Comprehensive Protocol to Simultaneously Study Protein Phosphorylation, Acetylation, and N-Linked Sialylated Glycosylation
| Autor: | Martin R. Larsen, Marcella Nunes Melo-Braga, Katarzyna Kulej, María Ibáñez-Vea |
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| Přispěvatelé: | Posch, Anton |
| Rok vydání: | 2015 |
| Předmět: |
Proteomics
Liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) Spectrometry Mass Electrospray Ionization Glycan Glycosylation Peptide TiSH Hydrophilic interaction liquid chromatography (HILIC)/high pH reversed phase chromatography (HpH) Sequential elution from immobilized metal affinity chromatography (SIMAC) Chromatography Affinity Workflow chemistry.chemical_compound Tandem Mass Spectrometry Immunoprecipitation Protein phosphorylation Phosphorylation Protein posttranslational modification (PTM) enrichment Proteins/analysis chemistry.chemical_classification Chromatography Reverse-Phase Immunoprecipitation (IP) biology Molecular mass Elution Chemistry Hydrophilic interaction chromatography Acetylation Titanium/chemistry Biochemistry Sialic acid (SA) N-linked glycosylation biology.protein Comprising of titanium dioxide (TiO2) Protein Processing Post-Translational |
| Zdroj: | Methods in Molecular Biology ISBN: 9781493925490 Methods in Molecular Biology ISBN: 9781071611852 Melo-Braga, M N, Ibáñez-Vea, M, Kulej, K & Larsen, M R 2021, Comprehensive Protocol to Simultaneously Study Protein Phosphorylation, Acetylation, and N-Linked Sialylated Glycosylation . in A Posch (ed.), Proteomic Profiling : Methods and Protocols . Humana Press, Methods in molecular biology (Clifton, N.J.), vol. 2261, pp. 55-72 . https://doi.org/10.1007/978-1-0716-1186-9_5 |
| DOI: | 10.1007/978-1-4939-2550-6_21 |
| Popis: | Posttranslational modifications (PTMs) such as phosphorylation, acetylation, and glycosylation are an essential regulatory mechanism of protein function and interaction, and they are associated with a wide range of biological processes. Since most PTMs alter the molecular mass of a protein, mass spectrometry (MS) is the ideal analytical tool for studying various PTMs. However, PTMs are often present in substoichiometric levels, and therefore their unmodified counterpart often suppresses their signal in MS. Consequently, PTM analysis by MS is a challenging task, requiring highly specialized and sensitive PTM-specific enrichment methods. Currently, several methods have been implemented for PTM enrichment, and each of them has its drawbacks and advantages as they differ in selectivity and specificity toward specific protein modifications. Unfortunately, for the vast majority of more than 400 known modifications, we have no or poor tools for selective enrichment.Here, we describe a comprehensive workflow to simultaneously study phosphorylation, acetylation, and N-linked sialylated glycosylation from the same biological sample. The protocol involves an initial titanium dioxide (TiO2) step to enrich for phosphopeptides and sialylated N-linked glycopeptides followed by glycan release and post-fractionation using sequential elution from immobilized metal affinity chromatography (SIMAC) to separate mono-phosphorylated and deglycosylated peptides from multi-phosphorylated ones. The IMAC flow-through and acidic elution are subsequently subjected to a next round of TiO2 enrichment for further separation of mono-phosphopeptides from deglycosylated peptides. Furthermore, the lysine-acetylated peptides present in the first TiO2 flow-through fraction are enriched by immunoprecipitation (IP) after peptide cleanup. Finally, the samples are fractionated by high pH reversed phase chromatography (HpH) or hydrophilic interaction liquid chromatography (HILIC ) to reduce sample complexity and increase the coverage in the subsequent LC-MS /MS analysis. This allows the analysis of multiple types of modifications from the same highly complex biological sample without decreasing the quality of each individual PTM study. |
| Databáze: | OpenAIRE |
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