Application of 2-D free-flow electrophoresis/RP-HPLC for proteomic analysis of human plasma depleted of multi high-abundance proteins
Application of 2-D free-flow electrophoresis/RP-HPLC for proteomic analysis of human plasma depleted of multi high-abundance proteins
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DOI：
10.1002/pmic.200500096
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发表时间：
2005-08-01
期刊：
影响因子：
3.4
通讯作者：
Simpson, RJ
中科院分区：
文献类型：
--
作者：
Moritz, RL;Clippingdale, AB;Simpson, RJ
采用自由流动电泳（FFE）和快速（6 min）反相高效液相色谱（RP-HPLC）对枸橼酸处理后的血浆进行分离。分析前，用免疫亲和层析法去除血浆中最丰富的6种蛋白；将耗尽血浆和含有6种丰富蛋白质的部分进行质谱分析。分离得到的蛋白质用胰蛋白酶消化，生成的多肽进行质谱测序。迄今为止，通过人工验证，从收集的FFE池的16% （15/96 FFE总组分）中明确鉴定了78种血浆蛋白；55个鉴定基于>= 2个色氨酸肽，23个鉴定基于单肽。通过这种方法分离的蛋白质和肽的分子量范围从类似于190 K（例如，补体C3和C4）到类似于4-6 K（例如，CRISPP和载脂蛋白C1）。这种FFE/RP-HPLC方法揭示了低丰度的蛋白质和肽（例如，类似于17 ng/mL的l -选择素和类似于0.5-1 ng/mL的癌症相关的scm识别、免疫防御抑制和丝氨酸蛋白酶保护肽（CRISPP）），其中CRISPP被发现与α -1-抗胰蛋白酶作为非共价复合物相关联，在含有减少的高丰度蛋白质的部分中。与散弹枪蛋白质组学方法相比，本文描述的FFE/RP-HPLC方法允许鉴定潜在的有趣肽，并追溯到它们的起源蛋白，并且首次证实了“蛋白质海绵”假说，其中35个残基CRISPP多肽与主要循环血浆蛋白α -1-抗trypsin非共价络合。
Free-flow electrophoresis (FFE) and rapid (6 min) RP-HPLC was used to fractionate human citrate-treated plasma. Prior to analysis, the six most abundant proteins in plasma were removed by immunoaffinity chromatography; both depleted plasma and the fraction containing the six abundant proteins depleted were taken for MS-based analysis. Fractionated proteins were digested with trypsin and the generated peptides were subjected to MS-based peptide sequencing. To date, 78 plasma proteins have been unambiguously identified by manual validation from 16% (15/96 FFE total fractions) of the collected FFE pools; 55 identifications were based on >= 2 tryptic peptides and 23 using single peptides. The molecular weight range of proteins and peptides isolated by this method ranged from similar to 190 K (e.g., Complement C3 and C4) to similar to 4-6 K (e.g., CRISPP and Apolipoprotein C1). This FFE/RP-HPLC approach reveals low-abundance proteins and peptides (e.g., L-Selectin similar to 17 ng/mL and the cancer-associated SCM-recognition, immunodefense suppression, and serine protease protection peptide (CRISPP) at similar to 0.5-1 ng/mL), where CRISPP was found in association with alpha-1-antitrypsin as a non-covalent complex, in the fraction containing the depleted high-abundance proteins. In contrast to shotgun proteomic approaches, the FFE/RP-HPLC method described here allows the identification of potentially interesting peptides to be traced back to their protein of origin, and for the first time, has confirmed the "protein sponge" hypothesis where the 35 residue CRISPP polypeptide is non-covalently complexed with the major circulating plasma protein alpha-1-antitrypsin.