Differences in protein distribution between human plasma preparations, EDTA-plasma and heparin-plasma, analyzed by non-denaturing micro-2-DE and MALDI-MS PMF.
The effects of plasma preparation methods on the status of human plasma proteins were analyzed by non-denaturing micro-2-DE followed with polypeptide assignment with MALDI-TOF MS and PMF. In order to facilitate the separation of high-molecular-mass plasma proteins (up to ca. 2x10(3) kDa) in short separation time, agarose micro-IEF gels were employed. The comparisons of the 2-DE patterns between EDTA-plasma (1.0 mg EDTA/mL blood) and heparin-plasma (0.025 mg heparin/mL blood) revealed the differences in protein distribution around pI 5.4-6.5 and apparent molecular mass of ca. 1x10(3) kDa, which were mainly attributed to the presence of the complexes of complement C4b and C4b-binding protein in heparin-plasma and their absence in EDTA-plasma. The distribution of several spots around pI 5.0-5.6 and apparent molecular mass 1.2-1.5x10(2) kDa was also found to be different; the fragments of complement C3 and C4 were detected in heparin-plasma but not in EDTA-plasma. The 2-DE pattern of high-heparin-plasma (0.50 mg heparin/mL blood) showed pI changes of three plasma proteins, fibronectin, complement factor B, and pre-alpha-inhibitor when compared with that of heparin-plasma, suggesting the interactions between heparin and the proteins. These results demonstrated that subtle changes in plasma proteins, caused by the different plasma preparation procedures, can be analyzed by non-denaturing agarose-IEF/micro-2-DE followed by MALDI-MS and PMF.
Jin
Ya
Y
Department of Chemistry, Faculty of Science, Ehime University, Matsuyama, Japan.
Manabe
Takashi
T
D000328:Adult; D001798:Blood Proteins / Q000032:analysis / Q000737:chemistry / Q000378:metabolism; D001800:Blood Specimen Collection; D015935:Complement C4b / Q000737:chemistry / Q000378:metabolism; D050716:Complement C4b-Binding Protein / Q000737:chemistry / Q000378:metabolism; D004492:Edetic Acid / Q000737:chemistry*; D015180:Electrophoresis, Gel, Two-Dimensional; D005260:Female; D006493:Heparin / Q000737:chemistry*; D006801:Humans; D046912:Multiprotein Complexes / Q000737:chemistry / Q000378:metabolism; D010449:Peptide Mapping; D010949:Plasma / Q000737:chemistry*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
D001798:Blood Proteins; D050716:Complement C4b-Binding Protein; D046912:Multiprotein Complexes; D015935:Complement C4b; D006493:Heparin; D004492:Edetic Acid