Combining label-free and label-based accurate quantifications with SWATH-MS: Comparison with SRM and PRM for the evaluation of bovine muscle type effects.

Joanna Bons,Sarah Cianférani,François Delalande,Christine Carapito,Marie Chion,Muriel Bonnet,Frédéric Bertrand,Gauthier Husson,Myriam Maumy‐Bertrand,Brigitte Picard

doi:10.1002/pmic.202000214

Abstract

Mass spectrometry has proven to be a valuable tool for the accurate quantification of proteins. In this study, the performances of three targeted approaches, namely selected reaction monitoring (SRM), parallel reaction monitoring (PRM) and sequential windowed acquisition of all theoretical fragment ion mass spectra (SWATH-MS), to accurately quantify ten potential biomarkers of beef meat tenderness or marbling in a cohort of 64 muscle samples were evaluated. So as to get the most benefit out of the complete MS2 maps that are acquired in SWATH-MS, an original label-free quantification method to estimate protein amounts using an I-spline regression model was developed. Overall, SWATH-MS outperformed SRM in terms of sensitivity and dynamic range, while PRM still performed the best, and all three strategies showed similar quantification accuracies and precisions for the absolute quantification of targets of interest. This targeted picture was extended by 585 additional proteins for which amounts were estimated using the label-free approach on SWATH-MS; thus, offering a more global profiling of muscle proteomes and further insights into muscle type effect on candidate biomarkers of beef meat qualities as well as muscle metabolism.

Highlights

LC-MS/MS-based quantitative proteomics has become a valuable tool to explore proteomes and gain insights into the biological systems [1]
Among the 15 common quantifiable peptides, 87% (13) of the lower LOQ (LLOQ) were lower in parallel reaction monitoring (PRM) only, and 13% (2) in both PRM and SWATH-MS
SWATH-MS is reported in the literature to be generally less sensitive than Selected reaction monitoring (SRM) and PRM, [3,4,10,12,13] especially for low-abundant peptides, it appeared more sensitive than SRM in this study, with a median 3-fold increase

Summary

Introduction

LC-MS/MS-based quantitative proteomics has become a valuable tool to explore proteomes and gain insights into the biological systems [1]. With the introduction of high resolution/accurate mass (HR/AM) instruments – Q-Orbitrap and Q-TOF hybrids, additional targeted approaches have emerged, including parallel reaction monitoring (PRM) [8,9] and sequential windowed acquisition of all theoretical fragment ion spectra-mass spectrometry (SWATH-MS) based on data-independent acquisition (DIA) [10]. As SWATH-MS is not a targeted acquisition method [11], multiplexing capabilities are improved This offers the possibility to obtain accurate quantification of beforehand selected proteins using stable isotope-labelled (SIL) standards [4,5,10,13,14,15,16] , as with SRM and PRM, and to estimate proteome-wide absolute quantification in a label-free manner . This offers the possibility to obtain accurate quantification of beforehand selected proteins using stable isotope-labelled (SIL) standards [4,5,10,13,14,15,16] , as with SRM and PRM, and to estimate proteome-wide absolute quantification in a label-free manner . [14,17,18]

Methods

Results

Conclusion