Post-translational modifications (PTMs)

Identify and localize protein post-translational modifications (PTMs) such as phosphorylation, glycosylation, acetylation, methylation, ubiquitination, and oxidation with or without enrichment approach prior to mass spec analysis. We identify protein PTMs through digesting proteins into peptides, analyzing the modified peptides using LC-MS/MS, and then searching the PTM via proteomics search software. PTM Enrichment(s) may or may not be needed in sample prep procedure, depending on the complexity of samples.

In the case of phosphorylation, if you are able to purify the protein in vitro in many cases mass spec is able to identify and localize it without enrichment. In the case of global phosphoproteomics analysis, we use Fe-IMAC and TiO2 StageTip to enrich phosphopeptides. Because the relatively low abundance of phosphoproteins or other PTM-modified proteins, large amounts of starting materials are needed for proteome-wide analysis. The chance of detecting the PTMs you are studying of is largely dependent on the expression level of that protein, the occupancy rate and the stability of the modification, and the fragmentation efficiency in a mass spec. Basically, we don’t know whether we can identify it or not, so it usually needs to spend time on optimizing the enrichment procedure. You also may be interested in specific modifications required specialized expertise and instrument that we do not have (such as carbohydrates). We can suggest you contact experts if you need that type of analysis.

The PTM ID services include:

• Enzymatic digestion (trypsin or other proteases available)
• PTM enrichment (depends on the project)
• Peptide purification and concentration (C18 desalting)
• A quality control run (digested BSA or Hela cells)
• LC-MS/MS analysis (Q-Exactive MS)
• Database and PTM search (PTM-modified peptides and PTM localization score)
• Results returned via Excel file