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
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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, Marcel; Krösser, Dennis; Wurlitzer, Marcus; Steffen, Pascal; Barcaru, Andrei; Krisp, Christoph; Horvatovich, Péter; Bischoff, Rainer; Schlüter, Hartmut.
in: ANAL CHEM, Jahrgang 90, Nr. 16, 21.08.2018, S. 9951-9958.Publikationen: SCORING: Beitrag in Fachzeitschrift/Zeitung › SCORING: Zeitschriftenaufsatz › Forschung › Begutachtung
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TY - JOUR
T1 - 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
AU - Kwiatkowski, Marcel
AU - Krösser, Dennis
AU - Wurlitzer, Marcus
AU - Steffen, Pascal
AU - Barcaru, Andrei
AU - Krisp, Christoph
AU - Horvatovich, Péter
AU - Bischoff, Rainer
AU - Schlüter, Hartmut
PY - 2018/8/21
Y1 - 2018/8/21
N2 - 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.
AB - 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.
KW - Journal Article
U2 - 10.1021/acs.analchem.8b02189
DO - 10.1021/acs.analchem.8b02189
M3 - SCORING: Journal article
C2 - 30014690
VL - 90
SP - 9951
EP - 9958
JO - ANAL CHEM
JF - ANAL CHEM
SN - 0003-2700
IS - 16
ER -