High-resolution Mass Spectrometry-based Proteomics Research Articles

Species identification of ivory and bone museum objects using minimally invasive proteomics.

Ivory is a highly prized material in many cultures since it can be carved into intricate designs and have a highly polished surface. Due to its popularity, the animals from which ivory can be sourced are under threat of extinction. Identification of ivory species is not only important for CITES compliance, it can also provide information about the context in which a work was created. Here, we have developed a minimally invasive workflow to remove minimal amounts of material from precious objects and, using high-resolution mass spectrometry-based proteomics, identified the taxonomy of ivory and bone objects from The Metropolitan Museum of Art collection dating from as early as 4000 B.C. We built a proteomic database of underrepresented species based on exemplars from the American Museum of Natural History, and proposed alternative data analysis workflows for samples containing inconsistently preserved organic material. This application demonstrates extensive ivory species identification using proteomics to unlock sequence uncertainties, e.g., Leu/Ile discrimination.

Lactate- and immunomagnetic-purified hiPSC-derived cardiomyocytes generate comparable engineered cardiac tissue constructs.

Three-dimensional engineered cardiac tissue (ECT) using purified human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) has emerged as an appealing model system for the study of human cardiac biology and disease. A recent study reported widely used metabolic (lactate) purification of monolayer hiPSC-CM cultures results in an ischemic cardiomyopathy-like phenotype compared with magnetic antibody-based cell sorting (MACS) purification, complicating the interpretation of studies using lactate-purified hiPSC-CMs. Herein, our objective was to determine if use of lactate relative to MACS-purified hiPSC-CMs affects the properties of resulting hiPSC-ECTs. Therefore, hiPSC-CMs were differentiated and purified using either lactate-based media or MACS. Global proteomics revealed that lactate-purified hiPSC-CMs displayed a differential phenotype over MACS hiPSC-CMs. hiPSC-CMs were then integrated into 3D hiPSC-ECTs and cultured for 4 weeks. Structurally, there was no significant difference in sarcomere length between lactate and MACS hiPSC-ECTs. Assessment of isometric twitch force and Ca2+ transient measurements revealed similar functional performance between purification methods. High-resolution mass spectrometry-based quantitative proteomics showed no significant difference in protein pathway expression or myofilament proteoforms. Taken together, this study demonstrates that lactate- and MACS-purified hiPSC-CMs generate ECTs with comparable structural, functional, and proteomic features, and it suggests that lactate purification does not result in an irreversible change in a hiPSC-CM phenotype.

Potential relevance of salivary legumain for the clinical diagnostic of hand, foot, and mouth disease.

The fight against hand, foot, and mouth disease (HFMD) remains an arduous challenge without existing point-of-care (POC) diagnostic platforms for accurate diagnosis and prompt case quarantine. Hence, the purpose of this salivary biomarker discovery study is to set the fundamentals for the realization of POC diagnostics for HFMD. Whole salivary proteome profiling was performed on the saliva obtained from children with HFMD and healthy children, using a reductive dimethylation chemical labeling method coupled with high-resolution mass spectrometry-based quantitative proteomics technology. We identified 19 upregulated (fold change = 1.5-5.8) and 51 downregulated proteins (fold change = 0.1-0.6) in the saliva samples of HFMD patients in comparison to that of healthy volunteers. Four upregulated protein candidates were selected for dot blot-based validation assay, based on novelty as biomarkers and exclusions in oral diseases and cancers. Salivary legumain was validated in the Singapore (n = 43 healthy, 28 HFMD cases) and Taiwan (n = 60 healthy, 47 HFMD cases) cohorts with an area under the receiver operating characteristic curve of 0.7583 and 0.8028, respectively. This study demonstrates the feasibility of a broad-spectrum HFMD POC diagnostic test based on legumain, a virus-specific host systemic signature, in saliva.

