The Influence of pH on Elution Order of Tryptic Peptides on Reversed-Phase Columns: A Unique Approach to Multidimensional Liquid Chromatography Separations for Proteomics

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Paul Rainville, Kefei Zheng, Claude Mallet, Uwe Neue, Jeff Mazzeo, Reb Russell II
ASMS 2002; Orlando; 3 June 2002
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Recent developments in two-dimensional chromatography in which anion exchange is used first followed by reversed-phase in the second dimension coupled with mass spectrometry has already been utilized successfully to separate and detect peptides from yeast proteomes. However, this current method is limited in the first dimension by the low peak capacity offered by IEX columns and further requires a desalting step before introduction into the reversed-phase second dimension. An evaluation of different reversed-phase stationary phases was carried out to determine and compare retention time, peak shape and peak capacity for peptide samples obtained from a tryptic digest of cytochrome c. Symmetry, Spherisorb, Resolve, Nova-Pak and XTerra packings were all tested. The purpose of this experiment is to utilize this evaluation to develop a two-dimensional chromatography system where high pH reversed-phase separation is used first followed by low pH reversed-phase separation in the second dimension. The advantage of this technique would be to generate a salt-free, high capacity, high resolving separation technique in both dimensions that can readily be introduced into the mass spectrometer.

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