Mass spectrometry has proven to be a valuable tool for the accurate quantification of proteins. In this study, we have evaluated the performances of three targeted approaches, namely Selected Reaction Monitoring (SRM), Parallel Reaction Monitoring (PRM) and Sequential Windowed Acquisition of Theoretical Mass Spectra (SWATH-MS), to accurately quantify ten potential biomarkers of beef meat tenderness or marbling in a cohort of 64 muscle samples. Besides and so as to get the most benefit out of the complete M2 maps that are acquired in SWATH-MS, we developed an original label-free quantification method to estimate protein amounts using an I-spline regression model. Overall, we showed that 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.

The performances of label-based SWATH-MS, SRM and PRM to accurately quantify ten candidate biomarkers of beef meat tenderness or marbling, in a cohort of 64 bovine muscle tissues expected to cover a wide biological range of these traits, were evaluated. Limits of quantification, dynamic range and quantification performances were assessed. Moreover, protein amounts for all proteins detected in SWATH-MS were estimated in a label-free manner.

Sixty-four muscle samples consisting of 23 samples of Semimembranosus (SM, fast oxido-glycoltyic with intermediate intramuscular fat content), 33 samples of Longissimus thoracis (LT, mixt oxidative muscle with high intramuscular fat content), and 8 samples of Semitendinosus (ST, fast glycolytic with low intramuscular fat content) muscles were used. These muscle samples were collected on cows and young bulls from several breeds (Rouge des Prés, Limousine, Blonde d’Aquitaine) to be representative of cattle used in the French beef production. In addition, they have been chosen to represent a wide variety of tenderness and marbling. Proteins were extracted and 30-µg samples were prepared in triplicate using a tube-gel protocol slightly adapted from Muller et al., 2016 (doi: 10.1002/pmic.201600288). Eleven samples were randomly chosen as representative matrix for method development, external quality control and generating a spectral library necessary for SWATH-MS data interpretation. The matrix pool was prepared in tube-gel and fractionated by SDS-PAGE A concentration-balanced mixture of 20 accurately quantified SIL peptides (Spike Tides™ TL, JPT Peptide Technologies, Berlin, Germany) was spiked in each sample. Retention time standards (iRT; Biognosys, Schlieren, Switzerland) were additionally spiked in samples analysed in DDA and SWATH-MS modes. PRM analyses were performed on a nanoAcquity UPLC device (Waters, Milford, MA) coupled to a Q-Exactive Plus mass spectrometer (Thermo Fisher Scientific, Bremen, Germany). A scheduled PRM method consisting of one full MS scan and 16 targeted MS2 scans was developed. The full MS scan was collected from 300-1800 m/z at a resolution of 17,500 at 200 m/z (AGC target: 3e6, maximum IT: 50 ms). Targeted MS2 scans were collected at a resolution of 35,000 at 200 m/z (AGC target: 1e6, maximum IT: 128 ms) and scheduled for 3 min around the expected retention time (RT). SRM analyses were performed on a Dionex UltiMate 3000 system coupled with a TSQ Vantage (both from Thermo Fisher Scientific). A scheduled SRM method with 6-min time windows, 3s cycle time and 376 transitions was developed using crude stable isotope-labelled peptides (PEPotec SRM Peptides, Thermo Fisher Scientific). DDA and SWATH-MS analyses were performed on an ekspert nanoLC 400 system coupled to a TripleTOF 6600 mass spectrometer (both from Sciex, Concord, Canada). A SWATH-MS method consisting of 100 variable windows covering the 200-1,600 m/z range with an overlap of 1 m/z was developed. MS1 spectra were collected for 150 ms, and MS2 spectra for 45 ms in high-sensitivity mode.