Use of peptide retention time prediction for protein identification by off-line reversed-phase HPLC-MALDI MS/MS.
A new algorithm, sequence-specific retention calculator, was developed to predict retention time of tryptic peptides during RP HPLC fractionation on C18, 300-A pore size columns. Correlations of up to approximately 0.98 R2 value were obtained for a test library of approximately 2000 peptides and approximately 0.95-0.97 for a variety of real samples. The algorithm was applied in conjunction with an exclusion protocol based on mass (15 ppm tolerance) and retention time (2-min tolerance for 0.66% acetonitrile/min gradient), MART criteria to significantly reduce the instrument time required for complete MS/MS analysis of a digest separated by RP HPLC. This was confirmed by reanalyzing the set of HPLC-MALDI MS/MS data with no loss in protein identifications, despite the number of virtually executed MS/MS analyses being decreased by 57%.
Krokhin
Oleg V
OV
Department of Physics and Astronomy, University of Manitoba, Winnipeg, Manitoba R3T 2N2, Canada. krokhino@cc.umanitoba.ca
Ying
Stephen
S
Cortens
John P
JP
Ghosh
Dhiman
D
Spicer
Victor
V
Ens
Werner
W
Standing
Kenneth G
KG
Beavis
Ronald C
RC
Wilkins
John A
JA
D045744:Cell Line, Tumor; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D030562:Databases, Protein; D006801:Humans; D007477:Ions / Q000737:chemistry; D009862:Online Systems; D010455:Peptides / Q000737:chemistry*; D011506:Proteins / Q000737:chemistry*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods*; D053719:Tandem Mass Spectrometry / Q000379:methods*; D013997:Time Factors
D007477:Ions; D010455:Peptides; D011506:Proteins