A Novel Strategy for the Analysis of Phosphopeptides, Coupling N-terminal Peptide Derivatization and Hplc Separation with Mass Spectrometry

• Here we present an N-terminal derivatization approach, using TMPP-Ac-Osu that improves the HPLC performance for small hydrophilic phosphopeptides. • This approach is compatible with LC/MS and LC/MALDI. • The chromatographic enhancement was evaluated by on-line nanoscale HPLC coupled with Electrospray on a Q-Tof PremierTM. • LC-MALDI/MS/MS analysis was also investigated. Both MS/MS on a Q-Tof UltimaTM MALDI and PSD MXTM on a MALDI micro MXTM were used. • The use of TMPP-Ac-Osu derivatization results in a fixed charge on the N-terminus of the peptide, directing fragmentation for CID and PSD experiments.