Investigation of Scrambled Ions in Tandem Mass Spectra. Part 1. Statistical Characterization

Abstract

Scrambled ions have become the focus of recent investigations of peptide fragmentation. Here, an investigation of more than 390,000 high quality CID mass spectra is presented to explore the extent of scrambled ions in mass spectra and the possible fragmentation rules during scramble reactions. For the former, scrambled ions generally make up more than 10 % of mass spectra in number, although the abundances are less than 0.1 of the base peak. For the latter, relatively preferential re-opening sites were found for aliphatic residues Ala, Ile, Leu, and other residues such as Met, Gln, Ser, Phe, and Thr, whereas disfavored sites were found for basic residues Arg, Lys, and His, and Trp for both scrambled b and a ions. Similar preferential order in re-opening reaction was found in the reaction of losing internal residues when cleavage occurs at C-terminal side of 20 residues. However, when cleavage occurs at N-terminal side, Glu, Phe, and Trp become the most preferential sites. These results provide a deep insight into cleavage rules during scramble reactions for prediction of peptide mass spectra. Also, an additional investigation of whether scrambled ions could help discriminate false identifications from correct identifications was performed. Probing the number fraction of scrambled ions in falsely and correctly interpreted spectra and analyzing the correlation between scrambled ions and SEQUEST scores XCorr and Sp showed scrambled ions could at some extent help improve the discrimination in singly charged identifications, whereas no improvement was found for multiply charged results.

Chemometrics in instrumental analysis of complex systems—in honor and memory of Yi‐Zeng Liang

“…It has been shown that based on the analysis of very large‐scale independent mass spectral sets derived from various tandem mass spectrometric platforms, MS 2 PBPI has gained the highest prediction accuracy. To address the problem that peptide ion scrambling during CID may influence the identification of peptides for early peptide identification tools that do not consider these newly observed ions Liang's group provides statistical analysis of the fragmentation behaviors of these ions under CID and then evaluates the influence of these ions on peptide identification using various tools and mass spectral datasets . All these works provide valuable insights into the analysis of high‐throughput mass spectral data.…”

Gas‐phase fragmentation reactions of a7 ions containing a glutamine residue

“…IRMPD studies have provided strong evidence for forming macrocyclic structures in the gas phase 22–24 . Previous studies have been conducted to investigate the influences of different parameters (i.e., peptide length, amino acid type, and side‐chain functionality) on the formation of macrocyclic structures 23,25–33 …”

Effect of the sarcosine residue on sequence scrambling in peptide b5 ions

“…By contrast, Polfer and co‐workers reported that an analysis of a significant body of data on protonated tryptic peptides reported earlier showed that there were significant signals for non‐direct sequence ions, particularly at low m/z ratios, but that these products did not interfere with the sequencing of the peptides. Recently, Liang and co‐workers have examined a large body of peptide CID spectra available in accessible data bases. They reported that non‐direct sequence ions were generally observed but in relatively low abundance.…”