Immobilized metal affinity chromatography optimized for the analysis of extracellular phosphorylation
Phosphorylation is the most widely studied posttranslational modification. Its role within the cell has been the focus of numerous large-scale studies. Recently there is growing evidence on the biological significance of extracellular phosphorylation. The analysis of these phosphopeptides is complic...
Elmentve itt :
| Szerzők: | |
|---|---|
| Dokumentumtípus: | Cikk |
| Megjelent: |
Wiley
2016
|
| Sorozat: | PROTEOMICS
16 No. 13 |
| doi: | 10.1002/pmic.201500520 |
| mtmt: | 3101860 |
| Online Access: | http://publicatio.bibl.u-szeged.hu/13115 |
| Tartalmi kivonat: | Phosphorylation is the most widely studied posttranslational modification. Its role within the cell has been the focus of numerous large-scale studies. Recently there is growing evidence on the biological significance of extracellular phosphorylation. The analysis of these phosphopeptides is complicated by the abundance of glycosylation in the extracellular space, since glycopeptides are also enriched by the methods used for phosphopeptide isolation. Thus, we optimized IMAC for phosphorylation analysis of secreted proteins, specifically in human serum. Selectivity and efficiency of different enrichment conditions used in earlier large-scale phosphoproteomic studies were evaluated. We found that minimizing hydrophilic interactions in the enrichment allowed selective phosphopeptide isolation. Using a two-step IMAC enrichment protocol under these conditions led to the identification of similar to 100 phosphorylation sites from the tryptic digest of as little as 40 mu L human serum. |
|---|---|
| Terjedelem/Fizikai jellemzők: | 1858-1862 |
| ISSN: | 1615-9853 |