Cleavage of fibrinogen alpha chains during isoelectric focusing of human plasma under non-denaturing conditions analyzed by micro two-dimensional gel electrophoresis and matrix-assisted laser desorption/ionization mass spectrometry

T. Manabe, Ya Jin, Nao Yamaguchi, T. Sugiyama, Kohei Ikari
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引用次数: 6

Abstract

The identity of low-molecular-weight and minor protein spots, appeared in 2-DE patterns of human plasma, was examined. They were not obvious in the patterns of “Type-I” 2-DE (non-denaturing IEF followed by non-denaturing gel electrophoresis), but clearly detected in the patterns of “Type II” 2-DE (non-denaturing IEF followed by SDS gel electrophoresis) at pI 5.5-7.5 and apparent mass 8-40 kDa1). The spots were not obviously detected when the IEF gels were kept at low temperature (around 4°C) during electrophoresis, suggesting that they are the proteolysis products of plasma proteins. The minor spots were more obviously detected when human plasma was subjected to ammonium sulfate (AS) fractionation and the 0-35% saturated AS fraction was dialyzed and subjected to Type-II 2-DE. Then the 116 spots on the 2-DE pattern, detected at pI 5-7.5 and apparent mass 8-60 kDa, were excised and subjected to MALDI-MS measurements and the mass spectra were analyzed using the software of peptide mass fingerprinting (PMF) Mascot and ProFound to assign the proteins. Many of the spots were assigned to contain fibrinogen α chain, especially those at pI 5.5-7.5 and apparent mass 8-40 kDa, suggesting that these spots are its fragments. The distribution of the MS-detected peptide fragments suggested that the molecular-mass heterogeneity might be caused by the cleavage of multiple sites on the α chain. Care must be taken to keep the temperature of IEF gels at around 4°C during electrophoresis, when human plasma proteins are subjected to non-denaturing IEF. The absence of the spots of fibrinogen fragments on Type-II 2-DE gels would validate the intactness of plasma proteins. The advantages of micro gel system for the analysis of intact protein mixtures are suggested.
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微二维凝胶电泳和基质辅助激光解吸/电离质谱分析了非变性条件下人血浆等电聚焦过程中纤维蛋白原α链的断裂
研究了人血浆2-DE模式中出现的低分子量和少量蛋白斑点的一致性。它们在“i型”2-DE(非变性IEF +非变性凝胶电泳)模式中不明显,但在“II型”2-DE(非变性IEF + SDS凝胶电泳)模式中明显存在,在pI 5.5 ~ 7.5,表观质量8 ~ 40 kDa1)。电泳时低温保存(4℃左右)未发现明显斑点,提示其为血浆蛋白水解产物。人血浆经硫酸铵(AS)分馏、0-35%饱和AS馏分透析及ii型2-DE处理时,小斑点更为明显。选取pI 5 ~ 7.5、表观质量8 ~ 60 kDa的2-DE图谱上的116个点,进行MALDI-MS测定,并利用peptide mass fingerprinting (PMF) Mascot和ProFound软件进行质谱分析,确定蛋白质的定位。许多斑点被认为含有纤维蛋白原α链,特别是在pI 5.5 ~ 7.5和表观质量8 ~ 40 kDa的斑点,表明这些斑点是其片段。ms检测到的肽片段的分布表明,分子质量的不均匀性可能是由于α链上多个位点的断裂引起的。在电泳过程中,当人血浆蛋白受到非变性IEF时,必须注意将IEF凝胶的温度保持在4°C左右。ii型2-DE凝胶上没有纤维蛋白原片段斑点,证明血浆蛋白是完整的。指出了微凝胶体系分析完整蛋白混合物的优点。
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