established novel model to simulate the coexisting risk factors in the austere environment. The results of this study offer the direct evidences, for the first time, that the austere environment can substantially cause learning and memory deficits by altering plasticity of the synaptic transmission in a rodent model via proteomic strategy, PET imaging, oxidative stress and behavioral assessments. These findings provide valuable information to better understand the mechanisms of cognitive impairment.

A gas phase fractionation acquisition scheme integrating ion mobility for rapid diaPASEF library generation.

Data independent acquisition (DIA/SWATH) MS is a primary strategy in quantitative proteomics. diaPASEF is a recent adaptation using trapped ion mobility spectrometry (TIMS) to improve selectivity/sensitivity. Complex DIA spectra are typically analyzed with reference to spectral libraries. The best-established method for generating libraries uses offline fractionation to increase depth of coverage. More recently strategies for spectral library generation based on gas phase fractionation (GPF), where a representative sample is injected serially using narrow DIA windows that cover different mass ranges of the complete precursor space, have been introduced that performed comparably to deep offline fractionation-based libraries. We investigated whether an analogous GPF-based approach that accounts for the ion mobility (IM) dimension is useful for the analysis of diaPASEF data. We developed a rapid library generation approach using an IM-GPF acquisition scheme in the m/z versus 1/K0 space requiring seven injections of a representative sample and compared this with libraries generated by direct deconvolution-based analysis of diaPASEF data or by deep offline fractionation. We found that library generation by IM-GPF outperformed direct library generation from diaPASEF and had performance approaching that of the deep library. This establishes the IM-GPF scheme as a pragmatic approach to rapid library generation for analysis of diaPASEF data.

Contents: Proteomics 5'23

PROTEOMICSVolume 23, Issue 5 2370033 CONTENTSFree Access Contents: Proteomics 5'23 First published: 01 March 2023 https://doi.org/10.1002/pmic.202370033AboutRelatedInformationPDFPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessClose modalShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL No abstract is available for this article. Volume23, Issue5March 20232370033 RelatedInformation RecommendedCurrent two‐dimensional electrophoresis technology for proteomicsAngelika Görg, Walter Weiss, Michael J. Dunn, PROTEOMICSA novel strategy for quantitative proteomics using isotope‐coded protein labelsAlexander Schmidt, Josef Kellermann, Friedrich Lottspeich, PROTEOMICSAffinity prefractionation for MS‐based plasma proteomicsMaria Pernemalm, Rolf Lewensohn, Janne Lehtiö Dr., PROTEOMICSFront CoverPROTEOMICSQuantitative proteomics of parotid saliva in primary Sjögren's syndromeKiran S. Ambatipudi, Stephen Swatkoski, James J. Moresco, Patricia G. Tu, Andreea Coca, Jennifer H. Anolik, Marjan Gucek, Igñacio Sanz, John R. Yates, James E. Melvin, PROTEOMICS

Identification of PRTN3 as a novel biomarker for the diagnosis of early gastric cance

Gastric cancer (GC) remains one of the most common types of cancer worldwide and has a high mortality rate. However, tools for the early detection of gastric cancer are still lacking. Isobaric tagging for relative and absolute quantitation (iTRAQ) proteomic assays were conducted to identify and quantify the differentially expressed proteins (DEPs) in the gastric mucosal tissues of GC patients at different stages. Bioinformatics analysis was used to identify the pathways enriched among the DEPs and candidate marker proteins. The expression levels and distribution of candidate proteins were confirmed by parallel reaction monitoring (PRM) analysis. In this study, by using the iTRAQ quantitative proteomic strategy, we identified 727 and 502 DEPs that were upregulated in EGC vs. PGC and EGC vs. NGC, respectively. These DEPs were mainly involved in the innate immune response and RNA binding. PRTN3 was identified as a marker of early gastric cancer by Gene Ontology enrichment analysis. Furthermore, the PRM assay confirmed the significant overexpression of PRTN3 in EGC gastric mucosa compared to PGC and NGC mucosa. Our data demonstrated that PRTN3 in the gastric mucosa could be used as a novel biomarker to identify patients with early gastric cancer via endoscopy. SignificanceGastric cancer remains one of the most common types of cancer worldwide and has a high mortality rate. Patients with progressive gastric cancer and gastroesophageal junction cancer have a poor prognosis, with a 5-year relative survival rate of 6%. Therefore, early detection and diagnosis of gastric cancer is a key step toward improving the survival rate.The present study identified PRTN3 as a marker of early gastric cancer by an iTRAQ quantitative proteomic strategy. The PRM assay confirmed the significant overexpression of PRTN3 in EGC gastric mucosa compared to PGC and NGC mucosa. This study discovered that PRTN3 in the gastric mucosa could be used as a novel biomarker to identify patients with early gastric cancer via endoscopy.

