Integrated Solid-Phase Extraction–Capillary Liquid Chromatography (speLC) Interfaced to ESI–MS/MS for Fast Characterization and Quantification of Protein and Proteomes
2014-12-05T00:00:00Z (GMT) by
The high peptide sequencing speed provided by modern hybrid tandem mass spectrometers enables the utilization of fast liquid chromatographic (LC) separation techniques. We present a robust solid-phase extraction/capillary LC system (speLC) for 5–10 min separation of semicomplex peptide mixtures prior to ESI–MS/MS for peptide sequencing. This speLC–MS/MS system eliminates sample-to-sample carry-over by using disposable micropipette solid-phase extraction tips (StageTips) for peptide sample loading, concentration, and desalting. Automated analysis of 192 replicates of E. coli peptide mixtures in 30 h demonstrated the throughput, stability, and reproducibility of the system. The speLC–MS/MS system detected low-femtomole amounts of peptides and allowed sequencing of 1 μg of HeLa cells protein extracts at a rate of ∼90 peptides/min, identifying more than 1500 peptides (>500 proteins) in a 10 min speLC–MS/MS experiment. Analysis by selected reaction monitoring by speLC–SRM–MS/MS of distinct peptides derived from the blood proteins IGF1, IGF2, IBP2, and IBP3 demonstrated protein quantification with CV values below 10% across 96 replicates. The speLC–MS/MS system is ideally suited for fast screening and characterization of large numbers of peptide-containing samples in biological, biomedical, and clinical laboratories.