Inhibitory Mechanism of EGCG on Advanced Glycation End Products Formation During Low-Temperature Vacuum Heating of Russian Sturgeon

ABSTRACT Advanced glycation end products (AGEs) are generated in all stages of food thermal treatment. In order to reveal the inhibitory mechanism of AGEs, a range of concentration of (−)-epigallocatechin-3-gallate (EGCG) has been added during low-temperature vacuum heating (LTVH), and high-resolution mass spectrometry-based proteomics was applied for identifying and quantifying N-glycosylated proteins, N-glycosylated peptides, and N-glycosylated sites. The results indicated that, compared with atmospheric cooking, LTVH significantly decreased the generation of AGEs. The addition of EGCG has a positive effect on the inhibition of AGEs when the concentration is lower than 2,000 μg/mL, and 200 μg/mL was the optimal concentration.

Molecular Characterization of Esophageal Squamous Cell Carcinoma Using Quantitative Proteomics

Esophageal squamous cell carcinoma (ESCC) is a heterogeneous cancer associated with a poor prognosis in advanced stages. In India, it is the sixth most common cause of cancer-related mortality. In this study, we employed high-resolution mass spectrometry-based quantitative proteomics to characterize the differential protein expression pattern associated with ESCC. We identified several differentially expressed proteins including PDPN, TOP2A, POSTN and MMP2 that were overexpressed in ESCC. In addition, we identified downregulation of esophagus tissue-enriched proteins such as SLURP1, PADI1, CSTA, small proline-rich proteins such as SPRR3, SPRR2A, SPRR1A, KRT4, and KRT13, involved in squamous cell differentiation. We identified several overexpressed proteins mapped to the 3q24-29 chromosomal region, aligning with CNV alterations in this region reported in several published studies. Among these, we identified overexpression of SOX2, TP63, IGF2BP2 and RNF13 that are encoded by genes in the 3q26 region. Functional enrichment analysis revealed proteins involved in cell cycle pathways, DNA replication, spliceosome, and DNA repair pathways. We identified the overexpression of multiple proteins that play a major role in alleviating ER stress, including SYVN1 and SEL1L. The SYVN1/SEL1L complex is an essential part of the ER quality control machinery clearing misfolded proteins from the ER. SYVN1 is an E3 ubiquitin ligase that ubiquitinates ER-resident proteins. Interestingly, there are also other non-canonical substrates of SYVN1 which are known to play a crucial role in tumor progression. Thus, SYVN1 could be a potential therapeutic target in ESCC.

Regional vulnerability of brain white matter in vanishing white matter

Vanishing white matter (VWM) is a leukodystrophy that primarily manifests in young children. In this disease, the brain white matter is differentially affected in a predictable pattern with telencephalic brain areas being most severely affected, while others remain allegedly completely spared. Using high-resolution mass spectrometry-based proteomics, we investigated the proteome patterns of the white matter in the severely affected frontal lobe and normal appearing pons in VWM and control cases to identify molecular bases underlying regional vulnerability. By comparing VWM patients to controls, we identified disease-specific proteome patterns. We showed substantial changes in both the VWM frontal and pons white matter at the protein level. Side-by-side comparison of brain region-specific proteome patterns further revealed regional differences. We found that different cell types were affected in the VWM frontal white matter than in the pons. Gene ontology and pathway analyses identified involvement of region specific biological processes, of which pathways involved in cellular respiratory metabolism were overarching features. In the VWM frontal white matter, proteins involved in glycolysis/gluconeogenesis and metabolism of various amino acids were decreased compared to controls. By contrast, in the VWM pons white matter, we found a decrease in proteins involved in oxidative phosphorylation. Taken together, our data show that brain regions are affected in parallel in VWM, but to different degrees. We found region-specific involvement of different cell types and discovered that cellular respiratory metabolism is likely to be differentially affected across white matter regions in VWM. These region-specific changes help explain regional vulnerability to pathology in VWM.

