Chen Zhuo Zhen, Dufresne Jaimie, Bowden Peter, Celej Dominika, Miao Ming, Marshall John G
Research Analytical Biochemistry Laboratory, Department of Chemistry and Biology, Toronto Metropolitan University, Canada.
Anal Biochem. 2025 Feb;697:115694. doi: 10.1016/j.ab.2024.115694. Epub 2024 Oct 22.
Organic precipitation of proteins with acetonitrile demonstrated complete protein recovery and improved chromatography of human plasma proteins. The separation of 25 μL of human plasma into 22 fractions on a QA SAX resin facilitated more effective protein discovery despite the limited sample size. Micro chromatography of plasma proteins over quaternary amine (QA) strong anion exchange (SAX) resins performed best, followed by diethylaminoethyl (DEAE), heparin (HEP), carboxymethyl cellulose (CMC), and propyl sulfate (PS) resins. Two independent statistical methods, Monte Carlo comparison with random MS/MS spectra and the rigorous X!TANDEM goodness of fit algorithm protein p-values corrected to false discovery rate q-values (q ≤ 0.01) agreed on at least 12,000 plasma proteins, each represented by at least three fully tryptic corrected peptide observations. There was qualitative agreement on 9393 protein/gene symbols between the linear quadrupole versus orbital ion trap but also quantitative agreement with a highly significant linear regression relationship between log observation frequency (F value 4,173, p-value 2.2e-16). The use of a QA resin showed nearly perfect replication of all the proteins that were also found using DEAE-, HEP-, CMC-, and PS-based chromatographic methods combined and together estimated the size of the size of the plasma proteome as ≥12,000 gene symbols.
用乙腈对蛋白质进行有机沉淀可实现蛋白质的完全回收，并改善人血浆蛋白的色谱分离效果。尽管样品量有限，但在QA SAX树脂上把25μL人血浆分离成22个馏分有助于更有效地发现蛋白质。血浆蛋白在季胺（QA）强阴离子交换（SAX）树脂上的微色谱分析效果最佳，其次是二乙氨基乙基（DEAE）、肝素（HEP）、羧甲基纤维素（CMC）和丙基硫酸盐（PS）树脂。两种独立的统计方法，即与随机MS/MS谱的蒙特卡洛比较以及将蛋白质p值校正为错误发现率q值（q≤0.01）的严格X!TANDEM拟合优度算法，在至少12000种血浆蛋白上达成一致，每种蛋白至少由三个完全胰蛋白酶消化校正后的肽段观察值代表。线性四极杆与轨道离子阱之间在9393个蛋白质/基因符号上存在定性一致，而且在对数观察频率之间存在高度显著的线性回归关系（F值4173，p值2.2e - 16）也存在定量一致。使用QA树脂几乎能完美复制所有用基于DEAE、HEP、CMC和PS的色谱方法联合检测到的蛋白质，并且共同估计血浆蛋白质组的大小≥12000个基因符号。
