MassIVEapplication/xmlftp://massive-ftp.ucsd.edu/v02/MSV000085318/primaryOK2000000ProteomicsManfred WuhrerLTQ Orbitrap VelosQ Exactive HF-X hybrid quadrupole-OrbitrapCho Cellshttps://massive.ucsd.edu/ProteoSAFe/dataset.jsp?task=c7b37e5668db4931b4aa30e97085f157Leiden University Medical Centerm.wuhrer@lumc.nlMassIVE19UNIMOD:4 - "Iodoacetamide derivative."O-glycosylationN-glycosylationCysteine aminoethylationRecombinant human erythropoietin (rhEPO) is an important biopharmaceutical for which glycosylation is a critical quality attribute. Therefore, robust analytical methods are needed for the in-depth characterization of rhEPO glycosylation. Currently, the protease GluC is widely established for the site-specific glycosylation analysis of rhEPO. However, this enzyme shows disadvantages, such as its specificity and the characteristics of the resulting (glyco)peptides. The use of trypsin, the gold standard protease in proteomics, as the sole protease for rhEPO is compromised, as no natural tryptic cleavage site is located between the glycosylation sites Asn24 and Asn38. Here, cysteine aminoethylation using 2-bromoethylamine was applied as an alternative alkylation strategy to introduce artificial tryptic cleavage sites at Cys29 and Cys33 in rhEPO. The (glyco)peptides resulting from a subsequent digestion using trypsin were analyzed by reverse-phase liquid chromatography-mass spectrometry. The new trypsin-based workflow was easily implemented by adapting the alkylation step in a conventional workflow and was directly compared to an established approach using GluC. The new method shows an improved specificity, a significantly reduced chromatogram complexity, allows for shorter analysis times, and simplifies data evaluation. Furthermore, the method allows for the monitoring of additional attributes, such as oxidation and deamidation at specific sites in parallel to the site-specific glycosylation analysis of rhEPO.Cysteine Aminoethylation Enables the Site-Specific Glycosylation Analysis of Recombinant Human Erythropoietin using Trypsin.Lippold Steffen S, Büttner Alexander A, Choo Matthew S F MSF, Hook Michaela M, de Jong Coen J CJ, Nguyen-Khuong Terry T, Haberger Markus M, Reusch Dietmar D, Wuhrer Manfred M, de Haan Noortje NErythropoietin, EPO, Cysteine Hydrochloride, E 920, human being, O-linked Glycosylations, GlcNAcylation, Protein Glycosylation, determination, Zinc Cysteinate, Galactosylation, Modern, L-Zystein, Galactosylations, Glycosylations, N-linked Glycosylations, Cys, Fucosylations, Human, O linked Glycosylation, N-linked, O-linked Glycosylation, glycosylation, Homo sapiens, Glycosylation, Phosphoglycosylations, Protein, chemical analysis, GlcNAcylations, 2-amino-3-mercaptopropanoic acid, Cystein, Half-Cystine, (2R)-2-amino-3-sulfanylpropanoic acid, Man, Zystein, region, beta Trypsin., C, PRSS, Man (Taxonomy), 2-amino-3-sulfanylpropanoic acid, cisteina, Protein Glycosylations, L Cysteine, E-920, N linked Glycosylation, beta-Trypsin, Tripcellim, N-Acetylglucosaminylation, Sialylations, L-2-Amino-3-mercaptopropionic acid, L-Cysteine, (R)-2-amino-3-mercaptopropanoic acid, Hcys, 2-Amino-3-mercaptopropionic acid, Sialylation, human, L-Cystein, erythropoietin, Trypure, CYSTEINE, N Acetylglucosaminylation, Phosphoglycosylation, O-linked, Modern Man, FREE CYSTEINE, E920, site, erythropoietin receptor ligand, assay, Half Cystine, (2R)-2-amino-3-mercaptopropanoic acid, Fucosylation, epoetin, N-Acetylglucosaminylations, N-linked Glycosylation, humansErythropoietin, EPO, human being, Cysteine Hydrochloride, E 920, artificial sequence, O-linked Glycosylations, GRP1/cytohesin 1, GlcNAcylation, Procedures, GBA1, determination, Peptidomics, 2-bromoethanamine hydrobromide, Biocatalysts, Galactosylation, GLUC, cleavage, L-Zystein, Galactosylations, N-linked Glycosylations, Spectrum Analyses, GCase, Human, Alkylations, CG11633, N-linked, cytohesin/GRP1, Polypeptides, Techniques, method, O-linked Glycosylation, peptido, Homo sapiens, glycosylation, reduced, Glycosylation, Method, GRP1, method used in an experiment, Grp1, Studies, Mass, 2-amino-3-mercaptopropanoic acid, Cystein, Half-Cystine, Analysis, tiny, Work Flow, Man, Technique, Mass