P-516 Defective decidualization secretome in severe preeclampsia

Abstract Study question How defective decidualization (DD) induces a differential secretome compromising embryo invasion in severe preeclampsia (sPE). Summary answer DD endometrial stromal cells isolated from sPE patients secrete a differential secretome in response to hormonal stimuli. What is known already Endometrial decidualization is a primary driver of embryo invasion and placentation. This process is highly coordinated by estradiol and progesterone, which transform the endometrial stromal cells (ESCs) into decidualized cells specialized in the secretion of specific molecules. The secretome of ESCs regulates the endometrial differentiation into the decidua, including immune system and endothelial function regulation. Previously, we have demonstrated the existence of in vitro and in vivo defective decidualization (DD) in women who suffered sPE due to an impaired response to hormonal stimuli. However, the molecular mechanism that connects DD with a hostile maternal environment for embryo invasion remains unexplored. Study design, size, duration Human ESCs from women who suffered sPE (n = 3) and women with normal pregnancies as controls (n = 3) were isolated from endometrial biopsies collected at the time of late-secretory phase. ESCs were cultured using an established in vitro decidualization model, including three technical replicates per sample and condition. Culture media (n = 36) were analysed by mass spectrometry to characterize their global secretome, unveiling the differential secreted factors in DD underlaying sPE. The study duration was 12 months. Participants/materials, setting, methods For in vitro decidualization, isolated human ESCs were cultured for three days in the absence or presence of hormonal stimuli (0.5mM cAMP + 1μM MPA) in serum-free media. Culture media from decidualized and non-decidualized conditions were collected to analyse the secreted proteins by high-resolution mass spectrometry-based proteomics. Protein quantification was performed in the MaxQuant and Perseus software. This approach enables the characterization of the global secretory program as well as the discovery of proteins. Main results and the role of chance We identified and quantified a total of 1.117 proteins secreted by ESCs. In the transition from non-decidualized to decidualized status, the secretome of the control group showed significant differences in the abundance of 83 proteins (p-value<0.05), including two classical markers of decidualization (IGFBP1 and PRL), as well as other hormonally regulated proteins (e.g. VEGFA, WNT4 and MFAP2), supporting the validity of our experimental method. Then, we compared the secretome of decidualized ESCs from controls versus sPE patients, unveiling the altered differential secretome linked to DD. We detected 160 proteins significantly deregulated in sPE compared controls (p-value<0.05) during DD, including proteins reported as risk factors of sPE (e.g. LPA and TF), decidualization modulators (e.g. MFAP2 and TRAP1), mediators of maternal-fetal communication (NPTX1) and immune response regulators (e.g. C3, C5, C7, and SERPIN). Downregulated proteins were associated with cytoskeleton organization and small GTPase mediated signal transduction, consistent with defective decidualization. In contrast, upregulated proteins were implicated in complement activation, regulation of humoral and inflammatory response. A heatmap based on the 160 deregulated proteins successfully classified samples into sPE and controls, supporting the existence of a differential secretome in DD linked to sPE. Limitations, reasons for caution Sample size for in vitro decidualization was based on six patients, analysing two conditions and three technical replicates per patient (N total samples=36) to ensure robustness and reproducibility. Samples clustering in the heatmap support the biological variance was higher than the technical variance, supporting the effectiveness of the hallmark identified. Wider implications of the findings ESCs isolated from women who suffered sPE show an aberrant differential secretome linked to DD. These secreted factors into the decidual microenvironment might impact in embryo invasion, supporting the maternal contribution to sPE. Understanding the mechanisms underlying DD might provide new biomarkers for sPE risk prediction. Trial registration number Not applicable

Quantitative Aspects of the Human Cell Proteome.

The number and identity of proteins and proteoforms presented in a single human cell (a cellular proteome) are fundamental biological questions. The answers can be found with sophisticated and sensitive proteomics methods, including advanced mass spectrometry (MS) coupled with separation by gel electrophoresis and chromatography. So far, bioinformatics and experimental approaches have been applied to quantitate the complexity of the human proteome. This review analyzed the quantitative information obtained from several large-scale panoramic experiments in which high-resolution mass spectrometry-based proteomics in combination with liquid chromatography or two-dimensional gel electrophoresis (2DE) were used to evaluate the cellular proteome. It is important that even though all these experiments were performed in different labs using different equipment and calculation algorithms, the main conclusion about the distribution of proteome components (proteins or proteoforms) was basically the same for all human tissues or cells. It follows Zipf's law and has a formula N = A/x, where N is the number of proteoforms, A is a coefficient, and x is the limit of proteoform detection in terms of abundance.

