Efficient and clean charge derivatization of peptides for analysis by mass spectrometry

Charge derivatization with succinimidyloxycarbonylmethyl tris(2,4,6-trimethoxyphenyl)phosphonium bromide (TMPP-Ac-OSu) has great potential in several aspects of proteomics, such as peptide de novo sequencing, PTM analysis, etc. However, the excess reagent and its side products greatly limited its scope of use. Here, we report an improved method to perform charge derivatization of peptides by TMPP-Ac-OSu without interference from the excess reagent and corresponding side products. Briefly, the protein was first separated on sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) or coagulated with the gel. The protein in-gel was then incubated with a high concentration of reagent, followed by extensive washing. Afterwards, the protein was in-gel digested with trypsin according to the routine protocol. The mainly resultant peptides were attached with one positive tag on the N-termini or Lys-epsilon-NH(2). The process has been successfully applied to 2-DE resolved protein spots. Compared to the native proteins, the derivatized counterparts have higher rates of PMF identification and more straightforward tandem mass spectra. This promising method should pave the way for the practical use of charge derivatization in proteomics.