on 11-14-201407:28 AM - edited on 10-15-202111:36 AM by Closed Account
Falkenby LG, Such-Sanmartín G, Larsen MR, Vorm O, Bache N, Jensen ON. J Proteome Res. 2014 Oct 3. The high peptide sequencing speed provided by modern hybrid tandem mass spectrometers enable the utilisation of fast liquid chromatographic (LC) separation techniques. We present a robust solid phase extraction/capillary LC system (speLC) for 5-10 minute separation of semi-complex 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 hours 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 microgram of HeLa cells protein extracts at a rate of approximately 90 peptides/minute, 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 characterisation of large numbers of peptide-containing samples in biological, biomedical and clinical laboratories.