Radiotherapy Side Effects: Comprehensive Proteomic Study Unraveled Neural Stem Cell Degenerative Differentiation upon Ionizing Radiation.

Cranial radiation therapy is one of the most effective treatments for childhood brain cancers. Despite the ameliorated survival rate of juvenile patients, radiation exposure-induced brain neurogenic region injury could markedly impair patients' cognitive functions and even their quality of life. Determining the mechanism underlying neural stem cells (NSCs) response to irradiation stress is a crucial therapeutic strategy for cognitive impairment. The present study demonstrated that X-ray irradiation arrested NSCs' cell cycle and impacted cell differentiation. To further characterize irradiation-induced molecular alterations in NSCs, two-dimensional high-resolution mass spectrometry-based quantitative proteomics analyses were conducted to explore the mechanism underlying ionizing radiation's influence on stem cell differentiation. We observed that ionizing radiation suppressed intracellular protein transport, neuron projection development, etc., particularly in differentiated cells. Redox proteomics was performed for the quantification of cysteine thiol modifications in order to profile the oxidation-reduction status of proteins in stem cells that underwent ionizing radiation treatment. Via conjoint screening of protein expression abundance and redox status datasets, several significantly expressed and oxidized proteins were identified in differentiating NSCs subjected to X-ray irradiation. Among these proteins, succinate dehydrogenase [ubiquinone] flavoprotein subunit, mitochondrial (sdha) and the acyl carrier protein, mitochondrial (Ndufab1) were highly related to neurodegenerative diseases such as Parkinson's disease, Alzheimer's disease, and Huntington's disease, illustrating the dual-character of NSCs in cell differentiation: following exposure to ionizing radiation, the normal differentiation of NSCs was compromised, and the upregulated oxidized proteins implied a degenerative differentiation trajectory. These findings could be integrated into research on neurodegenerative diseases and future preventive strategies.

Engineering of succinyl-CoA metabolism in view of succinylation regulation to improve the erythromycin production

As a novel protein post-translational modification (PTM), lysine succinylation is widely involved in metabolism regulation by altering the activity of catalytic enzymes. Inactivating succinyl-CoA synthetase in Saccharopolyspora erythraea HL3168 E3 was proved significantly inducing the global protein hypersuccinylation. To investigate the effects, succinylome of the mutant strain E3ΔsucC was identified by using a high-resolution mass spectrometry-based proteomics approach. PTMomics analyses suggested the important roles of succinylation on protein biosynthesis, carbon metabolism, and antibiotics biosynthesis in S. erythraea. Enzymatic experiments in vivo and in vitro were further conducted to determine the succinylation regulation in the TCA cycle. We found out that the activity of aconitase (SACE_3811) was significantly inhibited by succinylation in E3ΔsucC, which probably led to the extracellular accumulation of pyruvate and citrate during the fermentation. Enzyme structural analyses indicated that the succinylation of K278 and K373, conservative lysine residues locating around the protein binding pocket, possibly affects the activity of aconitase. To alleviate the metabolism changes caused by succinyl-CoA synthetase inactivation and protein hypersuccinylation, CRISPR interference (CRISPRi) was applied to mildly downregulate the transcription level of gene sucC in E3. The erythromycin titer of the CRISPRi mutant E3-sucC-sg1 was increased by 54.7% compared with E3, which was 1200.5mg/L. Taken together, this work not only expands our knowledge of succinylation regulation in the TCA cycle, but also validates that CRISPRi is an efficient strategy on the metabolic engineering of S. erythraea. KEY POINTS: • We reported the first systematic profiling of the S. erythraea succinylome. • We found that the succinylation regulation on the activity of aconitase. • We enhanced the production of erythromycin by using CRISPRi to regulate the transcription of gene sucC.

