Bachi Angela, Simó Carolina, Restuccia Umberto, Guerrier Luc, Fortis Frederic, Boschetti Egisto, Masseroli Marco, Righetti Pier Giorgio
San Raffaele Scientific Institute, 20132 Milano, Italy.
Anal Chem. 2008 May 15;80(10):3557-65. doi: 10.1021/ac8001353. Epub 2008 Apr 15.
Sixteen different amino acids (Arg, Asn, Asp, Gln, Glu, Gly, His, Ile, Lys, Phe, Pro, Ser, Thr, Trp, Tyr, Val) have been separately linked to chromatographic beads and used for studying the mechanism of binding of such baits to proteins, as represented by the cytoplasmic proteome of the human red blood cell (RBC). The 16 different amino acid columns were confronted with equal amounts of RBC lysate, washed to remove unbound material, and eluted with denaturing agents. All eluates were analyzed by nanoLC-MS/MS.
there appears to be a dichotomy between a class of "Grand Catchers" (Arg, His, Ile, Lys, Phe, Trp, Tyr, Val), all able to bind from 330 up to 441 unique gene products, and the "Petite Catchers" (Asn, Asp, Gln, Glu, Gly, Pro, Ser, Thr), that bind in general half as much, with the notable exception of Glu that under the described conditions seems to bind only traces of proteins. By comparing homogeneous classes of amino acids (e.g., the basic, the hydrophobic aromatic, the neutral hydrophilic, etc.), it is found that, in general, more than half as many proteins are held in common among the members of each family. In a 16-way comparison, 72 proteins (less than 10% of the total amount, which amounts to 800 unique, nonredundant, identified proteins) appear to be the common catch of all 16 amino acids, suggesting that such proteins might have either multiple binding sites or general motifs recognized by any generic bait. By far, it would appear that the strongest interactions and thus the strongest catches occur with the three aromatic moieties of Phe, Trp, and Tyr, all able to capture a practically identical number of proteins. Ionic interactions, which in principle should be the strongest ones, appear to behave in a peculiar way: they are quite strong with the three basic amino acids (Arg, His, Lys) but almost inexistent with their acidic counterparts. This suggests a peculiar mechanism of interaction: upon formation of the ion pair, the linkage between the protein and the bait is stabilized by the hydrophobicity of the underlying chain (e.g., a butyl in the case of Lys).
已将16种不同的氨基酸（精氨酸、天冬酰胺、天冬氨酸、谷氨酰胺、谷氨酸、甘氨酸、组氨酸、异亮氨酸、赖氨酸、苯丙氨酸、脯氨酸、丝氨酸、苏氨酸、色氨酸、酪氨酸、缬氨酸）分别连接到色谱珠上，并用于研究此类诱饵与蛋白质的结合机制，以人类红细胞（RBC）的细胞质蛋白质组为代表。将16种不同的氨基酸柱与等量的RBC裂解物接触，洗涤以去除未结合的物质，并用变性剂洗脱。所有洗脱液均通过纳升液相色谱-串联质谱进行分析。
在一类“大捕获者”（精氨酸、组氨酸、异亮氨酸、赖氨酸、苯丙氨酸、色氨酸、酪氨酸、缬氨酸）之间似乎存在二分法，它们都能够结合330至441种独特的基因产物，而“小捕获者”（天冬酰胺、天冬氨酸、谷氨酰胺、谷氨酸、甘氨酸、脯氨酸、丝氨酸、苏氨酸）通常结合量只有前者的一半，显著的例外是谷氨酸，在所描述的条件下它似乎只结合痕量蛋白质。通过比较同类氨基酸（例如碱性氨基酸、疏水性芳香族氨基酸、中性亲水性氨基酸等）发现，一般来说，每个家族成员之间共有超过一半的蛋白质。在16向比较中，72种蛋白质（占总量不到10%，总量为800种独特的、非冗余的、已鉴定的蛋白质）似乎是所有16种氨基酸的共同捕获物，这表明此类蛋白质可能具有多个结合位点或任何通用诱饵都能识别的通用基序。到目前为止，似乎苯丙氨酸、色氨酸和酪氨酸的三个芳香部分之间的相互作用最强，因此捕获能力也最强，它们都能够捕获几乎相同数量的蛋白质。离子相互作用原则上应该是最强的，但表现出一种奇特的方式：它们与三种碱性氨基酸（精氨酸、组氨酸、赖氨酸）的相互作用很强，但与它们的酸性对应物几乎不存在相互作用。这表明存在一种奇特的相互作用机制：在形成离子对时，蛋白质与诱饵之间的连接通过底层链的疏水性（例如赖氨酸情况下的丁基）得以稳定。
