Journal
ANALYTICAL CHEMISTRY
Volume 80, Issue 18, Pages 7036-7042Publisher
AMER CHEMICAL SOC
DOI: 10.1021/ac800984n
Keywords
-
Categories
Funding
- Technology Transfer Office of the University of Manitoba
- Natural Sciences and Engineering Research Council of Canada
- Canadian Institute for Health Research
Ask authors/readers for more resources
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 similar to 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.
Authors
I am an author on this paper
Click your name to claim this paper and add it to your profile.
Reviews
Recommended
No Data Available