Quantitative proteomics approach reveals novel biomarkers and pathological mechanism of keloid.

BackgroundKeloid is a pathological skin scar formation with complex and unclear molecular pathology mechanism. Novel biomarkers and associated mechanisms are needed to improve current therapies.ObjectivesTo identify novel biomarkers and underlying pathological mechanisms of keloids.MethodsSix pairs of keloid scar tissues and corresponding normal skin tissues were quantitatively analyzed by a high‐resolution label‐free mass spectrometry‐based proteomics approach. Differential protein expression data was further analyzed by a comprehensive bioinformatics approach to identify novel biomarkers and mechanistic pathways for keloid formation. Candidate biomarkers were validated experimentally.ResultsIn total, 1359 proteins were identified by proteomic analysis. Of these, 206 proteins exhibited a significant difference in expression between keloid scar and normal skin tissues. RCN3 and CALU were significantly upregulated in keloids. RCN1 and PDGFRL were uniquely expressed in keloids. Pathway analysis suggested that the XBP1‐mediated unfolded protein response (UPR) pathway was involved in keloid formation. Moreover, a PDGFRL centric gene coexpression network was constructed to illustrate its function in skin.Conclusions and Clinical RelevanceOur study proposed four novel biomarkers and highlighted the role of XBP1‐mediated UPR pathway in the pathology of keloids. It provided novel biological insights that contribute to develop novel therapeutic strategies for keloids.

Abundance of plasma proteins in response to divergent ratios of dietary ω6:ω3 fatty acids in gestating and lactating sows using a quantitative proteomics approach

This study aimed to investigate the characteristic proteomic pattern of plasma from sows supplemented with low dietary ω6:ω3 fatty acids (FAs) ratio during gestation and lactation. Two dietary treatments (n = 8 each) comprised either a control ratio of ω6:ω3 FAs (CR, 13:1 during gestation and 10:1 during lactation) or a low ratio (LR, 4:1 during gestation and lactation) by adding soybean oil or linseed oil, respectively. High-resolution mass spectrometry-based quantitative proteomics was applied on plasma (n = 5 each) at day 108 of gestation (G108) and at the end of lactation (L-End), and a total of 379 proteins and 202 master proteins were identified. Out of these, four differentially abundant proteins between LR and CR samples at G108 may relate to serine-type endopeptidase inhibitor activity. Differentially abundant proteins in L-End versus G108 (12 up-regulated and 10 down-regulated) were positively correlated with the events that regulate plasma lipoproteins, stimulus- and defence-responses. These findings demonstrate the benefit of increased dietary ω3 FAs in modifying proteins involved in protective mechanisms against increased stresses in key life cycle phases in pigs. In addition, proteome changes from late gestation to late lactation disclosed the underlying mechanism of pigs in response to reproduction-related stimuli. SignificanceThis study aimed to provide a proteomics insight into the beneficial effects of maternal diet supplementation with a low ω6:ω3 fatty acids ratio, based on previously reported performance and zootechnical data. The results suggest that a low dietary ω6:ω3 fatty acids ratio could enhance the cellular defence mechanisms against increased stresses and in particular to oxidative stress in sows during gestation and lactation, as reflected in proteomic changes of haptoglobin (HP), alpha-1-antitrypsin (SERPINA1) and serum amyloid P-component (APCS). Furthermore, significantly changed proteome profiles in sow plasma between late gestation and lactation phases have been revealed for the first time. This finding identified the adaptation mechanisms of sows to changing physiological events during reproduction.

Lysine Acetylation Stoichiometry Analysis at the Proteome Level.

