Ultra-high-efficiency strong cation exchange LC/RPLC/MS/MS for high dynamic range characterization of the human plasma proteome
Ultra-high-efficiency strong cation exchange LC/RPLC/MS/MS for high dynamic range characterization of the human plasma proteome
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DOI：
10.1021/ac034869m
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发表时间：
2004-02-15
影响因子：
7.4
通讯作者：
Tompkins, RG
中科院分区：
文献类型：
--
作者：
Shen, YF;Jacobs, JM;Tompkins, RG
高效纳滤膜反相液相色谱法（色谱峰容量接近1000）赵，R.;伯杰，S。J.道：安德森，G.一、Rodriguez，N.;史密斯河D. Anal. Chem.2002，74，4235。沈，Y.;摩尔河J.道：赵，R.; Blonder，J.; Auberry，D. L.的; Masselon，C.; Pasa-Tolic，L.;希克森，K K;史密斯河D. Anal. Chem.2003，75，3596）。强阳离子交换LC用于获得超高效分离（组合色谱峰容量> 10（4）），并结合串联质谱（MS/MS）用于人血浆蛋白质组的表征。使用保守SEQUEST肽鉴定标准（即，不考虑胰凝乳蛋白酶肽或弹性肽）和肽LC标准化洗脱时间限制，分离质量使得能够使用离子阱MS/MS仪器在相对丰度大于8个数量级的动态范围内鉴定蛋白质。根据用于鉴定的标准，从总共365 μ g人血浆中鉴定出800至1682种人蛋白质。分析鉴定了相对低水平（类似topg/mL）的蛋白质（例如，细胞因子）与高丰度蛋白质（例如，mg/mL水平的血清白蛋白）。
High-efficiency nanoseale reversed-phase liquid chromatography (chromatographic peak capacities of similar to1000: Shen, Y.; Zhao, R.; Berger, S. J.; Anderson, G. A.; Rodriguez, N.; Smith, R. D. Anal. Chem. 2002, 74, 4235. Shen, Y.; Moore, R. J.; Zhao, R.; Blonder, J.; Auberry, D. L.; Masselon, C.; Pasa-Tolic, L.; Hixson, K K; Auberry, K J.; Smith, R. D. Anal. Chem. 2003, 75, 3596.) and strong cation exchange LC was used to obtain ultra-high-efficiency separations (combined chromatographic peak capacities of > 10(4)) in conjunction with tandem mass spectrometry (MS/MS) for characterization of the human plasma proteome. Using conservative SEQUEST peptide identification criteria (i.e., without considering chymotryptic or elastic peptides) and peptide LC normalized elution time constraints, the separation quality enabled the identification of proteins over a dynamic range of greater than 8 orders of magnitude in relative abundance using ion trap MS/MS instrumentation. Between 800 and 1682 human proteins were identified, depending on the criteria used for identification, from a total of 365 mug of human plasma. The analyses identified relatively low-level (similar topg/mL) proteins (e.g., cytokines) coexisting with high-abundance proteins (e.g., mg/mL-level serum albumin).