Integrated Solid-Phase Extraction–Capillary Liquid Chromatography (speLC) Interfaced to ESI–MS/MS for Fast Characterization and Quantification of Protein and Proteomes

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.