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Quantifying Peptides in Complex Mixtures with High Sensitivity and Precision Using a Targeted Approach with a Hybrid Linear Ion Trap Orbitrap Mass Spectrometer

Reputable Mentor II
Reputable Mentor II
Kiyonami R, Prakash A, Hart B, Cunniff J, Zabrouskov V.
Application Note 500
A workflow for simultaneous quantification and identity verification of targeted peptides using the LTQ Orbitrap Velos hybrid mass spectrometer and Pinpoint software was developed and evaluated. The workflow employs wide mass range SIM scanning to improve the sensitivity and scheduled MS/MS for peptide verification. The high-resolution MS analysis (100,000 resolution FWHM) enables detection of co-eluting isobars that would interfere with quantitation. The detection limits were investigated by spiking isotopically labeled yeast peptides into a complex yeast digest matrix at different concentrations. The peptides were detected as low as 10 amol for all but one spiked heavy peptide and the quantitative dynamic range was 4 orders of magnitude. The 77 peptides selected to represent the 25 targeted yeast proteins in the optimized instrument method were successfully quantified and verified with high analytical precision. Ninety-seven percent of the targeted peptides gave a CV of less than 10%, and the average CV was 4.7%. Workflow detection limits, precision and specificity were enhanced using wide SIM isolations and a time-scheduled global MS/MS list.

Thermo Fisher Scientific
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Last update:
‎10-15-2021 11:45 AM
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