Differential Glycosite Profiling—A Versatile Method to Compare Membrane Glycoproteomes
Glycosylation is the most prevalent and varied form of post-translational protein modifications. Protein glycosylation regulates multiple cellular functions, including protein folding, cell adhesion, molecular trafficking and clearance, receptor activation, signal transduction, and endocytosis. In p...
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| Format: | Article |
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MDPI AG
2021-06-01
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| Series: | Molecules |
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| Online Access: | https://www.mdpi.com/1420-3049/26/12/3564 |
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| author | Malwina Michalak Martin Simon Kalteis Aysel Ahadova Matthias Kloor Mark Kriegsmann Katharina Kriegsmann Uwe Warnken Dominic Helm Jürgen Kopitz |
| author_facet | Malwina Michalak Martin Simon Kalteis Aysel Ahadova Matthias Kloor Mark Kriegsmann Katharina Kriegsmann Uwe Warnken Dominic Helm Jürgen Kopitz |
| author_sort | Malwina Michalak |
| collection | DOAJ |
| description | Glycosylation is the most prevalent and varied form of post-translational protein modifications. Protein glycosylation regulates multiple cellular functions, including protein folding, cell adhesion, molecular trafficking and clearance, receptor activation, signal transduction, and endocytosis. In particular, membrane proteins are frequently highly glycosylated, which is both linked to physiological processes and of high relevance in various disease mechanisms. The cellular glycome is increasingly considered to be a therapeutic target. Here we describe a new strategy to compare membrane glycoproteomes, thereby identifying proteins with altered glycan structures and the respective glycosites. The workflow started with an optimized procedure for the digestion of membrane proteins followed by the lectin-based isolation of glycopeptides. Since alterations in the glycan part of a glycopeptide cause mass alterations, analytical size exclusion chromatography was applied to detect these mass shifts. N-glycosidase treatment combined with nanoUPLC-coupled mass spectrometry identified the altered glycoproteins and respective glycosites. The methodology was established using the colon cancer cell line CX1, which was treated with 2-deoxy-glucose—a modulator of N-glycosylation. The described methodology is not restricted to cell culture, as it can also be adapted to tissue samples or body fluids. Altogether, it is a useful module in various experimental settings that target glycan functions. |
| first_indexed | 2024-03-10T10:30:07Z |
| format | Article |
| id | doaj.art-3905173813f440df958d63df45b737ae |
| institution | Directory Open Access Journal |
| issn | 1420-3049 |
| language | English |
| last_indexed | 2024-03-10T10:30:07Z |
| publishDate | 2021-06-01 |
| publisher | MDPI AG |
| record_format | Article |
| series | Molecules |
| spelling | doaj.art-3905173813f440df958d63df45b737ae2023-11-21T23:40:01ZengMDPI AGMolecules1420-30492021-06-012612356410.3390/molecules26123564Differential Glycosite Profiling—A Versatile Method to Compare Membrane GlycoproteomesMalwina Michalak0Martin Simon Kalteis1Aysel Ahadova2Matthias Kloor3Mark Kriegsmann4Katharina Kriegsmann5Uwe Warnken6Dominic Helm7Jürgen Kopitz8Department of Applied Tumor Biology, Institute of Pathology, Heidelberg University Hospital, Im Neuenheimer Feld 224, 69120 Heidelberg, GermanyDepartment of Applied Tumor Biology, Institute of Pathology, Heidelberg University Hospital, Im Neuenheimer Feld 224, 69120 Heidelberg, GermanyDepartment of Applied Tumor Biology, Institute of Pathology, Heidelberg University Hospital, Im Neuenheimer Feld 224, 69120 Heidelberg, GermanyDepartment of Applied Tumor Biology, Institute of Pathology, Heidelberg University Hospital, Im Neuenheimer Feld 224, 69120 Heidelberg, GermanyInstitute of Pathology, Heidelberg University Hospital, Im Neuenheimer Feld 224, 69120 Heidelberg, GermanyDepartment of Hematology, Oncology and Rheumatology, Heidelberg University Hospital, Im Neuenheimer Feld 410, 69120 Heidelberg, GermanyClinical Cooperation Unit Neurooncology, DKFZ (German Cancer Research Center), Im Neuenheimer Feld 280, 69120 Heidelberg, GermanyGenomics and Proteomics Core Facility, MS-based Protein Analysis Unit, DKFZ (German Cancer Research Center) Heidelberg, Im Neuenheimer Feld 280, 69120 Heidelberg, GermanyDepartment of Applied Tumor Biology, Institute of Pathology, Heidelberg University Hospital, Im Neuenheimer Feld 224, 69120 Heidelberg, GermanyGlycosylation is the most prevalent and varied form of post-translational protein modifications. Protein glycosylation regulates multiple cellular functions, including protein folding, cell adhesion, molecular trafficking and clearance, receptor activation, signal transduction, and endocytosis. In particular, membrane proteins are frequently highly glycosylated, which is both linked to physiological processes and of high relevance in various disease mechanisms. The cellular glycome is increasingly considered to be a therapeutic target. Here we describe a new strategy to compare membrane glycoproteomes, thereby identifying proteins with altered glycan structures and the respective glycosites. The workflow started with an optimized procedure for the digestion of membrane proteins followed by the lectin-based isolation of glycopeptides. Since alterations in the glycan part of a glycopeptide cause mass alterations, analytical size exclusion chromatography was applied to detect these mass shifts. N-glycosidase treatment combined with nanoUPLC-coupled mass spectrometry identified the altered glycoproteins and respective glycosites. The methodology was established using the colon cancer cell line CX1, which was treated with 2-deoxy-glucose—a modulator of N-glycosylation. The described methodology is not restricted to cell culture, as it can also be adapted to tissue samples or body fluids. Altogether, it is a useful module in various experimental settings that target glycan functions.https://www.mdpi.com/1420-3049/26/12/3564glycoproteomicsglycositecolorectal cancerglycosylationglycoproteinmass spectrometry |
| spellingShingle | Malwina Michalak Martin Simon Kalteis Aysel Ahadova Matthias Kloor Mark Kriegsmann Katharina Kriegsmann Uwe Warnken Dominic Helm Jürgen Kopitz Differential Glycosite Profiling—A Versatile Method to Compare Membrane Glycoproteomes Molecules glycoproteomics glycosite colorectal cancer glycosylation glycoprotein mass spectrometry |
| title | Differential Glycosite Profiling—A Versatile Method to Compare Membrane Glycoproteomes |
| title_full | Differential Glycosite Profiling—A Versatile Method to Compare Membrane Glycoproteomes |
| title_fullStr | Differential Glycosite Profiling—A Versatile Method to Compare Membrane Glycoproteomes |
| title_full_unstemmed | Differential Glycosite Profiling—A Versatile Method to Compare Membrane Glycoproteomes |
| title_short | Differential Glycosite Profiling—A Versatile Method to Compare Membrane Glycoproteomes |
| title_sort | differential glycosite profiling a versatile method to compare membrane glycoproteomes |
| topic | glycoproteomics glycosite colorectal cancer glycosylation glycoprotein mass spectrometry |
| url | https://www.mdpi.com/1420-3049/26/12/3564 |
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