Technical Research on Removal of Excess TMPP in Sample Preparation of TMPP-Labeled Peptides

The labeling reagent (N-Succinimidyloxycarbonylmethyl)tris(2,4,6-trimethoxyphenyl)phosphonium bromide (TMPP-Ac-Osu, abbreviated as TMPP) effectively enhances peptide ionization efficiency and mass spectrometric (MS) detection sensitivity, enabling deep-coverage immunopeptidomic analysis. However, the efficient removal of excess TMPP remains challenging and severely interferes with liquid chromatography (LC) separation and subsequent MS analysis. In this study, an efficient MS sample preparation strategy was established to address residual TMPP contamination during the purification of TMPP-labeled peptides. Using a labeling system with a TMPP-to-peptide mass ratio of 2:1, peptide retention and TMPP removal efficiencies of three commonly used desalting materials, C18, strong cation exchange (SCX), and strong anion exchange (SAX) columns, were systematically compared. Residual TMPP was quantitatively evaluated by extracting characteristic chromatographic peaks and comparing signal intensities and peak areas. SAX achieved a peptide retention efficiency of 84.1%, second only to C18 (88.9%), while exhibiting the most effective TMPP removal, with a 184-fold reduction in residual TMPP signal relative to C18. Moreover, SAX-processed peptides showed lower background noise, higher signal-to-noise ratios, improved spectral quality, and higher peptide confidence scores. Collectively, the results indicated that SAX was an optimal sample preparation method for TMPP-labeled peptides, enabling efficient removal of excess labeling reagent and ensuring high-quality immunopeptide identification. This strategy provided a robust methodological foundation for the scalable application of TMPP labeling in immunopeptidomics and related fields.