Obtaining high sequence coverage in matrix-assisted laser desorption time-of-flight mass spectrometry for studies of protein modification: analysis of human serum albumin as a model.
Obtaining high sequence coverage in matrix-assisted laser desorption time-of-flight mass spectrometry for studies of protein modification: analysis of human serum albumin as a model.
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在基质辅助激光解吸飞行时间质谱中获得高序列覆盖率，用于蛋白质修饰研究：以人血清白蛋白为模型进行分析。
DOI：
10.1016/j.ab.2005.11.015
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发表时间：
2006
期刊：
影响因子：
--
通讯作者：
Hage,DavidS
中科院分区：
文献类型：
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作者：
Wa,Chunling;Cerny,Ron;Hage,DavidS
在基质辅助激光解吸/电离飞行时间质谱（MALDI-TOF MS）进行的蛋白质修饰研究中，探索了几种获得高序列覆盖率的方法。人血清白蛋白（HSA，66.5kDa）被用作这项工作的模型蛋白。在这项研究中考虑的实验因素包括用于MALDI-TOF MS的基质类型，蛋白质消化方法，以及在MALDI-TOF MS分析之前对肽段进行分级分离。采用α-氰基-4-羟基肉桂酸和2，5-二羟基苯甲酸的混合物作为HSA的最终基质。当与胰蛋白酶消化物一起使用时，这给出了HSA一级结构中仅一半肽的独特信息。然而，基于胰蛋白酶、内切蛋白酶Lys-C和内切蛋白酶Glu-C的三种酶的组合使用使该序列覆盖率增加到72.8%。在分析之前使用ZipTip柱将这些肽中的肽片段化，将序列覆盖率增加到97.4%。这些条件使得有可能检查来自几乎所有HSA结构的独特肽并鉴定对该蛋白质的特异性修饰（例如，糖化位点）。例如，Lys199被确认为正常HSA上的糖化位点，而Lys536和Lys389被鉴定为最低糖化HSA上的额外修饰位点。
Several approaches were explored for obtaining high sequence coverage in protein modification studies performed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). Human serum albumin (HSA, 66.5kDa) was used as a model protein for this work. Experimental factors considered in this study included the type of matrix used for MALDI-TOF MS, the protein digestion method, and the use of fractionation for peptide digests prior to MALDI-TOF MS analysis. A mixture of α-cyano-4-hydroxycinnamic acid and 2,5-dihydroxybenzoic acid was employed as the final matrix for HSA. When used with a tryptic digest, this gave unique information on only half of the peptides in the primary structure of HSA. However, the combined use of three enzyme digests based on trypsin, endoproteinase Lys-C, and endoproteinase Glu-C increased this sequence coverage to 72.8%. The use of a ZipTip column to fractionate peptides in these digests prior to analysis increased the sequence coverage to 97.4%. These conditions made it possible to examine unique peptides from nearly all of the structure of HSA and to identify specific modifications to this protein (e.g., glycation sites). For instance, Lys199 was confirmed as a glycation site on normal HSA, whereas Lys536 and Lys389 were identified as additional modification sites on minimally glycated HSA.