pFind Studio: a computational solution for mass spectrometry-based proteomics
2021
Journal of Proteome Research2021. Schulze, S et al.
Univ Penn, Dept Biol, Philadelphia, PA 19104 USA.
ABSTRACT:The identification of peptide sequences and their posttranslational modifications (PTMs) is a crucial step in the analysis of bottom-up proteomics data. The recent development of open modification search (OMS) engines allows virtually all PTMs to be searched for. This not only increases the number of spectra that can be matched to peptides but also greatly advances the understanding of the biological roles of PTMs through the identification, and the thereby facilitated quantification, of peptidoforms (peptide sequences and their potential PTMs). Whereas the benefits of combining results from multiple protein database search engines have been previously established, similar approaches for OMS results have been missing so far. Here we compare and combine results from three different OMS engines, demonstrating an increase in peptide spectrum matches of 8-18%. The unification of search results furthermore allows for the combined downstream processing of search results, including the mapping to potential PTMs. Finally, we test for the ability of OMS engines to identify glycosylated peptides. The implementation of these engines in the Python framework Ursgal facilitates the straightforward application of the OMS with unified parameters and results files, thereby enabling yet unmatched high-throughput, large-scale data analysis.
Use: pGlyco
frontiers in Chemistry2021. Zhao, Y et al.
Natl Inst Metrol, Ctr Adv Measurement Sci, Beijing, Peoples R China.
ABSTRACT:Diabetes has become a major public health concern worldwide, most of which are type 2 diabetes (T2D). The diagnosis of T2D is commonly based on plasma glucose levels, and there are no reliable clinical biomarkers available for early detection. Recent advances in proteome technologies offer new opportunity for the understanding of T2D; however, the underlying proteomic characteristics of T2D have not been thoroughly investigated yet. Here, using proteomic and glycoproteomic profiling, we provided a comprehensive landscape of molecular alterations in the fasting plasma of the 24 Chinese participants, including eight T2D patients, eight prediabetic (PDB) subjects, and eight healthy control (HC) individuals. Our analyses identified a diverse set of potential biomarkers that might enhance the efficiency and accuracy based on current existing biological indicators of (pre)diabetes. Through integrative omics analysis, we showed the capability of glycoproteomics as a complement to proteomics or metabolomics, to provide additional insights into the pathogenesis of (pre)diabetes. We have newly identified systemic site-specific N-glycosylation alterations underlying T2D patients in the complement activation pathways, including decreased levels of N-glycopeptides from C1s, MASP1, and CFP proteins, and increased levels of N-glycopeptides from C2, C4, C4BPA, C4BPB, and CFH. These alterations were not observed at proteomic levels, suggesting new opportunities for the diagnosis and treatment of this disease. Our results demonstrate a great potential role of glycoproteomics in understanding (pre)diabetes and present a new direction for diabetes research which deserves more attention.
Use: pGlyco
frontiers in Chemistry2021. Shu, H et al.
Fudan Univ, Zhongshan Hosp, Liver Canc Inst, Shanghai, Peoples R China.
ABSTRACT:Haptoglobin (Hp) is one of the acute-phase response proteins secreted by the liver, and its aberrant N-glycosylation was previously reported in hepatocellular carcinoma (HCC). Limited studies on Hp O-glycosylation have been previously reported. In this study, we aimed to discover and confirm its O-glycosylation in HCC based on lectin binding and mass spectrometry (MS) detection. First, serum Hp was purified from patients with liver cirrhosis (LC) and HCC, respectively. Then, five lectins with Gal or GalNAc monosaccharide specificity were chosen to perform lectin blot, and the results showed that Hp in HCC bound to these lectins in a much stronger manner than that in LC. Furthermore, label-free quantification based on MS was performed. A total of 26 intact O-glycopeptides were identified on Hp, and most of them were elevated in HCC as compared to LC. Among them, the intensity of HYEGS(316)TVPEK (H1N1S1) on Hp was the highest in HCC patients. Increased HYEGS(316)TVPEK (H1N1S1) in HCC was quantified and confirmed using the MS method based on O-18/O-16 C-terminal labeling and multiple reaction monitoring. This study provided a comprehensive understanding of the glycosylation of Hp in liver diseases.
Use: pGlyco; pQuant
Frontiers in Immunology2021. Yang, LJ et al.
Fudan Univ, Inst Biomed Sci, Shanghai, Peoples R China.
