Long-Gradient Separations Coupled with Selected Reaction Monitoring for Highly Sensitive, Large Scale Targeted Protein Quantification in a Single Analysis

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• 作者：Tujin Shi ; Thomas L. Fillmore ; Yuqian Gao ; Rui Zhao ; Jintang He ; Athena A. Schepmoes ; Carrie D. Nicora ; Chaochao Wu ; Justin L. Chambers ; Ronald J. Moore ; Jacob Kagan ; Sudhir Srivastava ; Alvin Y. Liu ; Karin D. Rodland ; Tao Liu ; David G. Camp ; II ; Richard D. Smith ; Wei-Jun Qian
• 刊名：Analytical Chemistry
• 出版年：2013
• 出版时间：October 1, 2013
• 年：2013
• 卷：85
• 期：19
• 页码：9196-9203
• 全文大小：366K
• 年卷期：v.85,no.19(October 1, 2013)
• ISSN：1520-6882

文摘

Long-gradient separations coupled to tandem mass spectrometry (MS) were recently demonstrated to provide a deep proteome coverage for global proteomics; however, such long-gradient separations have not been explored for targeted proteomics. Herein, we investigate the potential performance of the long-gradient separations coupled with selected reaction monitoring (LG-SRM) for targeted protein quantification. Direct comparison of LG-SRM (5 h gradient) and conventional liquid chromatography (LC)-SRM (45 min gradient) showed that the long-gradient separations significantly reduced background interference levels and provided an 8- to 100-fold improvement in limit of quantification (LOQ) for target proteins in human female serum. On the basis of at least one surrogate peptide per protein, an LOQ of 10 ng/mL was achieved for the two spiked proteins in nondepleted human serum. The LG-SRM detection of seven out of eight endogenous plasma proteins expressed at ng/mL or subng/mL levels in clinical patient sera was also demonstrated. A correlation coefficient of >0.99 was observed for the results of LG-SRM and enzyme-linked immunosorbent assay (ELISA) measurements for prostate-specific antigen (PSA) in selected patient sera. Further enhancement of LG-SRM sensitivity was achieved by applying front-end IgY14 immunoaffinity depletion. Besides improved sensitivity, LG-SRM potentially offers much higher multiplexing capacity than conventional LC-SRM due to an increase in average peak widths (3-fold) for a 300 min gradient compared to a 45 min gradient. Therefore, LG-SRM holds great potential for bridging the gap between global and targeted proteomics due to its advantages in both sensitivity and multiplexing capacity.