Identification of proteins and phosphoproteins using pulsed Q collision induced dissociation (PQD)

Abstract

Pulsed Q collision induced dissociation (PQD) was developed to facilitate detection of low-mass reporter ions from labeling reagents (e.g., iTRΑQ) in peptide quantification using an LTQ mass spectrometer (MS). Despite the large number of linear ion traps worldwide, the use and optimization of PQD for protein identification have been limited, in part due to less effective ion fragmentation relative to the collision induced dissociation (CID). PQD expands the m/z coverage of fragment ions to the lower m/z range by circumventing the typical low mass cut-off of an ion trap MS. Since database searching relies on the matching between theoretical and observed spectra, it is not clear how ion intensity and peak number might affect the outcomes of a database search. In this report, we systematically evaluated the attributes of PQD mass spectra, performed intensity optimization, and assessed the benefits of using PQD on the identification of peptides and phosphopeptides from an LTQ. Based on head-to-head comparisons between CID (higher intensity) and PQD (better m/z coverage), peptides identified using PQD generally have Xcorr scores lower than those using CID. Such score differences were considerably diminished by the use of 0.1% m-nitrobenzyl alcohol (m-NBA) in mobile phases. The ion intensities of both CID and PQD were adversely affected by increasing m/z of the precursor, with PQD more sensitive than CID. In addition to negating the 1/3 rule, PQD enhances direct bond cleavage and generates patterns of fragment ions different from those of CID, particularly for peptides with a labile functional group (e.g., phosphopeptides). The higher energy fragmentation pathway of PQD on peptide fragmentation was further compared to those of CID and the quadrupole-type activation in parallel experiments.

Keywords: Pulsed Q collision induced dissociation (PQD); linear ion trap; protein identification; triple quadrupole (QqQ).

Figure 1

(a) Total ion current (TIC) profile with increasing collision energy (CE_PQD) on iTRAQ-labeled Glu-Fib B (0.5 pmol/μl). Samples were infused at 0.5 pmol/μl and the energy level was varied every 0.5 min. (b) Effect of increasing CE_PQD on TIC of the precursor ion, a selected product ion, and an iTRAQion.

Figure 2

The levels of TIC of full-scan MSMS using CID (black diamond) or PQD (cross) at different precursor m/z (left y axis); and relative ratios of TIC_PQD/TIC_CID (red diamond), as indicated by the right y axis.

Figure 3

(a) CID and (b) PQD mass spectra of triply charged HTVLYISPPPEDLLDNSR (from ToneBP protein) acquired at collision energy of 35% on an LTQ. (c) mass spectrum acquired with triple quadrupole-type collision at CE 33 on a QTrap 4000 MS.

Figure 3

(a) CID and (b) PQD mass spectra of triply charged HTVLYISPPPEDLLDNSR (from ToneBP protein) acquired at collision energy of 35% on an LTQ. (c) mass spectrum acquired with triple quadrupole-type collision at CE 33 on a QTrap 4000 MS.

Figure 4

Tandem mass spectra of (a) conventional CID, (b) PQD, and (c) triple quadrupole-type collision, respectively, of VNQIGTLS(phos)ES(phos)IK. Some fragment ions are labeled. The collision energy were 35%, 28%, and 41 for CID, PQD, and QqQ (based on rolling collision energy of Qtrap 4000), respectively.

Figure 4

Tandem mass spectra of (a) conventional CID, (b) PQD, and (c) triple quadrupole-type collision, respectively, of VNQIGTLS(phos)ES(phos)IK. Some fragment ions are labeled. The collision energy were 35%, 28%, and 41 for CID, PQD, and QqQ (based on rolling collision energy of Qtrap 4000), respectively.

Figure 5

Box and whisker plot of relative Xcorr scores (Xcorr_PQD/Xcorr_CID), using PQD_default and PQD_modified settings, for the phosphoproteins identified with at least two distinct p-peptides.

Figure 6

Box and whisker plot of the ratios of Ascores (PQD_modified/CID) of phosphopeptides having increasing m/z. Phosphopeptides were identified from wild-type S49 cell lysate.