Wang Yueju, Rudnick Paul A, Evans Erin L, Li Jie, Zhuang Zhengping, Devoe Don L, Lee Cheng S, Balgley Brian M
Department of Chemistry and Biochemistry, University of Maryland, College Park, Maryland 20742, USA.
Anal Chem. 2005 Oct 15;77(20):6549-56. doi: 10.1021/ac050491b.
This study demonstrates the ability to perform sensitive proteome analysis on the limited protein quantities available through tissue microdissection. Capillary isoelectric focusing combined with nano-reversed-phase liquid chromatography in an automated and integrated platform not only provides systematic resolution of complex peptide mixtures based on their differences in isoelectric point and hydrophobicity but also eliminates peptide loss and analyte dilution. In comparison with strong cation exchange chromatography, the significant advantages of electrokinetic focusing-based separations include high resolving power, high concentration and narrow analyte bands, and effective usage of electrospray ionization-tandem MS toward peptide identifications. Through the use of capillary isoelectric focusing-based multidimensional peptide separations, a total of 6866 fully tryptic peptides were detected, leading to the identification of 1820 distinct proteins. Each distinct protein was identified by at least one distinct peptide sequence. These high mass accuracy and high-confidence identifications were generated from three proteome runs of a single glioblastoma multiforme tissue sample, each run consuming only 10 microg of total protein, an amount corresponding to 20,000 selectively isolated cells. Instead of performing multiple runs of multidimensional separations, the overall peak capacity can be greatly enhanced for mining deeper into tissue proteomics by increasing the number of CIEF fractions without an accompanying increase in sample consumption.
本研究证明了对通过组织显微切割获得的有限蛋白量进行灵敏蛋白质组分析的能力。在自动化集成平台中，毛细管等电聚焦与纳米反相液相色谱相结合，不仅能基于复杂肽混合物的等电点和疏水性差异对其进行系统分离，还能消除肽损失和分析物稀释。与强阳离子交换色谱相比，基于电动聚焦的分离方法具有显著优势，包括高分辨率、高浓度和窄分析物带，以及在肽鉴定中有效利用电喷雾电离串联质谱。通过使用基于毛细管等电聚焦的多维肽分离方法，共检测到6866个完全胰蛋白酶消化的肽段，从而鉴定出1820种不同蛋白质。每种不同蛋白质至少由一个不同肽序列鉴定。这些高质量准确性和高可信度的鉴定结果来自对单个多形性胶质母细胞瘤组织样本的三次蛋白质组分析，每次分析仅消耗10微克总蛋白，这一量相当于20000个选择性分离的细胞。通过增加CIEF馏分数量，而不伴随样品消耗增加，无需进行多次多维分离运行，就可大大提高整体峰容量，从而更深入地挖掘组织蛋白质组学。
