Integrated Solid-Phase Extraction鈥揅apillary Liquid Chromatography (speLC) Interfaced to ESI鈥揗S/MS for Fast Characterization and Quantification of Protein and Proteomes
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- 作者：Lasse Gaarde Falkenby ; Gerard Such-Sanmart铆n ; Martin R. Larsen ; Ole Vorm ; Nicolai Bache ; Ole N. Jensen
- 刊名：Journal of Proteome Research
- 出版年：2014
- 出版时间：December 5, 2014
- 年：2014
- 卷：13
- 期：12
- 页码：6169-6175
- 全文大小：423K
- ISSN：1535-3907
文摘
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鈥?0 min separation of semicomplex peptide mixtures prior to ESI鈥揗S/MS for peptide sequencing. This speLC鈥揗S/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鈥揗S/MS system detected low-femtomole amounts of peptides and allowed sequencing of 1 渭g of HeLa cells protein extracts at a rate of 鈭?0 peptides/min, identifying more than 1500 peptides (>500 proteins) in a 10 min speLC鈥揗S/MS experiment. Analysis by selected reaction monitoring by speLC鈥揝RM鈥揗S/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鈥揗S/MS system is ideally suited for fast screening and characterization of large numbers of peptide-containing samples in biological, biomedical, and clinical laboratories.