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Effects of column and gradient lengths on peak capacity and peptide identification in nanoflow LC-MS/MS of complex proteomic samples.

ABSTRACT: Reversed-phase liquid chromatography is the most commonly used separation method for shotgun proteomics. Nanoflow chromatography has emerged as the preferred chromatography method for its increased sensitivity and separation. Despite its common use, there are a wide range of parameters and conditions used across research groups. These parameters have an effect on the quality of the chromatographic separation, which is critical to maximizing the number of peptide identifications and minimizing ion suppression. Here we examined the relationship between column lengths, gradient lengths, peptide identifications, and peptide peak capacity. We found that while longer column and gradient lengths generally increase peptide identifications, the degree of improvement is dependent on both parameters and is diminished at longer column and gradients. Peak capacity, in comparison, showed a more linear increase with column and gradient lengths. We discuss the discrepancy between these two results and some of the considerations that should be taken into account when deciding on the chromatographic conditions for a proteomics experiment.

SUBMITTER: Hsieh EJ

PROVIDER: S-EPMC3554873 | biostudies-literature | 2013 Jan

REPOSITORIES: biostudies-literature

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Effects of column and gradient lengths on peak capacity and peptide identification in nanoflow LC-MS/MS of complex proteomic samples.

Hsieh Edward J EJ Bereman Michael S MS Durand Stanley S Valaskovic Gary A GA MacCoss Michael J MJ

Journal of the American Society for Mass Spectrometry 20121130 1

Reversed-phase liquid chromatography is the most commonly used separation method for shotgun proteomics. Nanoflow chromatography has emerged as the preferred chromatography method for its increased sensitivity and separation. Despite its common use, there are a wide range of parameters and conditions used across research groups. These parameters have an effect on the quality of the chromatographic separation, which is critical to maximizing the number of peptide identifications and minimizing io ...[more]

PMID: 23197307

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Project description:Mass spectrometry (MS)-based analysis of complex biological samples is essential for biomedical research and clinical diagnostics. The separation prior to MS plays a key role in the overall analysis, with separations having larger peak capacities often leading to more identified species and improved confidence in those identifications. High-resolution ion mobility (IM) separations enabled by Structures for Lossless Ion Manipulation (SLIM) can provide extremely rapid, high-resolution separations and are well suited as a second dimension of separation following nanoscale liquid chromatography (nanoLC). However, existing sample handling approaches for offline coupling of separation modes require microliter-fraction volumes and are thus not well suited for analysis of trace biological samples. We have developed a novel nanowell-mediated fractionation system that enables nanoLC-separated samples to be efficiently preconcentrated and directly infused at nanoelectrospray flow rates for downstream analysis. When coupled with SLIM IM-MS, the platform enables rapid and high-peak-capacity multidimensional separations of small biological samples. In this study, peptides eluting from a 100 nL/min nanoLC separation were fractionated into ~?60 nanowells on a microfluidic glass chip using an in-house-developed robotic system. The dried samples on the chip were individually reconstituted and ionized by nanoelectrospray for SLIM IM-MS analysis. Using model peptides for characterization of the nanowell platform, we found that at least 80% of the peptide components of the fractionated samples were recovered from the nanowells, providing up to ~tenfold preconcentration for SLIM IM-MS analysis. The combined LC-SLIM IM separation peak capacities exceeded 3600 with a measurement throughput that is similar to current one-dimensional (1D) LC-MS proteomic analyses. Graphical abstract A nanowell-mediated multidimensional separation platform that combines nanoLC with SLIM IM-MS enables rapid, high-peak-capacity proteomic analyses.

Dataset Information

Effects of column and gradient lengths on peak capacity and peptide identification in nanoflow LC-MS/MS of complex proteomic samples.

Publications

Effects of column and gradient lengths on peak capacity and peptide identification in nanoflow LC-MS/MS of complex proteomic samples.

Similar Datasets

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