Spectroscopy, Mass Spectrum Analysis, C, Man (Taxonomy), peptides, Analyses, Protein Glycosylations, cisteina, L Cysteine, E-920, bromoethyleneamine hydrobromide, PTPSTEP, beta-Trypsin, hypoplasia, N-Acetylglucosaminylation, l(2)SH2 0323, chemical analysis., procedures, Hcys, Sialylation, Study, Enzyme, Neural-specific protein-tyrosine phosphatase, Methodological Studies, CYSTEINE, Phosphoglycosylation, O-linked, l(2)k08110, site, FREE CYSTEINE, peptidos, erythropoietin receptor ligand, Half Cystine, Fucosylation, (2R)-2-amino-3-mercaptopropanoic acid, epoetin, N-Acetylglucosaminylations, N-linked Glycosylation, GPH, small, Protein Glycosylation, Zinc Cysteinate, Modern, Liquid Chromatography, 2-bromoethylamine hydrobromide, Glycosylations, 2-BEA, artificial gene, stepk, Procedure, synthetic DNA, Cys, Spectrum Analysis, Fucosylations, Peptide, Spectroscopy, O linked Glycosylation, Workflows, MS, Phosphoglycosylations, Protein, chemical analysis, 3.1.3.48, synthetic, GlcNAcylations, techniques, 2-bromoethanamine, (2R)-2-amino-3-sulfanylpropanoic acid, l(2)SH0323, region, Zystein, Mass Spectrum Analyses, CYH1, Mass Spectrum, PRSS, underdeveloped, Step, CG11628, betaGC, 2-amino-3-sulfanylpropanoic acid, N linked Glycosylation, Tripcellim, Spectrometry, Sialylations, L-2-Amino-3-mercaptopropionic acid, L-Cysteine, (R)-2-amino-3-mercaptopropanoic acid, Striatum-enriched protein-tyrosine phosphatase, Methodological, Methodological Study, beta Trypsin, synthetic constructs, GC, human, 2-Amino-3-mercaptopropionic acid, L-Cystein, DmelCG11628, erythropoietin, plan specification, Trypure, SYNTHETIC CONSTRUCT sequences, GCB, STEP, N Acetylglucosaminylation, Modern Man, Biocatalyst, E920, artificial, Peptid, assay, Polypeptide, Work Flows, humans, methodologyC, EPP, Supplementary Material, EPO, EPX-PEN, Cysteine Hydrochloride, E 920, determination, Zinc Cysteinate, 2-amino-3-sulfanylpropanoic acid, cisteina, MVCD2, L Cysteine, E-920, L-Zystein, 1.11.1.7, CLGI, L-2-Amino-3-mercaptopropionic acid, Glycopeptide, L-Cysteine, (R)-2-amino-3-mercaptopropanoic acid, Hcys, Eosinophil peroxidase heavy chain, Cys, 2-Amino-3-mercaptopropionic acid, L-Cystein, CYSTEINE, Eosinophil peroxidase light chain, EP, Supporting Information, chemical analysis, HCI, Zystein., FREE CYSTEINE, E920, Electronic Supplementary Material, Supplementary Information, assay, 2-amino-3-mercaptopropanoic acid, Cystein, Half-Cystine, Half Cystine, EPA, (2R)-2-amino-3-mercaptopropanoic acid, epoetin, (2R)-2-amino-3-sulfanylpropanoic acid, TIMPEPP, EPO, Cysteine Hydrochloride, E 920, human being, O-linked Glycosylations, GlcNAcylation, Protein Glycosylation, determination, Zinc Cysteinate, Galactosylation, Modern, L-Zystein, Galactosylations, 1.11.1.7, CLGI, Glycosylations, N-linked Glycosylations, Eosinophil peroxidase heavy chain, Cys, Fucosylations, Human, O linked Glycosylation, N-linked, O-linked Glycosylation, glycosylation, Homo sapiens, Glycosylation, Phosphoglycosylations, Eosinophil peroxidase light chain, EP, Protein, chemical analysis, HCI, GlcNAcylations, 2-amino-3-mercaptopropanoic acid, Cystein, Half-Cystine, (2R)-2-amino-3-sulfanylpropanoic acid, Man, Zystein, region, beta Trypsin., C, EPX-PEN, PRSS, Man (Taxonomy), 2-amino-3-sulfanylpropanoic acid, cisteina, Protein Glycosylations, L Cysteine, MVCD2, E-920, N linked Glycosylation, beta-Trypsin, Tripcellim, N-Acetylglucosaminylation, Sialylations, L-2-Amino-3-mercaptopropionic acid, L-Cysteine, (R)-2-amino-3-mercaptopropanoic acid, Hcys, 2-Amino-3-mercaptopropionic acid, Sialylation, human, L-Cystein, Trypure, CYSTEINE, N Acetylglucosaminylation, Phosphoglycosylation, O-linked, Modern Man, FREE CYSTEINE, E920, site, assay, Half Cystine, (2R)-2-amino-3-mercaptopropanoic acid, Fucosylation, EPA, epoetin, N-Acetylglucosaminylations, N-linked Glycosylation, humans, TIMP0falseSupporting information for tryptic EPO glycopeptide LC-MS analysis via cysteine aminoethylationSupplementary information for a technical note describing the application of cysteine aminoethylation for the site-specific glycosylation analysis of recombinant human EPO using trypsin as sole protease.Tue Apr 21 14:32:00 BST 2020MSV00008531832578997