Comparison of Multiple Liquid Partition Chromatography to Fractionate Human Serum for LC-MALDI Mass Spectrometry and LC-ESI Tandem Mass Spectrometry

摘要

Mass spectrometry may be an ideal tool for the identification and quantification of peptides in blood. However, mass spectrometers detect some abundant ions with greater intensity than other less abundant sample components. In order to identify the less abundant ions it is necessary to carry our various sample preparation techniques that will reduce or isolate the abundant ions. Also in order to select the best column for HPLC analysis it will be necessary to compare chromatography resins under the same conditions. Here we compared the fractionation of human serum proteins by 11 different forms of chromatography including Propyl Sulfate (PS), Quaternary Amine (QA), DiEthylAmino Ethanol (DEAE) and DEAE Blue, Phenol Sepharose, carboxy methyl sepharose, Hydroxy Apatite, Heparin (HEP), Concanavalin A (CONA), Cibacron Blue and Protein G chromatography. We analyzed the intact serum protein fractions obtained from each chromatographic support prior to digestion by SDS-PAGE and MALDI-TOF and the tryptic peptides by LC-ESI-MS/MS. The concentration of proteins in each fraction was determined by dot blotting on filter paper along side BSA standards. Fractions were resolved and compared by SDS-PAGE on 7percent tricine gels stained with coomasie brilliant blue or diamine silver staining and often showed different protein profiles consistent with selective chromatography. Fractions from the various columns were compared by MALDI-TOF and again showed the differential presence of ions indicating the column separations were selective. The selectivity of the chromatographic supports were also compared by LC-ESI-MS/MS with an ion trap. Proteins fractionated over each resin were digested with trypsin and the peptides were collected and desalted over C18. At least five and typically between 10-20 replicates from each column were collected. We settled on four columns, Concanavalin A, Propyl Sulfate, Quanternary Amine, and Heparin to analyze more than 100 fractions. All MS/MS spectra was correlated to the human transcripts in the Reference Sequence Data base (RefSeq) using X-TANDEM. The resulting list of proteins identified was reduced to distinct full-length sequences using SQL.