An Improved Model for Prediction of Retention Times of Tryptic Peptides in Ion Pair Reversed-phase HPLC

摘要

The proposed model is based on the measurement of the retention times of 346 tryptic peptides in the 560- to 4,000-Da mass range, derived from a mixture of 17 protein digests. These peptides were measured in HPLC-MALDI MS runs, with peptide identities confirmed by MS/MS. The model relies on summation of the retention coefficients of the individual amino acids, as in previous approaches, but additional terms are introduced that depend on the retention coefficients for amino acids at the N-terminal of the peptide. In the 17-protein mixture, optimization of two sets of coefficients, along with additional compensation for peptide length and hydrophobicity, yielded a linear dependence of retention time on hydrophobicity, with an R2 value about 0.94. The predictive capability of the model was used to distinguish peptides with close m/z values and for detailed peptide mapping of selected proteins. Its applicability was tested on columns of different sizes, from nano- to narrow-bore, and for direct sample injection, or injection via a pre-column. It can be used for accurate prediction of retention times for tryptic peptides on reversed-phase (300-Å pore size) columns of different sizes with a linear water-ACN gradient and with TFA as the ion-pairing modifier. The proposed model is based on the measurement of the retention times of 346 tryptic peptides in the 560- to 4,000-Da mass range, derived from a mixture of 17 protein digests. These peptides were measured in HPLC-MALDI MS runs, with peptide identities confirmed by MS/MS. The model relies on summation of the retention coefficients of the individual amino acids, as in previous approaches, but additional terms are introduced that depend on the retention coefficients for amino acids at the N-terminal of the peptide. In the 17-protein mixture, optimization of two sets of coefficients, along with additional compensation for peptide length and hydrophobicity, yielded a linear dependence of retention time on hydrophobicity, with an R2 value about 0.94. The predictive capability of the model was used to distinguish peptides with close m/z values and for detailed peptide mapping of selected proteins. Its applicability was tested on columns of different sizes, from nano- to narrow-bore, and for direct sample injection, or injection via a pre-column. It can be used for accurate prediction of retention times for tryptic peptides on reversed-phase (300-Å pore size) columns of different sizes with a linear water-ACN gradient and with TFA as the ion-pairing modifier. The application of MS to biomolecular analysis has revolutionized protein research within the past decade (1Mann M. Hendrickson R.C. Pandey A. Analysis of proteins and proteomes by mass spectrometry..Annu. Rev. Biochem. 2001; 70: 437-473Google Scholar). This can be mostly attributed to the development of ionization techniques that are compatible with biomolecules, i.e. MALDI (2Karas M. Hillenkamp F. Laser desorption ionization of proteins with molecular masses exceeding 10,000 daltons..Anal. Chem. 1988; 60: 2299-2301Google Scholar, 3Hillenkamp F. Karas M. Beavis R.C. Chait B.T. Matrix-assisted laser desorption/ionization mass spectrometry of biopolymers..Anal. Chem. 1991; 63: 1193A-1203AGoogle Scholar) and ESI (4Fenn J.B. Mann M. Meng C.K. Wong S.F. Whitehouse C.M. Electrospray ionization for mass spectrometry of large biomolecules..Science. 1989; 246: 64-71Google Scholar), as well as improved instrumentation. However, although modern mass spectrometers provide high mass accuracy and sensitivity, the protein complexity and concentration range usually found in biological samples still present a challenge. The problem has been traditionally attacked by separation of complex protein mixtures by two-dimensional gel electrophoresis, with subsequent protein in-gel digestion, followed by ESI or MALDI MS. This remains one of the most popular sample preparation procedures, especially suitable for protein identification and quantitation. Ho