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Quantifying Peptides in Complex Mixtures with High Sensitivity and Precision Using Targeted Approach with a Hybrid Linear Ion Trap Orbitrap Mass Spectrometer
Author(s) -
Kiyonami Reiko,
Prakash Amol,
Zabrouskov Vlad
Publication year - 2011
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.25.1_supplement.926.3
Subject(s) - orbitrap , mass spectrometry , chromatography , chemistry , isobaric labeling , peptide , quadrupole ion trap , quantitative proteomics , ion trap , resolution (logic) , analytical chemistry (journal) , proteomics , protein mass spectrometry , tandem mass spectrometry , computer science , biochemistry , gene , artificial intelligence
High‐resolution, accurate‐mass (HR/AM) mass spectrometry is routinely used for identification and relative quantitation of peptides present in complex mixtures using stable‐isotope labeling, isobaric tags, or label‐free techniques. However, HR/AM application for targeted peptide analysis is less explored. Due to the complexity of biological samples, accurate mass and retention time alone are not sufficient for verifying identity of a targeted peptide. Therefore, sequence confirmation via MS/MS is essential during the early verification and validation stages of development of a targeted quantitative assay. Here, a workflow was developed for targeted protein/peptide quantitation on the LTQ Orbitrap Velos to use high resolution selected ion monitoring (SIM) for quantitation followed by targeted MS/MS for sequence confirmation. A standard peptide mixture spiked into a yeast digest mixture at different concentration as quantitative targets and LOD was as low as 10 amol. An additional yeast digest was used to further evaluate the precision and throughput and the workflow by 77 peptides (25 yeast proteins). All the peptides were successfully quantified and verified with high analytical precision in a single LC/MS run.