ABSTRACT:Macrophages are important immune cells that participate in both innate and adaptive immune responses, such as phagocytosis, recognition of molecular patterns, and activation of the immune response. In this study, murine peritoneal macrophages were isolated and then activated by LPS, HSV and VSV. Integrative proteomic and precision N-glycoproteomic profiling were conducted to assess the underlying macrophage activation. We identified a total of 587 glycoproteins, including 1239 glycopeptides, 526 monosaccharide components, and 8326 intact glycopeptides in glycoproteomics, as well as a total of 4496 proteins identified in proteomic analysis. These glycoproteins are widely involved in important biological processes, such as antigen presentation, cytokine production and glycosylation progression. Under the stimulation of the different pathogens, glycoproteins showed a dramatic change. We found that receptors in the Toll-like receptor pathway, such as Tlr2 and CD14, were increased under LPS and HSV stimulation. Glycosylation of those proteins was proven to influence their subcellular locations.
Use: pGlyco
JOURNAL OF ORGANIC CHEMISTRY2021. Zhao, JF et al.
Fudan Univ, Dept Chem, Shanghai 200433, Peoples R China.
ABSTRACT:Oligosaccharide natural products have diverse biological activities and represent a potentially important source for drug development. In this study, we focus on the glycosylation pathway in the biosynthesis of saccharomicin A (SA-A), an oligosaccharide antibiotic containing 17 sugar moieties. By extensive gene-knockout studies with comparative metabolic profile analysis, we established a complete pathway in assembling the heptadecasaccharide chain of SA-A, the longest saccharide chain found in natural products.
Use: pGlyco
Analytical Chemistry2021. Kong, Siyuan et al.
Fudan Univ, Dept Chem, Shanghai 200032, Peoples R China; Fudan Univ, NHC Key Lab Glycoconjugates Res, Shanghai 200032, Peoples R China; Fudan Univ, Peoples Hosp 5, Shanghai 200032, Peoples R China; Fudan Univ, Shanghai Key Lab Med Epigenet, Int Colab Med Epigenet & Metab, Minist Sci & Technol,Inst Biomed Sci, Shanghai 200032, Peoples R China
ABSTRACT:The heterogeneity and low abundance of protein glycosylation present challenging barriers to the analysis of intact glycopeptides, which is key to comprehensively understanding the role of glycosylation in an organism. Efficient and specific enrichment of intact glycopeptides could help greatly with this problem. Here, we propose a new enrichment strategy using a boronic acid (BA)-functionalized mesoporous graphene-silica composite (denoted as GO@mSiO(2)-GLYMO-APB) for isolating intact glycopeptides from complex biological samples. The merits of this composite, including high surface area and synergistic effect from size exclusion functionality of mesoporous material, hydrophilic interaction of silica, and the reversible covalent binding with BA, enable the effective and specific enrichment of both intact N- and O-glycopeptides. The results from the enrichment performance of the strategy evaluated by standard glycoproteins and the application to global N- and O-glycosylation analyses in human serum indicate the robustness and potential of the strategy for intact glycopeptide analysis.
Use: pGlyco
Nature Communications2021. Yang, Y et al.
Fudan Univ, Dept Chem, Shanghai 200000, Peoples R China.
ABSTRACT:Data independent acquisition (DIA) proteomics provides deep coverage and high quantitative accuracy, but is not yet well established in glycoproteomics. Here, the authors develop a DIA-based glycoproteomics workflow with stringent statistical controls to enable accurate glycopeptide identification. Large-scale profiling of intact glycopeptides is critical but challenging in glycoproteomics. Data independent acquisition (DIA) is an emerging technology with deep proteome coverage and accurate quantitative capability in proteomics studies, but is still in the early stage of development in the field of glycoproteomics. We propose GproDIA, a framework for the proteome-wide characterization of intact glycopeptides from DIA data with comprehensive statistical control by a 2-dimentional false discovery rate approach and a glycoform inference algorithm, enabling accurate identification of intact glycopeptides using wide isolation windows. We further utilize a semi-empirical spectrum prediction strategy to expand the coverage of spectral libraries of glycopeptides. We benchmark our method for N-glycopeptide profiling on DIA data of yeast and human serum samples, demonstrating that DIA with GproDIA outperforms the data-dependent acquisition-based methods for glycoproteomics in terms of capacity and data completeness of identification, as well as accuracy and precision of quantification. We expect that this work can provide a powerful tool for glycoproteomic studies.