Lysine acetylation is a widespread posttranslational modification (PTM) in all kingdoms of live. A large number of proteins involved in most of biological pathways are targets of this PTM. The lysine acetylation is a reversible modification controlled by two main groups of enzymes, lysine acetyltransferases responsible for transferring the acetyl group of acetylCoA to the side chain of lysine residues and lysine deacetylases which effectively remove the acetyl tag. Dysregulation of enzymes that control acetylation and/or target proteins have been associated with a growing number of human pathologies. Lysine acetylation is largely a modification that occurs at low stoichiometry at its target sites. Here we describe a method to identify lysine acetylation sites and estimate their site occupancy at the proteome scale. The method relies on a high-resolution mass spectrometry-based proteomics approach, which includes a specific chemical acetylation reaction on unmodified lysine residues that carry heavy isotopes. The procedures described here have been applied to cell line cultures and to clinically relevant samples stored as both snap-frozen and formalin-fixed paraffin-embedded (FFPE) tissues.

Large-scale and high-resolution mass spectrometry-based proteomics profiling defines molecular subtypes of esophageal cancer for therapeutic targeting

Esophageal cancer (EC) is a type of aggressive cancer without clinically relevant molecular subtypes, hindering the development of effective strategies for treatment. To define molecular subtypes of EC, we perform mass spectrometry-based proteomic and phosphoproteomics profiling of EC tumors and adjacent non-tumor tissues, revealing a catalog of proteins and phosphosites that are dysregulated in ECs. The EC cohort is stratified into two molecular subtypes—S1 and S2—based on proteomic analysis, with the S2 subtype characterized by the upregulation of spliceosomal and ribosomal proteins, and being more aggressive. Moreover, we identify a subtype signature composed of ELOA and SCAF4, and construct a subtype diagnostic and prognostic model. Potential drugs are predicted for treating patients of S2 subtype, and three candidate drugs are validated to inhibit EC. Taken together, our proteomic analysis define molecular subtypes of EC, thus providing a potential therapeutic outlook for improving disease outcomes in patients with EC.

Global analysis of lysine 2-hydroxyisobutyrylation in wheat root

Lysine 2-hydroxyisobutyrylation (Khib) is a novel naturally occurring post-translational modification. The system Khib identification at proteomics level has been performed in various species and tissues to characterize the role of Khib in biological activities. However, the study of Khib in plant species is relatively less. In the present study, the first plant root tissues lysine 2-hydroxyisobutyrylome analysis was performed in wheat with antibody immunoprecipitation affinity, high resolution mass spectrometry-based proteomics and bioinformatics analysis. In total, 6328 Khib sites in 2186 proteins were repeatedly identified in three replicates. These Khib proteins showed a wide subcellular location distribution. Function and pathways characterization of these Khib proteins indicated that many cellular functions and metabolism pathways were potentially affected by this modification. Protein and amino acid metabolism related process may be regulated by Khib, especially ribosome activities and proteins biosynthesis process. Carbohydrate metabolism and energy production related processes including glycolysis/gluconeogenesis, TCA cycle and oxidative phosphorylation pathways were also affected by Khib modification. Besides, root sulfur assimilation and transformation related enzymes exhibited Khib modification. Our work illustrated the potential regulation role of Khib in wheat root physiology and biology, which could be used as a useful reference for Khib study in plant root.

A Cytological Atlas of Human Liver Proteome from PROTEOME <sup>SKY</sup>-LIVER <sup>Hu</sup> 2.0, A Publicly Available Database

