Mass spectrometry global analysis of phosphorylated proteins in a complex sample is an important challenge among the post-translational modifications analysis because phosphoproteins are low abundant proteins and are present at substoichiometric levels. Moreover, this modification is labile leading to a difficult detection by mass spectrometry. In order to identify the maximum number of phosphoproteins in a sample, it is necessary to optimize the protein extraction, the phosphopeptides enrichment and the mass spectrometry analysis steps.
The method we use for phosphoproteome analysis is based on the enrichment of mono- and multiphosphorylated peptides according to the SIMAC (Sequential Elution from IMAC) method and on a combination of 3 different mass spectrometry acquisition methods: a CID mode, a Neutral Loss mode and a Multistage Activation mode.
Example of study performed:
Colinet et al., 2017, Scientific Reports. 2017; 7(1): 1713. doi: 10.1038/s41598-017-01974-z.