Comparison Of Alternative Proteomic Techniques For The Characterization Of The Human Serum Proteome.

Of all the proteomes, the proteins of the blood are perhaps of the greatest biological, medicinal importance. Serum or plasma is of unusual biological complexity, reflecting its communication with all cells, tissues and organs. Thus, the task of the HUPO Plasma Proteome Project (PPP) has been distributed among collaborating labs around the world.During the first stage of participating in and carrying out the HUPO PPP research, a varies of serum protein preparation methods for depleting high abundant proteins or fractionating proteins, which were Bio-rad Aurum Serum kit, Agilent multiple affinity removal system(MARS), preparative solution IEF (Rotofor) and organic solvent precipitation, were investigated and their applicable characteristics and limits were compared and summarized. On this basis, the Agilent MARS column with better reproducibility and specificity for depletion of high abundant proteins was applied for the sample preparation of HUPO PPP reference plasma or serum samples, which laid the solid ground for completing the analysis of the reference samples and comparing multiple proteomic techniques for their application in the research of serum protoeme.Based on the selection of MARS column for the depletion of six high a bundant proteins in plasma or serum samples, the high throughput two dimensional capillary liquid chromatography combined with electrospray ion trap MS/MS was established for the analysis of HUPO PPP reference plasma and serum samples. With optimized protein digestion method and improved mass spectra acquiring approach, the protein identification result of the reference samples was obviously improved and reproducible result was obtained. In average, about 110 proteins were identified in each of the reference samples. The protein identification level was medium among all the participating labs.At the workshop of HUPO PPP held in Montreal in 2003, the preliminary data sets submitted from different labs had little overlap in proteins identified. The lack of overlap can be attributed in part to the different technological strategies or methods of sample preparation used. In a systematic approach to this issue, we here compare the results from five different techniques using the same HUPO PPP reference serum specimen with the six proteins of highest abundance depleted by affinity chromatography. The approaches compared were: (1) Intact protein fractionation by anion exchange chromatography (WAX) followed by 2-dimensional electrophoresis (2-DE) and MALDI-TOF-MS-MS for protein identification (2DE strategy); (2) Intact protein fractionation by 2D-HPLC and then coupled with solution digestion of each fraction and micro-capillary RP-HPLC microESI-MS-MS identification (protein prefractionation strategy); (3) Digestion of mixed proteins by trypsin followed by automated online micro-capillary 2D-HPLC with ion trap micro-ESI-MS/MS; (online shotgun strategy); (4) Same as 3) with the SCX step performed offline (offline shotgun strategy) and (5) Same as 4) with the SCX fractions reanalysed by optimised nanoRP-HPLC-nanoESI-MS-MS. (offline shotgun-nanospray strategy). The protein identification results of each strategy were compared and their particular features were summarized. All five approaches yielded complementary sets of protein identifications. The total number of unique proteins identified by each of these five approaches was 1) 78, 2) 179, 3) 131, 4) 224, and 5) 330, respectively. In all 560 unique proteins were identified, only 37 proteins were identified by all five approaches which reflecting the complementary protein identification result from all these five strategies. The result could be attributed to obvious variations in the sample preparation process, differences in peptide separation efficiency, and the sensitivity of the mass spectrometer.In practice, the 2-DE approach yielded more information on the pi-altered isoforms of some serum proteins and the relative abundance of identified proteins. The protein 2D-HPLC prefractionation strategy slightly improved the capacity to detect proteins of lower abundance. Optimising the separation at the peptide level and improving the detection sensitivity of ESI-MS/MS were more effective than fractionation of intact proteins in increasing the total number of proteins identified with extended dynamic range . This is very important for the serum proteome research. The results also indicate that by increasing the number of well-separated fractions collected in offline SCX and optimization of RP-HPLC-ESI-MS/MS, more serum proteins of low abundance can be identified, such as Coagulation factor IX precursor (ug/mL), L-selectin precursor, and Hepatocyte growth factor-like protein precursor, identified through high-confidence MS-MS spectra of their peptides.All three strategies had similar distribution characteristics of mass range, isoelectric point and hydrophobicity of the identified proteins. It does appear that 2-D HPLC of intact proteins works best for proteins representing extremes in these properties while 2DE strategy is difficult to profiling the proteins with low molecular weight, or proteins with extremely acidic, basic or high hydrophobic characters.At last, when the whole dataset was compared with the high confident proteins list of HUPO PPP(N=3020), about 257 unique proteins were overlapped, 139 proteins of which were identified with 2 or more peptides; While compared with the reported non redundant list, totally 169 unique proteins were overlapped, 117 of which were identified with 2 or more peptides.