Technical Note pubs.acs.org/jpr
Integrated Solid-Phase Extraction−Capillary Liquid Chromatography (speLC) Interfaced to ESI−MS/MS for Fast Characterization and Quantification of Protein and Proteomes Lasse Gaarde Falkenby,†,‡,§ Gerard Such-Sanmartín,†,§ Martin R. Larsen,† Ole Vorm,‡,∥ Nicolai Bache,‡,∥ and Ole N. Jensen*,† †
Department of Biochemistry and Molecular Biology, University of Southern Denmark, Campusvej 55, DK-5230 Odense M, Denmark ‡ Thermo Scientific, Edisonvej 4, DK-5000 Odense C, Denmark S Supporting Information *
ABSTRACT: 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 solidphase 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. KEYWORDS: nanoliquid chromatography, solid-phase extraction, stagetips, mass spectrometry, protein analysis
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INTRODUCTION Liquid chromatography (LC)−tandem mass spectrometry (MS/MS) is the preferred technique for peptide sequencing for unambiguous identification and quantification of hundreds to thousands of proteins in a proteomics experiment. The capabilities and versatility of MS-based analysis are surpassing most traditional assays, and MS will likely become the preferred method for protein analysis in biological, biomedical, and clinical laboratories. However, the analysis of complex protein samples, such as whole cell extracts, tissue extracts, and body fluids, is challenging because of the extraordinary high number of different proteins and accompanying proteoforms due to post-translational modifications and processing as well as the large differences in protein concentrations.1 Hence, standard workflows for mass-spectrometry-based proteome analyses include protein isolation and separation and or peptide fractionation steps prior to peptide separation and sequencing by LC−MS/MS.2,3 Peptide separation in LC−MS/MS is best achieved by separating peptides at maximum chromatographic performance by applying long shallow gradients (2−8 h) at nanoliter/minute flow rates using long capillary columns (20− 100 cm). However, protein and peptide fractionation methods, © XXXX American Chemical Society
for example, SDS-PAGE, 2D-PAGE, HILIC, or SCX, are commonly used prior to LC−MS/MS, in effect producing series of fractions of lower complexity than the initial protein or peptide sample. Extended (1−4 h) chromatographic separation in LC−MS/MS then becomes time- and cost-inefficient. In such cases, fast (5−10 min) capillary LC separations would provide a higher throughput while still achieving acceptable identification rate. We present an integrated and automated solid-phase extraction (SPE) gradient LC system (speLC) optimized for fast (
