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Anal Chem. 2008 Sep 15;80(18):7036-42. doi: 10.1021/ac800984n. Epub 2008 Aug 8.

Practical implementation of 2D HPLC scheme with accurate peptide retention prediction in both dimensions for high-throughput bottom-up proteomics.

Author information

1
Manitoba Centre for Proteomics and Systems Biology, University of Manitoba, 799 JBRC, 715 McDermot Avenue, Winnipeg, MB, R3E 3P4, Canada.

Abstract

We describe the practical implementation of a new RP (pH 10 - pH 2) 2D HPLC-ESI/MS scheme for large-scale bottom-up analysis in proteomics. When compared to the common SCX-RP approach, it provides a higher separation efficiency in the first dimension and increases the number of identified peptides/proteins. We also employed the methodology of our sequence-specific retention calculator (SSRCalc) and developed peptide retention prediction algorithms for both LC dimensions. A diverse set of approximately 10,000 tryptic peptides from the soluble protein fraction of whole NK-type cells gave retention time versus hydrophobicity correlations, with R (2) values of 0.95 for pH 10 and 0.945 for pH 2 (formic acid) separation modes. The superior separation efficiency and the ability to use retention prediction to filter out false-positive MS/MS identifications gives promise that this approach will be a method of choice for large-scale proteomics analyses in the future. Finally, the "semi-orthogonal" separation selectivity permits the concatenation of fractions in the first dimension of separation before the final LC-ESI MS step, effectively cutting the analysis time in half, while resulting in a minimal reduction in protein identification.

PMID:
18686972
DOI:
10.1021/ac800984n
[Indexed for MEDLINE]

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