Definition and Characterization of a “Trypsinosome” from Specific Peptide Characteristics by Nano-HPLC−MS/MS and in Silico Analysis of Complex Protein Mixtures

Although HPLC−ESI−MS/MS is rapidly becoming an indispensable tool for the analysis of peptides in complex mixtures, the sequence coverage it affords is often quite poor. Low protein expression resulting in peptide signal intensities that fall below the limit of detection of the MS system in combination with differences in peptide ionization efficiency plays a significant role in this. A second important factor stems from differences in physicochemical properties of each peptide and how these properties relate to chromatographic retention and ultimate detection. To identify and understand those properties, we compared data from experimentally identified peptides with data from peptides predicted by in silico digest of all corresponding proteins in the experimental set. Three different complex protein mixtures extracted were used to define a training set to evaluate the amino acid retention coefficients based on linear regression analysis. The retention coefficients were also compared with other previous hydrophobic and retention scale. From this, we have constructed an empirical model that can be readily used to predict peptides that are likely to be observed on our HPLC−ESI−MS/MS system based on their physicochemical properties. Finally, we demonstrated that in silico prediction of peptides and their retention coefficients can be used to generate an inclusion list for a targeted mass spectrometric identification of low abundance proteins in complex protein samples. This approach is based on experimentally derived data to calibrate the method and therefore may theoretically be applied to any HPLC−MS/MS system on which data are being generated.

Keywords: proteomics • QStar • mass spectrometry • nano-HPLC−MS/MS • inclusion list • trypsinosome • membrane preparation • hydrophobicity