Preprocessing significantly improves the peptide/protein identification sensitivity of high-resolution isobarically labeled tandem mass spectrometry data

Abstract

Isobaric labeling techniques coupled with high-resolution mass spectrometry have been widely employed in proteomic workflows requiring relative quantification. For each high-resolution tandem mass spectrum (MS/MS), isobaric labeling techniques can be used not only to quantify the peptide from different samples by reporter ions, but also to identify the peptide it is derived from. Because the ions related to isobaric labeling may act as noise in database searching, the MS/MS spectrum should be preprocessed before peptide or protein identification. In this article, we demonstrate that there are a lot of high-frequency, high-abundance isobaric related ions in the MS/MS spectrum, and removing isobaric related ions combined with deisotoping and deconvolution in MS/MS preprocessing procedures significantly improves the peptide/protein identification sensitivity. The user-friendly software package TurboRaw2MGF (v2.0) has been implemented for converting raw TIC data files to mascot generic format files and can be downloaded for free from https://github.com/shengqh/RCPA.Tools/releases as part of the software suite ProteomicsTools. The data have been deposited to the ProteomeXchange with identifier PXD000994.

Fig. 1.

Identification improvement rank of 16 preprocessing methods in five searching engines and two isobaric labeling approaches. The size of spot indicates the rank of method based on identification performance. The bigger the spot, the better the identification performance. The red circle indicates the best performance method in the same identification level, same engine, and same isobaric labeling approach. Mascot, MyriMatch, OMSSA, and X! Tandem achieved the best two-hit protein identification with preprocessing isobaric related ions in iTRAQ4 data set. Mascot, OMSSA, and X! Tandem achieved the best two-hit protein identification with preprocessing isobaric related ions in iTRAQ8 data set. The preprocessing considering isobaric related ions did not significantly improve the Comet identification sensitivity in both iTRAQ4 and iTRAQ8 data sets.

Fig. 2.

The spectrum/peptide/two-hit protein identification improvement percentage of 16 preprocessing methods in five searching engines and two isobaric labeling approaches. Mascot achieved most identification improvement among five engines while Comet achieved smallest identification improvement.

Fig. 3.

Mascot score improvement after preprocessing tandem mass spectra. Both top two density plots and bottom two violin plots indicated that the majority of the spectra gained score improvement with data preprocessing in both iTRAQ4 and iTRAQ8 data sets. p value 2.2e-16 from Wilcoxon rank sum test indicates that the score improvement in iTRAQ8 data set was significantly higher than in iTRAQ4 data set.

Fig. 4.

C-terminal peptide identification improvement in iTRAQ4 and iTRAQ8 data sets after preprocessing tandem mass spectra. The top two Venn diagrams indicated that preprocessing also increased C-terminal peptide identification sensitivity in both iTRAQ4 and iTRAQ8 data sets. The bottom two scatter plots indicate that the Mascot scores of the majority of commonly identified C-terminal peptides were also increased after preprocessing.