title: 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.
authors.affiliation: Laboratory of Clinical Investigation, National Institute on Aging, National Institutes of Health, Baltimore, MD 21224, USA.
authors.forename: Wells W
authors.initials: WW
authors.lastname: Wu
authors.forename: Guanghui
authors.initials: G
authors.lastname: Wang
authors.forename: Paul A
authors.initials: PA
authors.lastname: Insel
authors.forename: Cheng-Te
authors.initials: CT
authors.lastname: Hsiao
authors.forename: Sige
authors.initials: S
authors.lastname: Zou
authors.forename: Stuart
authors.initials: S
authors.lastname: Maudsley
authors.forename: Bronwen
authors.initials: B
authors.lastname: Martin
authors.forename: Rong-Fong
authors.initials: RF
authors.lastname: Shen
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001592:Benzyl Alcohols; D002457:Cell Extracts; D045744:Cell Line, Tumor; D007477:Ions / Q000737:chemistry; D013058:Mass Spectrometry / Q000379:methods*; D051379:Mice; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry; D010750:Phosphoproteins / Q000737:chemistry*; D011506:Proteins / Q000737:chemistry*; D053719:Tandem Mass Spectrometry
chemical_list: D001592:Benzyl Alcohols; D002457:Cell Extracts; D007477:Ions; D010446:Peptide Fragments; D010750:Phosphoproteins; D011506:Proteins; C046886:3-nitrobenzyl alcohol