Benchmarking Bioinformatics Pipelines in Data-Independent Acquisition Mass Spectrometry for Immunopeptidomics

Immunopeptidomes are the peptide repertoires bound by the molecules encoded by the major histocompatibility complex [human leukocyte antigen (HLA) in humans]. These HLA–peptide complexes are presented on the cell surface for immune T-cell recognition. Immunopeptidomics denotes the utilization of tandem mass spectrometry to identify and quantify peptides bound to HLA molecules. Data-independent acquisition (DIA) has emerged as a powerful strategy for quantitative proteomics and deep proteome-wide identification; however, DIA application to immunopeptidomics analyses has so far seen limited use. Further, of the many DIA data processing tools currently available, there is no consensus in the immunopeptidomics community on the most appropriate pipeline(s) for in-depth and accurate HLA peptide identification. Herein, we benchmarked four commonly used spectral library-based DIA pipelines developed for proteomics applications (Skyline, Spectronaut, DIA-NN, and PEAKS) for their ability to perform immunopeptidome quantification. We validated and assessed the capability of each tool to identify and quantify HLA-bound peptides. Generally, DIA-NN and PEAKS provided higher immunopeptidome coverage with more reproducible results. Skyline and Spectronaut conferred more accurate peptide identification with lower experimental false-positive rates. All tools demonstrated reasonable correlations in quantifying precursors of HLA-bound peptides. Our benchmarking study suggests a combined strategy of applying at least two complementary DIA software tools to achieve the greatest degree of confidence and in-depth coverage of immunopeptidome data.

Site-specific quantitative cysteine profiling with data-independent acquisition-based mass spectrometry.

Chemical proteomics methods, such as activity-based protein profiling, have emerged as powerful and versatile tools to annotate the protein functions and targets of bioactive small molecules in complex biological systems. Incorporated with mass spectrometry (MS)-based quantitative proteomics method, changes of protein activities could be captured and investigated with site-specific precision. However, the semi-stochastic nature of data-dependent acquisition and high cost of the isotopic-labeled reagents make it challenging for chemical biology research to systematically and reproducibly analyze a large number of samples in multidimensional analysis and high-throughput screening. In this chapter, we describe an efficient quantitative chemical proteomic strategy, termed DIA-ABPP, with good reproducibility and high quantification accuracy. Cysteinome profiling was used as a proof-of-concept example with the detailed protocol to demonstrate the workflow of the DIA-ABPP method, including dose-dependent analysis of cysteines that are sensitive to modification by a reactive metabolite, screening of a cysteine-reactive fragment library, and profiling of circadian cysteinome fluctuation. This quantitative chemoproteomic strategy would provide an opportunity for in-depth multi-dimensional chemical proteomic profiling and illuminate the function of bioactive small molecules and proteins in complex biological systems.

Dynamic Interactome of PRC2-EZH1 Complex Using Tandem-Affinity Purification and Quantitative Mass Spectrometry.

The Polycomb repressive complex 2 (PRC2) is a well-characterized chromatin regulator of transcription programs acting through H3K27me3 deposition. In mammals, there are two main versions of PRC2 complexes: PRC2-EZH2, which is prevalent in cycling cells, and PRC2-EZH1 where EZH1 replaces EZH2 in post-mitotic tissues. Stoichiometry of PRC2 complex is dynamically modulated during cellular differentiation and various stress conditions. Therefore, unraveling unique architecture of PRC2 complexes under specific biological context through comprehensive and quantitative characterization could provide insight into the underlying mechanistic molecular mechanism in regulation of transcription process. In this chapter, we describe an efficient method which combines tandem-affinity purification (TAP) with label-free quantitative proteomics strategy for studying PRC2-EZH1 complex architecture alterations and identifying novel protein regulators in post-mitotic C2C12 skeletal muscle cells.

Quantitative Proteomic Analysis of Serum Reveals MST1 as a Potential Candidate Biomarker in Spontaneously Diabetic Cynomolgus Monkeys.

The prevalence of type 2 diabetes (T2DM) is increasing globally, creating essential demands for T2DM animal models for the study of disease pathogenesis, prevention, and therapy. A non-human primate model such as cynomolgus monkeys can develop T2DM spontaneously in an age-dependent way similar to humans. In this study, a data-independent acquisition-based quantitative proteomics strategy was employed to investigate the serum proteomic profiles of spontaneously diabetic cynomolgus monkeys compared with healthy controls. The results revealed significant differences in protein abundances. A total of 95 differentially expressed proteins (DEPs) were quantitatively identified in the current study, among which 31 and 64 proteins were significantly upregulated and downregulated, respectively. Bioinformatic analysis revealed that carbohydrate digestion and absorption was the top enriched pathway by the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis. Protein-protein interaction network analysis demonstrated that MST1 was identified as the most connected protein in the network and could be considered as the hub protein. MST1 was significantly and inversely associated with FSG and HbA1c. Furthermore, recent lines of evidence also indicate that MST1 acts as a crucial regulator in regulating hepatic gluconeogenesis to maintain metabolic homeostasis while simultaneously suppressing the inflammatory processes. In conclusion, our study provides novel insights into serum proteome changes in spontaneously diabetic cynomolgus monkeys and points out that the dysregulation of several DEPs may play an important role in the pathogenesis of T2DM.