Practical Implementation of 2D HPLC Scheme with Accurate Peptide Retention Prediction in Both Dimensions for High-Throughput Bottom-Up Proteomics

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 ∼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.