Application of Displacement Chromatography to Online Two-Dimensional Liquid Chromatography Coupled to Tandem Mass Spectrometry Improves Peptide Separation Efficiency and Detectability for the Analysis of Complex Proteomes

Kwiatkowski, M., Krösser, D., Wurlitzer, M., Steffen, P., Barcaru, A., Krisp, C., Horvatovich, P., Bischoff, R. & Schlüter, H., 21-Aug-2018, In : Analytical Chemistry. 90, 16, p. 9951-9958 8 p.

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The complexity of mammalian proteomes is a challenge in bottom-up proteomics. For a comprehensive proteome analysis, multidimensional separation strategies are necessary. Online two-dimensional liquid chromatography-tandem mass spectrometry (2D-LC-MS/MS) combining strong cation exchange (SCX) in the first dimension with reversed-phase (RP) chromatography in the second dimension provides a powerful approach to analyze complex proteomes. Although the combination of SCX with RP chromatography provides a good orthogonality, only a moderate separation is achieved in the first dimension for peptides with two (+2) or three (+3) positive charges. The aim of this study was to improve the performance of online SCX-RP-MS/MS by applying displacement chromatography to the first separation dimension. Compared to gradient chromatography mode (GCM), displacement chromatography mode (DCM) was expected to improve the separation of +2-peptides and +3-peptides, thus reducing complexity and increasing ionization and detectability. The results show that DCM provided a separation of +2-peptides and +3-peptides in remarkably sharp zones with a low degree of coelution, thus providing fractions with significantly higher purities compared to GCM. In particular, +2-peptides were separated over several fractions, which was not possible to achieve in GCM. The better separation in DCM resulted in a higher reproducibility and significantly higher identification rates for both peptides and proteins including a 2.6-fold increase for +2-peptides. The higher number of identified peptides in DCM resulted in significantly higher protein sequence coverages and a considerably higher number of unique peptides per protein. Compared to conventionally used salt based GCM, DCM increased the performance of online SCX-RP-MS/MS and enabled comprehensive proteome profiling in the low microgram range.

Original languageEnglish
Pages (from-to)9951-9958
Number of pages8
JournalAnalytical Chemistry
Issue number16
Early online date17-Jul-2018
Publication statusPublished - 21-Aug-2018



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