Optimized Nonlinear Gradients for Reversed-Phase Liquid Chromatography in Shotgun Proteomics
2013 (English)In: Analytical Chemistry, ISSN 0003-2700, E-ISSN 1520-6882, Vol. 85, no 16, 7777-7785 p.Article in journal (Refereed) Published
Reversed-phase liquid chromatography has become the preferred method for separating peptides in most of the mass spectrometry-based proteomics workflows of today. In the way the technique is typically applied, the peptides are released from the chromatography column by the gradual addition of an organic buffer according to a linear function. However, when applied to complex peptide mixtures, this approach leads to unequal spreads of the peptides over the chromatography time. To address this, we investigated the use of nonlinear gradients, customized for each setup at hand. We developed an algorithm to generate optimized gradient functions for shotgun proteomics experiments and evaluated it for two data sets consisting each of four replicate runs of a human complex sample. Our results show that the optimized gradients produce a more even spread of the peptides over the chromatography run, while leading to increased numbers of confident peptide identifications. In addition, the list of peptides identified using nonlinear gradients differed considerably from those found with the linear ones, suggesting that such gradients can be a valuable tool for increasing the proteome coverage of mass spectrometry-based experiments.
Place, publisher, year, edition, pages
2013. Vol. 85, no 16, 7777-7785 p.
Chromatography columns, Complex peptide mixtures, Complex samples, Linear functions, Optimized gradients, Peptide identification, Reversed-phase liquid chromatography, Shotgun proteomics
IdentifiersURN: urn:nbn:se:kth:diva-129458DOI: 10.1021/ac401145qISI: 000323471800024ScopusID: 2-s2.0-84882713295OAI: oai:DiVA.org:kth-129458DiVA: diva2:652540
FunderScience for Life Laboratory - a national resource center for high-throughput molecular bioscienceSwedish e‐Science Research CenterEU, European Research Council
QC 201310012013-10-012013-09-302013-10-01Bibliographically approved