pFind Studio: a computational solution for mass spectrometry-based proteomics
2022
ACS omega2022. Saraswat, M et al.
Mayo Clin, Dept Lab Med & Pathol, Rochester, MN 55905 USA; Inst Bioinformat, Bangalore 560066, Karnataka, India; Manipal Acad Higher Educ MAHE, Manipal 576104, Karnataka, India; Natl Inst Mental Hlth & Neurosci NIMHANS, Ctr Mol Med, Bangalore 560029, Karnataka, India; Mayo Clin, Ctr Individualized Med, Rochester, MN 55905 USA
ABSTRACT:Y Glycoproteomics, or the simultaneous characterization of glycans and their attached peptides, is increasingly being employed to generate catalogs of glycopeptides on a large scale. Nevertheless, quantitative glycoproteomics remains challenging even though isobaric tagging reagents such as tandem mass tags (TMT) are routinely used for quantitative proteomics. Here, we present a workflow that combines the enrichment or fractionation of TMT-labeled glycopeptides with size-exclusion chromatography (SEC) for an in-depth and quantitative analysis of the glycoproteome. We applied this workflow to study the cellular glycoproteome of an isogenic mammary epithelial cell system that recapitulated oncogenic mutations in the PIK3CA gene, which codes for the phosphatidylinositol-3-kinase catalytic subunit. As compared to the parental cells, cells with mutations in exon 9 (E545K) or exon 20 (H1047R) of the PIK3CA gene exhibited site-specific glycosylation alterations in 464 of the 1999 glycopeptides quantified. Our strategy led to the discovery of site-specific glycosylation changes in PIK3CA mutant cells in several important receptors, including cell adhesion proteins such as integrin beta-6 and CD166. This study demonstrates that the SEC-based enrichment of glycopeptides is a simple and robust method with minimal sample processing that can easily be coupled with TMT-labeling for the global quantitation of glycopeptides.
Use: pGlyco
Journal of Proteome Research2022. Bi, M et al.
Tongji Univ, Sch Chem Sci & Engn, Shanghai 200092, Peoples R China; Nanjing Univ, Ctr Precis Med, Dept Lab Med, Affiliated Hosp,Med Sch,Nanjing Drum Tower Hosp, Nanjing 210008, Jiangsu, Peoples R China
ABSTRACT:The characteristics of monoclonal antibodies (mAbs) cohering various function effectors show great expectation in therapy. Glycosylation, one of the common post-translational modifications, deeply influences cohesion. It is necessary to grasp monosaccharide composition/sequence and glycan structures in mAbs. There has been comprehensive mass spectrometry characterization of N-glycosylation of mAbs, and monosaccharide compositions are deduced according to known biosynthetic rules. Our recently developed intact N-glycopeptide search engine GPSeeker has made structure-specific characterization of N-glycosylation possible with structure-diagnostic fragment ions from selective fragmentation of N-glycan moieties. Here, we report our structure-specific N-glycoproteomics characterization of NIST monoclonal antibody reference material 8671 using GPSeeker, and 59 N-glycan structures (including 16 pairs of isomers) are characterized.
Use: pGlyco
Molecular metabolism2022. Verzijl, CRC et al.
