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Differences in protein distribution between human plasma preparations, EDTA‐plasma and heparin‐plasma, analyzed by non‐denaturing micro‐2‐DE and MALDI‐MS PMF
Author(s) -
Jin Ya,
Manabe Takashi
Publication year - 2009
Publication title -
electrophoresis
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.666
H-Index - 158
eISSN - 1522-2683
pISSN - 0173-0835
DOI - 10.1002/elps.200800663
Subject(s) - chemistry , heparin , molecular mass , blood proteins , blood plasma , agarose , complement system , human plasma , chromatography , agarose gel electrophoresis , fibronectin , biochemistry , antibody , enzyme , immunology , biology , gene , extracellular matrix
Abstract 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. 2×10 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 p I 5.4–6.5 and apparent molecular mass of ca. 1×10 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 p I 5.0–5.6 and apparent molecular mass 1.2–1.5×10 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 p I 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.