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.

Proteotranscriptomic Profiling of 231-BR Breast Cancer Cells: Identification of Potential Biomarkers and Therapeutic Targets for Brain Metastasis

“…Peptides were extracted from 231-BR and MDA-MD-231 cells as described above (see Protein preparation for mass spectrometry) but from a different batch of cultured cells (biological replicate). These peptides were loaded on C18-containing stage tips prior to reversed phase chromatography on a SPE-LC (modified EASY-nLC 1000, Thermo, Odense, Denmark), as described in Falkenby et al (77). Short gradients ranging from 4 to 35% in 5 min were used.…”

Robust, reproducible and quantitative analysis of thousands of proteomes by micro-flow LC–MS/MS

“…ano-flow liquid chromatography (nano-flow LC) has been the mainstay in proteome research for >20 years 1 , primarily because low flow rates improve peptide ionization by electrospray (ESI) for mass spectrometry (MS) and, hence, sensitivity 2,3 . However, this comes at the cost of the challenge of manufacturing reproducible and long-lasting columns, maintaining stable ESI over extended periods of time, rapid chromatographic overloading, mass spectrometric saturation and often long, unproductive overhead times for sample transfer at low flow rates [4][5][6] . These factors can limit the reproducibility of peptide identification and quantification as well as the comprehensiveness, robustness and throughput of proteome analysis, particularly when analyzing samples of high complexity or wide dynamic range of protein concentrations as represented by tissues and body fluids 7 .…”

Sample Clean‐up Strategies for ESI Mass Spectrometry Applications in Bottom‐up Proteomics: Trends from 2012 to 2016

“…The analytes eluted from the purification column were directed toward another column used for analytical separation, the outlet of which was connected directly to the MS. Falkenby et al. developed an integrated SPE‐capillary LC system interfaced to ESI‐MS/MS for the fast analysis of semicomplex and complex peptide mixtures derived from HeLa and Escherichia coli cells from human blood plasma. The research group employed disposable micropipette SPE tips (StageTips) for sample preconcentration and desalting.…”