Univ Med Ctr Groningen, Dept Pediat, Sect Mol Genet, Antonius Deusinglaan 1, NL-9713 AV Groningen, Netherlands
ABSTRACT:Objective: GALNT2, encoding polypeptide N-acetylgalactosaminyltransferase 2 (GalNAc-T2), was initially discovered as a regulator of highdensity lipoprotein metabolism. GalNAc-T2 is known to exert these effects through post-translational modification, i.e., O-linked glycosylation of secreted proteins with established roles in plasma lipid metabolism. It has recently become clear that loss of GALNT2 in rodents, cattle, nonhuman primates, and humans should be regarded as a novel congenital disorder of glycosylation that affects development and body weight. The role of GALNT2 in metabolic abnormalities other than plasma lipids, including insulin sensitivity and energy homeostasis, is poorly understood.Methods: GWAS data from the UK Biobank was used to study variation in the GALNT2 locus beyond changes in high-density lipoprotein metabolism. Experimental data were obtained through studies in Galnt2-/- mice and wild-type littermates on both control and high-fat diet.Results: First, we uncovered associations between GALNT2 gene variation, adiposity, and body mass index in humans. In mice, we identify the insulin receptor as a novel substrate of GalNAc-T2 and demonstrate that Galnt2-/- mice exhibit decreased adiposity, alterations in insulin signaling and a shift in energy substrate utilization in the inactive phase.Conclusions: This study identifies a novel role for GALNT2 in energy homeostasis, and our findings suggest that the local effects of GalNAc-T2 are mediated through posttranslational modification of the insulin receptor.(c) 2022 The Author(s). Published by Elsevier GmbH. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).
Use: pGlyco
Frontiers in Molecular Biosciences2022. Cao, Xinyi et al.
Fudan Univ, Dept Chem, Shanghai, Peoples R China; Guangxi Med Univ Canc Hosp, Dept Clin Lab, Nanning, Peoples R China; Fudan Univ, Inst Biomed Sci, Shanghai Canc Ctr, Shanghai, Peoples R China; Fudan Univ, NHC Key Lab Glycoconjugates Res, Shanghai, Peoples R China
ABSTRACT:Glycosylation inhibition has great potential in cancer treatment. However, the corresponding cellular response, protein expression and glycosylation changes remain unclear. As a cell-permeable small-molecule inhibitor with reduced cellular toxicity, N-linked glycosylation inhibitor-1 (NGI-1) has become a great approach to regulate glycosylation in mammalian cells. Here for the first time, we applied a nascent proteomic method to investigate the effect of NGI-1 in hepatocellular carcinoma (HCC) cell line. Besides, hydrophilic interaction liquid chromatography (HILIC) was adopted for the enrichment of glycosylated peptides. Glycoproteomic analysis revealed the abundance of glycopeptides from LAMP2, NICA, and CEIP2 was significantly changed during NGI-1 treatment. Moreover, the alterations of LAMP2 site-specific intact N-glycopeptides were comprehensively assessed. NGI-1 treatment also led to the inhibition of Cathepsin D maturation and the induction of autophagy. In summary, we provided evidence that NGI-1 repressed the expression of glycosylated LAMP2 accompanied with the occurrence of lysosomal defects and autophagy.
Use: pQuant
Nature Communications2022. Ferreira, RB et al.
UF Scripps Biomed Res, Dept Chem, Jupiter, FL 33458 USA
ABSTRACT:Fluorogenic detection of H2O2 in cells is established, but equivalent tools to monitor its cellular targets remain in their infancy. Here authors develop fluorogenic probes for detecting cysteine sulfenic acid, a redox modification inextricably linked to H2O2 signalling and oxidative stress.'Turn-on' fluorescence probes for detecting H2O2 in cells are established, but equivalent tools to monitor the products of its reaction with protein cysteines have not been reported. Here we describe fluorogenic probes for detecting sulfenic acid, a redox modification inextricably linked to H2O2 signaling and oxidative stress. The reagents exhibit excellent cell permeability, rapid reactivity, and high selectivity with minimal cytotoxicity. We develop a high-throughput assay for measuring S-sulfenation in cells and use it to screen a curated kinase inhibitor library. We reveal a positive association between S-sulfenation and inhibition of TK, AGC, and CMGC kinase group members including GSK3, a promising target for neurological disorders. Proteomic mapping of GSK3 inhibitor-treated cells shows that S-sulfenation sites localize to the regulatory cysteines of antioxidant enzymes. Our studies highlight the ability of kinase inhibitors to modulate the cysteine sulfenome and should find broad application in the rapidly growing field of redox medicine.
Use: pQuant
Bioorganic Chemistry2022. Wang, MR et al.
