Standard Mixtures for Proteome Studies

Abstract

Mixtures of moderate complexity were formed from 23 peptides and 12 proteins digested with trypsin, all individually characterized. These mixtures were analyzed with replicates in full and windowed m/z ranges using online high-performance reverse phase liquid chromatography coupled via electrospray ionization to an ion trap mass spectrometer. The resulting spectra were searched using SEQUEST against databases of different sizes and contents and confidences of the observed identifications were evaluated by our earlier statistical model. These data were then combined with biologically derived spectral data, searched, and further evaluated. All peptides but one and all proteins were identified with high confidence. Additionally, the presence and behavior of quadruply charged peptides was analyzed. The properties of the proposed peptide and protein mixtures as well as the performance of the statistical model were carefully investigated. These mixtures mimic the complexity seen in large-scale proteomics experiments, and are proposed to serve as quality assessment standards for future proteome studies.