Removal of Interference MS/MS Spectra for Accurate Quantification in Isobaric Tag-Based Proteomics

Abstract

Rapid progress in mass spectrometry (MS) has made comprehensive analyses of the proteome possible, but accurate quantification remains challenging. Isobaric tags for relative and absolute quantification (iTRAQ) is widely used as a tool to quantify proteins expressed in different cell types and various cellular conditions. The quantification precision of iTRAQ is quite high, but the accuracy dramatically decreases in the presence of interference peptides that are coeluted and coisolated with the target peptide. Here, we developed "removal of interference mixture MS/MS spectra (RiMS)" to improve the quantification accuracy of isobaric tag approaches. The presence of spectrum interference is judged by examining the overlap in the elution time of all scanned precursor ions. Removal of this interference decreased protein identification (11% loss) but improved quantification accuracy. Further, RiMS does not require any specialized equipment, such as MS 3 instruments or an additional ion separation mode. Finally, we demonstrated that RiMS can be used to quantitatively compare human-induced pluripotent stem cells and human dermal fibroblasts, as it revealed differential protein expressions that reflect the biological characteristics of the cells.

Multi-omics approach reveals posttranscriptionally regulated genes are essential for human pluripotent stem cells

“…Protein samples lysed with PTS buffer were subjected to reduction, alkylation, Lys-C/trypsin digestion (enzyme ratio 1/100), and desalting as previously described ( Iwasaki et al., 2019 ). Briefly, 1000 μL of an organic solvent was added to 1000 μL of the digested protein solution, and the mixture was acidified with 0.5% TFA (final concentration).…”

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“…False discovery rates were estimated by searching against a decoy sequence database (< 1%). For the peptide and protein quantification, we used RiMS approach to increase the accuracy 50 .…”

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“…False discovery rates (FDRs) were estimated by searching against a decoy sequence database (< 1%). For the peptide and protein quantification, the iTRAQ area was normalized by the total area of the whole proteome for each sample, and we used the RiMS method to increase the accuracy (Iwasaki et al, 2019).…”