title: Practical implementation of 2D HPLC scheme with accurate peptide retention prediction in both dimensions for high-throughput bottom-up proteomics.
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.
authors.affiliation: Manitoba Centre for Proteomics and Systems Biology, University of Manitoba, 799 JBRC, 715 McDermot Avenue, Winnipeg, MB, R3E 3P4, Canada.
authors.forename: Ravi C
authors.initials: RC
authors.lastname: Dwivedi
authors.forename: Vic
authors.initials: V
authors.lastname: Spicer
authors.forename: Michael
authors.initials: M
authors.lastname: Harder
authors.forename: Mihaela
authors.initials: M
authors.lastname: Antonovici
authors.forename: Werner
authors.initials: W
authors.lastname: Ens
authors.forename: Kenneth G
authors.initials: KG
authors.lastname: Standing
authors.forename: John A
authors.initials: JA
authors.lastname: Wilkins
authors.forename: Oleg V
authors.initials: OV
authors.lastname: Krokhin
mesh_terms: D045744:Cell Line, Tumor; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D006801:Humans; D010455:Peptides / Q000032:analysis*; D011506:Proteins / Q000032:analysis; D040901:Proteomics / Q000379:methods*; D012680:Sensitivity and Specificity; D021241:Spectrometry, Mass, Electrospray Ionization
chemical_list: D010455:Peptides; D011506:Proteins