A Strategy for the Establishment of Plasma Proteome Database Based on Stable Isotope Labeling and FT-MS

A new strategy for the establishment of plasma proteome core data combined immunoaffinity separation, acetic anhydride stable isotope labeling, high accuracy FTICR mass spectrometry with data analysis software ASMQ1.0 writen by ourselves was displayed.  First, The top-12 high abundant proteins were depleted with the immunoaffinity fractionation columns（Genway, Seppro）and the low abundant proteins were enriched in the flow through fraction (FF). Second, the FF fraction were digested by trypsin and then split equally and labeled by stable isotope reagent  d₀/d₆acetic anhydride (1:1) respectively. As a result, the labeled peptides display a pair of peaks with 3.0 Da mass shift (one charge) in the spectrum. Third, after multi-dimension chromatography separation, the eluted peptides were identified by high accuracy (<5 ppm), high dynamic range (single spectrum >5 000) and high sensitivity (sub-fmol) LTQFT mass spectrum. Last, the MS data was search by local Mascot software. Then the mascot search results (p<0.05) was connected with MS original raw file by software ASMQ1.0. All the observed peaks ratio of the labeled peptides could be calculated with the theory value equal to 1.0. Through the statistical analysis of normal distribution fit, the 99% confidence interval was defined.