Simó Carolina, Bachi Angela, Cattaneo Angela, Guerrier Luc, Fortis Frederic, Boschetti Egisto, Podtelejnikov Alexander, Righetti Pier Giorgio
Department of Chemistry, Materials and Chemical Engineering Giulio Natta, Polytechnic of Milano, 20131 Milano, Italy.
Anal Chem. 2008 May 15;80(10):3547-56. doi: 10.1021/ac702635v. Epub 2008 Apr 10.
For a better understanding of the behavior of solid-phase combinatorial peptide ligands for capturing the red blood cell low-abundance soluble proteome, combinatorial peptides of different lengths from a single amino acid up to a hexapeptide were evaluated. A red blood cell lysate (6 g total protein) was loaded in a cascade fashion to the six columns, which were individually eluted with 8 M urea, 2% 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (v/w), and 50 mM citric acid. Each eluate was analyzed via sodium dodecyl sulfate polyacrylamide gel electrophoresis, two-dimensional maps, and nanoLC-MS/MS.
mixed beads with a single amino acid attached showed the capture of a non-negligible portion of the proteome. A progressive increasing of the length of the peptide bait enlarges the pool of captured proteins. Above a length of four amino acids, a plateau is progressively reached, suggesting that not much could be gained with baits longer than six amino acids. Interestingly, whereas the beads laden with a single amino acid seem to be able to capture large-size proteins (>40 kDa), beads with progressively longer peptides capture additional proteins in the smaller size range (10-50 kDa). This suggests that interactions already begin with a single amino acid, but selectivity requires baits of proper length, at least above four amino acids. Plain beads, with a spacer arm carrying a primary amino terminal group for anchoring the baits, are essentially unable to capture proteins, suggesting that the peptide baits do not act by a mechanism of ion exchange but rather via a complex mixed mode, yielding a specific capture.
为了更好地理解用于捕获红细胞低丰度可溶性蛋白质组的固相组合肽配体的行为，评估了从单个氨基酸到六肽的不同长度的组合肽。将红细胞裂解物（总蛋白6克）以级联方式加载到六个柱上，每个柱分别用8M尿素、2% 3-[(3-胆酰胺丙基)二甲基铵]-1-丙烷磺酸盐（v/w）和50mM柠檬酸洗脱。通过十二烷基硫酸钠聚丙烯酰胺凝胶电泳、二维图谱和纳升液相色谱-串联质谱对每个洗脱液进行分析。
附着单个氨基酸的混合珠显示捕获了蛋白质组中不可忽略的一部分。肽诱饵长度的逐渐增加扩大了捕获蛋白质的库。当肽长度超过四个氨基酸时，逐渐达到一个平台期，这表明使用长度超过六个氨基酸的诱饵不会有太多收获。有趣的是，负载单个氨基酸的珠子似乎能够捕获大尺寸蛋白质（>40 kDa），而负载逐渐变长肽的珠子则捕获较小尺寸范围（10-50 kDa）内的其他蛋白质。这表明相互作用从单个氨基酸就已开始，但选择性需要适当长度的诱饵，至少超过四个氨基酸。带有用于锚定诱饵的伯氨基端基间隔臂的普通珠子基本上无法捕获蛋白质，这表明肽诱饵不是通过离子交换机制起作用，而是通过复杂的混合模式起作用，从而实现特异性捕获。