Liver plays a unique role as a metabolic center of the body, and also performs other important functions such as detoxification and immune response. The regulatory networks are formed between hepatocytes and non-parenchymal liver cells in human liver to maintain liver homeostasis. But an in-depth knowledge of the constituent proteins in different human liver cell types is still lacking. Here, we determine the healthy human liver proteome by measuring four major cell types that build classical liver lobule, hepatocytes (HCs), hepatic stellate cells (HSCs), Kupffer cells (KCs), and liver sinusoidal endothelial cells (LSECs) by high-resolution mass spectrometry-based proteomics. Overall, we quantify a total of 8,354 proteins for four cell types and over 6,000 proteins for each cell type. Analysis of this dataset and regulatory pathway reveals the cellular labor division in human liver follows the pattern that parenchymal cells make the main components of pathways, but non-parenchymal cells trigger the pathways. The cells in human liver show some novel molecular features, HCs maintain KC and LSEC homeostasis by producing cholesterol and ketone body; HSCs participate xenobiotics metabolism as an agent deliver; KCs and LSECs mediate immune response through MHC class II-TLRs and MHC class I-TGFβ cascade, respectively; KCs play a central role in diurnal rhythms regulation through sensing diurnal IGF and temperature flux . Together, this work expands our understandings of liver physiology on the basis of cell type resolved proteomics of healthy human liver and provides a useful resource for future analyses of normal and diseased livers. Funding: This work was partially supported by National Key R&D Program of China (Nos.2018YFA0507502, 2020YFE0202200), National Natural Science Foundation of China (Nos. 82090051, 81770581 and 81570526), and Open Project Program of the State Key Laboratory of Proteomics (Academy of Military Medical Sciences, SKLP-K201801, SKLP-K201901, SKLP-O201902 and SKLP-O202001) Declaration of Interest: There is no competing interest in this study. Ethical Approval: This study was approved by the Ethics Committee of The third medical 407 center of Chinese PLA general hospital (Beijing, China).

Quantitative acetylome analysis reveals involvement of glucosyltransferase acetylation in Streptococcus mutans biofilm formation.

Streptococcus mutans (S. mutans) effectively utilizes dietary sucrose for the exopolysaccharide productions, which are mostly synthesized by the effects of glucosyltransferases (Gtfs). In the present study, the acetylome of S. mutans was identified and quantitative acetylome analysis of the bacterial biofilm growth (SMB) was compared with that of planktonic growth (SMP). The dynamic changes of protein acetylation were quantified using the integrated approach involving TMT labeling and Kac affinity enrichment followed by high-resolution mass spectrometry-based quantitative proteomics. In total, 973 acetylation sites in 445 proteins were identified, among which 617 acetylation sites in 302 proteins were quantitated. The overall analysis indicated that 22.7% of proteins were acetylated. Among the quantified proteins in SMB, the acetylation degree of lysine in 56 sites increased, while that of lysine decreased in 52 sites. In the acetylome of S. mutans, six significantly enriched motifs were identified and obtained including Kac****K, KacF, Kac****R, KacY, KacH, F*Kac. In addition, KEGG pathway-based enrichment analysis indicated significant enrichments in glycolysis/gluconeogenesis, and RNA degradation. Particularly, most downregulated acetylated lysine proteins were glucosyltransferase-SI, glucosyltransferase-I, and glucosyltransferase-S in S. mutans biofilm, which probably reveals a switch-off mechanism for the regulation of glucosyltransferases function during the biofilm development.

Distinct hypertrophic cardiomyopathy genotypes result in convergent sarcomeric proteoform profiles revealed by top-down proteomics

Hypertrophic cardiomyopathy (HCM) is the most common heritable heart disease. Although the genetic cause of HCM has been linked to mutations in genes encoding sarcomeric proteins, the ability to predict clinical outcomes based on specific mutations in HCM patients is limited. Moreover, how mutations in different sarcomeric proteins can result in highly similar clinical phenotypes remains unknown. Posttranslational modifications (PTMs) and alternative splicing regulate the function of sarcomeric proteins; hence, it is critical to study HCM at the level of proteoforms to gain insights into the mechanisms underlying HCM. Herein, we employed high-resolution mass spectrometry-based top-down proteomics to comprehensively characterize sarcomeric proteoforms in septal myectomy tissues from HCM patients exhibiting severe outflow track obstruction (n = 16) compared to nonfailing donor hearts (n = 16). We observed a complex landscape of sarcomeric proteoforms arising from combinatorial PTMs, alternative splicing, and genetic variation in HCM. A coordinated decrease of phosphorylation in important myofilament and Z-disk proteins with a linear correlation suggests PTM cross-talk in the sarcomere and dysregulation of protein kinase A pathways in HCM. Strikingly, we discovered that the sarcomeric proteoform alterations in the myocardium of HCM patients undergoing septal myectomy were remarkably consistent, regardless of the underlying HCM-causing mutations. This study suggests that the manifestation of severe HCM coalesces at the proteoform level despite distinct genotype, which underscores the importance of molecular characterization of HCM phenotype and presents an opportunity to identify broad-spectrum treatments to mitigate the most severe manifestations of this genetically heterogenous disease.

