Selected Reaction Monitoring by Linear Ion-Trap Mass Spectrometry Can Effectively Be Applicable to Simultaneous Quantification of Multiple Peptides

Abstract

Selected reaction monitoring (SRM) mass spectrometry (MS) is becoming a popular approach for targeted quantitative proteomics. Triple-quadrupole mass spectrometers have been historically considered as the instrument of choice for this type of quantitative analysis. Recently, however, it has been reported that the SRM MS with a linear ion-trap (LIT) mass spectrometer is rather more appropriate for quantitative analysis of large peptides than the triple-quadrupole ones. In this study, we demonstrate that the SRM MS performed with a LIT mass spectrometer can simultaneously analyze multiple peptides and can quantify specific peptides in biological specimens without the use of stable isotope (SI)-labeled standard peptides. Firstly, a mixture of 10 synthesized peptides derived from yeast proteins and bovine serum albumin (BSA) was simultaneously analyzed by the LIT SRM. The ion peak areas of the 10 peptides were linearly correlated with the input amounts between 1 fmol and 10 pmol. Furthermore, the same peptide mixture spiked into human plasma was analyzed, and a linear response was found. Next, the amount of a BSA tryptic peptide was quantified by using an SI-labeled or a non SI-labeled peptide as an external reference standard. The difference in the quantified amounts of the BSA tryptic peptide was less than 10% between the 2 methods, suggesting that the "externally pulsed" non SI-labeled standard peptides derived from another species are useful. These results indicate that the SRM MS conducted with a LIT mass spectrometer is applicable to targeted quantitative proteomics of peptides at least up to 10 in number.