Posttranslational modifications (PTMs) of proteins represent fascinating extensions of the dynamic complexity of living cells' proteomes. The results of enzymatically catalyzed or spontaneous chemical reactions, PTMs form a fourth tier in the gene - transcript - protein cascade, and contribute not only to proteins' biological functions, but also to challenges in their analysis. There have been tremendous advances in proteomics during the last decade. Identification and mapping of PTMs in proteins have improved dramatically, mainly due to constant increases in the sensitivity, speed, accuracy and resolution of mass spectrometry (MS). However, it is also becoming increasingly evident that simple gel-free shotgun MS profiling is unlikely to suffice for comprehensive detection and characterization of proteins and/or protein modifications present in low amounts. Here, we review current approaches for enriching and separating posttranslationally modified proteins, and their MS-independent detection. First, we discuss general approaches for proteome separation, fractionation and enrichment. We then consider the commonest forms of PTMs (phosphorylation, glycosylation and glycation, lipidation, methylation, acetylation, deamidation, ubiquitination and various redox modifications), and the best available methods for detecting and purifying proteins carrying these PTMs. This article is part of a Special Issue entitled: Posttranslational Protein modifications in biology and Medicine.
- MeSH
- lidé MeSH
- posttranslační úpravy proteinů * MeSH
- proteom chemie izolace a purifikace metabolismus MeSH
- proteomika metody MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- přehledy MeSH
Rapid changes of protein phosphorylation play a crucial role in the regulation of many cellular processes. Being post-translationally modified, phosphoproteins are often present in quite low abundance and tend to co-exist with their unphosphorylated isoform within the cell. To make their identification more practicable, the use of enrichment protocols is often required. The enrichment strategies can be performed either at the level of phosphoproteins or at the level of phosphopeptides. Both approaches have their advantages and disadvantages. Most enriching strategies are based on chemical modifications, affinity chromatography to capture peptides and proteins containing negatively charged phosphate groups onto a positively charged matrix, or immunoprecipitation by phospho-specific antibodies.In this article, the most up-to-date enrichment techniques are discussed, taking into account their optimization, and highlighting their advantages and disadvantages. Moreover, these methods are compared to each other, revealing their complementary nature in providing comprehensive coverage of the phosphoproteome.
- MeSH
- barvení a značení MeSH
- chromatografie afinitní MeSH
- fosfoproteiny chemie izolace a purifikace metabolismus MeSH
- fosforylace MeSH
- hmotnostní spektrometrie MeSH
- imunoprecipitace MeSH
- peptidové fragmenty chemie izolace a purifikace metabolismus MeSH
- posttranslační úpravy proteinů MeSH
- proteom chemie izolace a purifikace metabolismus MeSH
- proteomika MeSH
- rostlinné proteiny chemie izolace a purifikace metabolismus MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- přehledy MeSH
- MeSH
- 2D gelová elektroforéza MeSH
- duktální karcinom prsu genetika imunologie patologie MeSH
- finanční podpora výzkumu jako téma MeSH
- lidé MeSH
- lobulární karcinom genetika imunologie patologie MeSH
- medulární karcinom genetika imunologie patologie MeSH
- metastázy nádorů MeSH
- mucinózní adenokarcinom genetika imunologie patologie MeSH
- nádory prsu klasifikace MeSH
- proteom izolace a purifikace MeSH
- spektrometrie hmotnostní - ionizace laserem za účasti matrice MeSH
- Check Tag
- lidé MeSH
- ženské pohlaví MeSH