Quantitative proteomics reveals stage-specific protein regulation of triple negative breast cancer.

Triple negative breast cancer (TNBC) is a heterogeneous disease with more aggressive clinical courses than other subtypes of breast cancer. In this study, we performed high-resolution mass spectrometry-based quantitative proteomics with TNBC clinical tissue specimens to explore the early and sensitive diagnostic signatures and potential therapeutic targets for TNBC patients. We performed an iTRAQ labeling coupled LC-MS/MS approach to explore the global proteome in tumor tissues and corresponding para-tumor tissues from 24 patients with grade I-II and grade III primary TNBC. Relative peptide quantification and protein identification were performed by Proteome Discoverer™ software with Mascot search engine. Differentially expressed proteins were analyzed by bioinformatic analyses, including GO function classification annotation and KEGG enrichment analysis. Pathway analyses for protein-protein interactions and upstream regulations of differentially expressed candidates were performed by Ingenuity Pathway Analysis (IPA) software. Totally, 5401 unique proteins were identified and quantified in different stage of TNBCs. 845 proteins were changed in patients with grade I or II TNBC, among which 304 were up-regulated and 541 were down-regulated. Meanwhile, for patients with grade III TNBC, 358 proteins were increased and 651 proteins were decreased. Comparing to para-cancerous tissues, various signaling pathways and metabolic processes, including PPAR pathways, PI3K-Akt pathway, one-carbon metabolism, amino acid synthesis, and lipid metabolism were activated in TNBC cancer tissues. Death receptor signaling was significantly activated in grade I-II TNBCs, however, remarkably inhibited in grade III TNBCs. Western blot experiments were conducted to validate expression levels of CYCS, HMGA1 and XIAP with samples from individual patients. Overall, our proteomic data presented precise quantification of potential signatures, signaling pathways, regulatory networks, and characteristic differences in each clinicopathological subgroup. The proteome provides complementary information for TNBC accurate subtype classification and therapeutic targets research.

Tandem Mass Tag (TMT)-based quantitative proteomics reveals potential targets associated with onset of Sub-clinical Mastitis in cows

Bovine milk is vital for infant nutrition and is a major component of the human diet. Bovine mastitis is a common inflammatory disease of mammary gland in cattle. It alters the immune profile of the animal and lowers the quality and yield of milk causing huge economic losses to dairy industry. The incidence of sub-clinical mastitis (SCM) is higher (25–65% worldwide) than clinical mastitis (CM) (>5%), and frequently progresses to clinical stage due to lack of sensitive and specific detection method. We used quantitative proteomics to identify changes in milk during sub-clinical mastitis, which may be potential biomarkers for developing rapid, non-invasive, sensitive detection methods. We performed comparative proteome analysis of the bovine milk, collected from the Indian hybrid cow Karan Fries. The differential proteome in the milk of Indian crossbred cows during sub-acute and clinical intramammary gland infection has not been investigated to date. Using high-resolution mass spectrometry-based quantitative proteomics of the bovine whey proteins, we identified a total of 1459 and 1358 proteins in biological replicates, out of which 220 and 157 proteins were differentially expressed between normal and infected samples. A total of 82 proteins were up-regulated and 27 proteins were down-regulated, having fold changes of ≥2 and ≤0.8 respectively. Among these proteins, overexpression of CHI3L1, LBP, GSN, GCLC, C4 and PIGR proteins was positively correlated with the events that elicit host defence system, triggering production of cytokines and inflammatory molecules. The appearance of these potential biomarkers in milk may be used to segregate affected cattle from the normal herd and may support mitigation measures for prevention of SCM and CM.