Evaluation of HCD- and CID-type fragmentation within their respective detection platforms for murine phosphoproteomics

Abstract

Protein phosphorylation modulates a myriad of biological functions, and its regulation is vital for proper cellular activity. Mass spectrometry is the enabling tool for phosphopeptide analysis, where recent instrumentation advances in both speed and sensitivity in linear ion trap and orbitrap technologies may yield more comprehensive phosphoproteomic analyses in less time. Protein phosphorylation analysis by MS relies on structural information derived through controlled peptide fragmentation. Compared with traditional, ion-trap-based collision-induced dissociation (CID), a more recent type of fragmentation termed HCD (higher energy collisional dissociation) provides beam type CID tandem MS with detection of fragment ions at high resolution in the orbitrap mass analyzer. Here we compared HCD to traditional CID for large-scale phosphorylation analyses of murine brain under three separate experimental conditions. These included a same-precursor analysis where CID and HCD scans were performed back-to-back, separate analyses of a phosphotyrosine peptide immunoprecipitation experiment, and separate whole phosphoproteome analyses. HCD generally provided higher search engine scores with more peptides identified, thus out-performing CID for back-to-back experiments for most metrics tested. However, for phosphotyrosine IPs and in a full phosphoproteome study of mouse brain, the greater acquisition speed of CID-only analyses provided larger data sets. We reconciled our results with those in direct contradiction from Nagaraj N, D'Souza RCJ et al. (J. Proteome Res. 9:6786, 2010). We conclude, for large-scale phosphoproteomics, CID fragmentation with rapid detection in the ion trap still produced substantially richer data sets, but the back-to-back experiments demonstrated the promise of HCD and orbitrap detection for the future.

Fig. 1.

Phosphopeptide analysis by a back-to-back, alternating CID- and HCD-type fragmentation for same-precursor ions. A, Workflow for the data-dependent scans using the hybrid LTQ Orbitrap Velos mass spectrometer. The five most abundant ions from each full MS cycle were subjected to sequential CID (ion-trap detection) and HCD (orbitrap detection) fragmentation. A titanium dioxide-enriched sample from 1 mg proteolyzed mouse brain was analyzed by LC-MS/MS techniques using this method. B, Scatter plot distributions of XCorr, ΔCn′, and Ascore values for phosphopeptides identified by both HCD (y axis) and CID (x axis) for 2+, 3+, and 4+ charged ions, showing generally higher trends for HCD. C, Table summarizing peptide and protein identifications from this experiment. Matches to reversed (decoy) sequences are shown in parentheses.

Fig. 2.

CID- and HCD- type fragmentation for phosphotyrosine antibody-enriched peptides. A, Workflow for phosphotyrosine analysis. B, Venn diagram of phosphotyrosine containing peptides and their overlap between CID and HCD. C, Table summarizing this experiment. Matches to reversed (decoy) sequences are in parentheses.

Fig. 3.

Full phosphoproteome analysis of mouse brain by CID- and HCD-type fragmentation. A, Workflow for phosphoproteomic analysis. Brain phosphopeptides were enriched with the SCX-IMAC approach (26), and ten fractions were analyzed by separate 85-min CID and HCD runs. B, Phosphopeptides identified in SCX fractions. C, Summary of these studies. Matches to reversed (decoy) sequences are in parentheses. D, E, Venn diagrams of site and phosphoprotein overlaps between CID and HCD experiments.

Fig. 4.

Cycle depth (A) and validation rate (B) for the CID and HCD methods. The data from Fig. 3 were analyzed for cycle depth by computing the number of MS/MS spectra triggered from each full MS scan across all ten SCX runs. A TOP10 and TOP20 method were used for HCD and CID analyses, respectively. When MS/MS were triggered, the vast majority of cycles contained either 10 or 20 MS/MS events, correspondingly. HCD spectra had higher validation rates at all MS/MS positions within a cycle. Fraction valid denotes the fraction containing phosphopeptides at the 1% FDR.