Unbiased Selective Isolation of Protein N-Terminal Peptides from Complex Proteome Samples Using Phospho Tagging PTAG) and TiO2-based Depletion
| Autor: | Ad P. J. M. de Jong, Geert P M Mommen, Hugo D. Meiring, Albert J. R. Heck, Gideon F.A. Kersten, Bas van de Waterbeemd |
|---|---|
| Jazyk: | angličtina |
| Rok vydání: | 2012 |
| Předmět: |
Proteomics
Bio Process Engineering Proteome Lysine Saccharomyces cerevisiae fractional diagonal chromatography Neisseria meningitidis Tandem mass spectrometry Biochemistry in-vivo Chromatography Affinity Analytical Chemistry Phosphates Fungal Proteins Affinity chromatography Bacterial Proteins Tandem Mass Spectrometry fragmentation Molecular Biology VLAG acetylation Titanium Fungal protein Chromatography cleavage products biology Staining and Labeling Chemistry phosphorylation Phosphoproteomics Technological Innovation and Resources proteolytic events phosphoproteomics biology.organism_classification Peptide Fragments Isotope Labeling identification Protein Processing Post-Translational Chromatography Liquid positional proteomics |
| Zdroj: | Molecular and Cellular Proteomics 11 (2012) 9 Molecular and Cellular Proteomics, 11(9), 832-842 |
| ISSN: | 1535-9476 |
| Popis: | A positional proteomics strategy for global N-proteome analysis is presented based on phospho tagging (PTAG) of internal peptides followed by depletion by titanium dioxide (TiO2) affinity chromatography. Therefore, N-terminal and lysine amino groups are initially completely dimethylated with formaldehyde at the protein level, after which the proteins are digested and the newly formed internal peptides modified with the PTAG reagent glyceraldhyde-3-phosphate in nearly perfect yields (> 99%). The resulting phosphopeptides are depleted through binding onto TiO2, keeping exclusively a set of N-acetylated and/or N-dimethylated terminal peptides for analysis by liquid chromatography-tandem MS. Analysis of peptides derivatized with differentially labeled isotopic analogs of the PTAG reagent revealed a high depletion efficiency (> 95%). The method enabled identification of 753 unique N-terminal peptides (428 proteins) in N. meningitidis and 928 unique N-terminal peptides (572 proteins) in S. cerevisiae. These included verified neo-N termini from subcellular-relocalized membrane and mitochondrial proteins. The presented PTAG approach is therefore a novel, versatile, and robust method for mass spectrometry-based N-proteome analysis and identification of protease-generated cleavage products. |
| Databáze: | OpenAIRE |
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