Use: pGlyco
Frontiers in Immunology2021. Yang, LJ et al.
Fudan Univ, Inst Biomed Sci, Shanghai, Peoples R China.
ABSTRACT:Macrophages are important immune cells that participate in both innate and adaptive immune responses, such as phagocytosis, recognition of molecular patterns, and activation of the immune response. In this study, murine peritoneal macrophages were isolated and then activated by LPS, HSV and VSV. Integrative proteomic and precision N-glycoproteomic profiling were conducted to assess the underlying macrophage activation. We identified a total of 587 glycoproteins, including 1239 glycopeptides, 526 monosaccharide components, and 8326 intact glycopeptides in glycoproteomics, as well as a total of 4496 proteins identified in proteomic analysis. These glycoproteins are widely involved in important biological processes, such as antigen presentation, cytokine production and glycosylation progression. Under the stimulation of the different pathogens, glycoproteins showed a dramatic change. We found that receptors in the Toll-like receptor pathway, such as Tlr2 and CD14, were increased under LPS and HSV stimulation. Glycosylation of those proteins was proven to influence their subcellular locations.
Use: pGlyco
Infectious Microbes & Diseases2021. Sun, ZY et al.
State Key Laboratory for the Diagnosis and Treatment of Infectious Diseases, The First Affiliated Hospital, Zhejiang University School of Medicine, No.79 Qingchun Road, Shangcheng District, Hangzhou 310003, China
ABSTRACT:The devastating coronavirus disease 2019 (COVID-19) pandemic has prompted worldwide efforts to study structural biological traits of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and its viral components. Compared to the Spike protein, which is the primary target for currently available vaccines or antibodies, knowledge about other virion structural components is incomplete. Using high-resolution mass spectrometry, we report a comprehensive post-translational modification (PTM) analysis of nucleocapsid phosphoprotein (NCP), the most abundant structural component of the SARS-CoV-2 virion. In addition to phosphoryl groups, we show that the SARS-CoV-2 NCP is decorated with a variety of PTMs, includingN-glycans and ubiquitin. Based on newly identified PTMs, refined protein structural models of SARS-CoV-2 NCP were proposed and potential immune recognition epitopes of NCP were aligned with PTMs. These data can facilitate the design of novel vaccines or therapeutics targeting NCP, as valuable alternatives to the current vaccination and treatment paradigm that is under threat of the ever-mutating SARS-CoV-2 Spike protein.
Use: pGlyco
Molecular & Cellular Proteomics2021. Chen, ZW et al.
Univ Wisconsin, Dept Chem, 1101 Univ Ave, Madison, WI 53706 USA.
ABSTRACT:As the body fluid that directly interchanges with the extracellular fluid of the central nervous system (CNS), cerebrospinal fluid (CSF) serves as a rich source for CNS-related disease biomarker discovery. Extensive proteome profiling has been conducted for CSF, but studies aimed at unraveling site-specific CSF N-glycoproteome are lacking. Initial efforts into site-specific N-glycoproteomics study in CSF yield limited coverage, hindering further experimental design of glycosylation-based disease biomarker discovery in CSF. In the present study, we have developed an N-glycoproteomic approach that combines enhanced N-glycopeptide sequential enrichment by hydrophilic interaction chromatography (HILIC) and boronic acid enrichment with electron transfer and higher-energy collision dissociation (EThcD) for large-scale intact N-glycopeptide analysis. The application of the developed approach to the analyses of human CSF samples enabled identifications of a total of 2893 intact N-glycopeptides from 511 N-glycosites and 285 N-glycoproteins. To our knowledge, this is the largest site-specific N-glycoproteome dataset reported for CSF to date. Such dataset provides molecular basis for a better understanding of the structure-function relationships of glycoproteins and their roles in CNS-related physiological and pathological processes. As accumulating evidence suggests that defects in glycosylation are involved in Alzheimer's disease (AD) pathogenesis, in the present study, a comparative indepth N-glycoproteomic analysis was conducted for CSF samples from healthy control and AD patients, which yielded a comparable N-glycoproteome coverage but a distinct expression pattern for different categories of glycoforms, such as decreased fucosylation in AD CSF samples. Altered glycosylation patterns were detected for a number of N-glycoproteins including alpha-1-antichymotrypsin, ephrin-A3 and carnosinase CN1 etc., which serve as potentially interesting targets for further glycosylation-based AD study and may eventually lead to molecular elucidation of the role of glycosylation in AD progression.
Use: pGlyco