The Plasma Proteome
Abstract INTODUCTIONThere is an urgent need for a simple and sensitive method to elucidate the human plasma proteome to find markers of disease, or therapeutic factors. Human plasma proteome may be obtained from tryptic peptides that results from native digestion using commonly available, sensitive and robust analytical instruments such as linear quadrupole, tandem mass spectrometers. METHODSThe human plasma proteome was elucidated from three independent human EDTA plasma populations analyzed by precipitation with acetonitrile (ACN) for quaternary amine (QA) micro-chromatography prior to native tryptic digestion for nano liquid chromatography, electrospray ionization and tandem mass spectrometry (LC-ESI-MS/MS). The LC-ESI-MS/MS results from authentic plasma and blank injection MS/MS noise controls were parsed into SQL Server along with the fit of the MS/MS spectra from the rigorous X!TANDEM for analysis with the R statistical system. A total of 13,408 gene symbols from tryptic (TRYP) and/or phosphor/tryptic (STYP) peptides showed ≥ 10 peptides with an FDR q ≤ 0.01 from fit of MS/MS spectra by X!TANDEM and were resolved from the null distribution of background noise showed a Chi Square value of χ2 ≥ 9 (p ≤ 0.005). RESULTSNative digestion of human EDTA plasma permitted the identification and quantification of ~ 13,408 protein gene symbols in plasma that showed low FDR (q≤0.01) from the fit of peptide MS/MS spectra and where observation frequency was resolved from the null distribution of random MS/MS spectra of source noise from recordings of blank injections. There was good agreement between the orbital ion trap (OIT) and the sensitive linear ion trap (LIT) as well as the tryptic versus phospho/tryptic peptides. A distinct subset of human cellular proteins showed a variety of specific interaction domains that formed a highly interconnected network in the plasma. DISCUSIONThe agreement between the fit of the peptide MS/MS spectra by the rigorous X!TANDEM algorithm versus random MS/MS spectra controls from blank noise injections demonstrated the reliability of the experimental approach. The highly interconnected network in the plasma confirmed that digestion of plasma under native conditions permitted the identification and quantification of the proteins in a population of human plasma samples. CONCLUSIONIt was feasible to identify more than ten thousand proteins from human plasma with high confidence using a simple linear ion trap after precipitation, quaternary amine chromatography, native digestion and nano spray analysis with a linear quadrupole ion trap.
