title: Integrated solid-phase extraction-capillary liquid chromatography (speLC) interfaced to ESI-MS/MS for fast characterization and quantification of protein and proteomes.
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 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.
authors.affiliation: Department of Biochemistry and Molecular Biology, University of Southern Denmark , Campusvej 55, DK-5230 Odense M, Denmark.
authors.forename: Lasse Gaarde
authors.initials: LG
authors.lastname: Falkenby
authors.forename: Gerard
authors.initials: G
authors.lastname: Such-Sanmartín
authors.forename: Martin R
authors.initials: MR
authors.lastname: Larsen
authors.forename: Ole
authors.initials: O
authors.lastname: Vorm
authors.forename: Nicolai
authors.initials: N
authors.lastname: Bache
authors.forename: Ole N
authors.initials: ON
authors.lastname: Jensen
mesh_terms: D000595:Amino Acid Sequence; D001798:Blood Proteins / Q000378:metabolism; D002853:Chromatography, Liquid / Q000295:instrumentation / Q000379:methods*; D006367:HeLa Cells; D006801:Humans; D010455:Peptides / Q000378:metabolism; D020543:Proteome / Q000302:isolation & purification / Q000378:metabolism*; D040901:Proteomics / Q000379:methods*; D015203:Reproducibility of Results; D052616:Solid Phase Extraction / Q000379:methods*; D021241:Spectrometry, Mass, Electrospray Ionization / Q000295:instrumentation / Q000379:methods*; D053719:Tandem Mass Spectrometry / Q000295:instrumentation / Q000379:methods*
chemical_list: D001798:Blood Proteins; D010455:Peptides; D020543:Proteome
keywords: mass spectrometry; nanoliquid chromatography; protein analysis; solid-phase extraction; stagetips