Northwest A&F Univ, Coll Chem & Pharm, Shaanxi Key Lab Nat Prod & Chem Biol, 3 Taicheng Rd, Yangling 712100, Shaanxi, Peoples R China; Hainan Univ, Sch Pharmaceut Sci, Key Lab Trop Biol Resources, Minist Educ, Haikou 570228, Hainan, Peoples R China
ABSTRACT:Neuroinflammation plays a key etiological role in the progressive neuronal damage of neurodegenerative dis-eases. Our phenotypic-based screening discovered 1,6-O,O-diacetylbritannilactone (OABL, 1) from Inula bri-tannica exhibited the potential anti-neuroinflammatory activity as well as a favorable blood-brain barrier penetration. 1 and its active derivative Br-OABL (2) with insert of Br at the C-14 position both modulated TLR4/ NF-kB/MAPK pathways. However, proteome-wide identification of 1 binding proteins remains unclear. Here, we employed an adapted isoTOP-ABPP, quantitative thiol reactivity profiling (QTRP) approach, to identify and quantify thiol reactivity binding proteins in murine microglia BV-2 cells. We screened out 15 proteins co-targeted by 1 and 2, which are involved in cellular response to oxidative stress and negative regulation NF-icB tran-scription factor in biological processes. In site-specific profiling, NLRP3 was identified as a covalent target of 1 and 2 for the first time, and the Cys483 of NLRP3 NACHT domain was identified as one active-site of NLRP3 cysteine residues that can be covalently modified by the alpha-methylene-gamma-lactone moiety. Furthermore, NLRP3 was validated to be directly binded by 1 and 2 by cellular thermo shift assay (CETSA) and activity-based protein profiling (ABPP), and NLRP3 functions were also verified by small interfering RNA approach. Notably, OABL treatment (i.p., 20 mg/kg/day) for 21 days reduced inflammation in 5XFAD mice brain. Together, we applied the QTRP to uncover the binding proteins of OABL in BV-2 cells, among which NLRP3 was revealed as a new co-valent target of 1 and 2 against neuroinflammation.
Use: pQuant
Frontiers in Molecular Biosciences2022. Cao, Xinyi et al.
Fudan Univ, Dept Chem, Shanghai, Peoples R China; Guangxi Med Univ Canc Hosp, Dept Clin Lab, Nanning, Peoples R China; Fudan Univ, Inst Biomed Sci, Shanghai Canc Ctr, Shanghai, Peoples R China; Fudan Univ, NHC Key Lab Glycoconjugates Res, Shanghai, Peoples R China
ABSTRACT:Glycosylation inhibition has great potential in cancer treatment. However, the corresponding cellular response, protein expression and glycosylation changes remain unclear. As a cell-permeable small-molecule inhibitor with reduced cellular toxicity, N-linked glycosylation inhibitor-1 (NGI-1) has become a great approach to regulate glycosylation in mammalian cells. Here for the first time, we applied a nascent proteomic method to investigate the effect of NGI-1 in hepatocellular carcinoma (HCC) cell line. Besides, hydrophilic interaction liquid chromatography (HILIC) was adopted for the enrichment of glycosylated peptides. Glycoproteomic analysis revealed the abundance of glycopeptides from LAMP2, NICA, and CEIP2 was significantly changed during NGI-1 treatment. Moreover, the alterations of LAMP2 site-specific intact N-glycopeptides were comprehensively assessed. NGI-1 treatment also led to the inhibition of Cathepsin D maturation and the induction of autophagy. In summary, we provided evidence that NGI-1 repressed the expression of glycosylated LAMP2 accompanied with the occurrence of lysosomal defects and autophagy.
Use: pQuant
2022. David L. Tabb et al.
Universit Paris Cit, Institut Pasteur, CNRS UAR 2024, Mass Spectrometry for Biology Unit, Paris 75015, France
ABSTRACT:
Use: pTop
2022. David L. Tabb et al.
Universit Paris Cit, Institut Pasteur, CNRS UAR 2024, Mass Spectrometry for Biology Unit, Paris 75015, France
ABSTRACT:
Use: pTop