Importance of Manual Validation for the Identification of Phosphopeptides Using a Linear Ion Trap Mass Spectrometer

Accurate determination of protein phosphorylation is challenging, particularly for researchers who lack access to a high-accuracy mass spectrometer. In this study, multiple protocols were used to enrich phosphopeptides, and a rigorous filtering workflow was used to analyze the resulting samples. Phosphopeptides were enriched from cultured rat renal proximal tubule cells using three commonly used protocols and a dual method that combines separate immobilized metal affinity chromatography (IMAC) and titanium dioxide (TiO₂) chromatography, termed dual IMAC (DIMAC). Phosphopeptides from all four enrichment strategies were analyzed by liquid chromatography-multiple levels of mass spectrometry (LC-MSⁿ) neutral-loss scanning using a linear ion trap mass spectrometer. Initially, the resulting MS² and MS³ spectra were analyzed using PeptideProphet and database search engine thresholds that produced a false discovery rate (FDR) of <1.5% when searched against a reverse database. However, only 40% of the potential phosphopeptides were confirmed by manual validation. The combined analyses yielded 110 confidently identified phosphopeptides. Using less-stringent initial filtering thresholds (FDR of 7–9%), followed by rigorous manual validation, 262 unique phosphopeptides, including 111 novel phosphorylation sites, were identified confidently. Thus, traditional methods of data filtering within widely accepted FDRs were inadequate for the analysis of low-resolution phosphopeptide spectra. However, the combination of a streamlined front-end enrichment strategy and rigorous manual spectral validation allowed for confident phosphopeptide identifications from a complex sample using a low-resolution ion trap mass spectrometer.