title: Long-gradient separations coupled with selected reaction monitoring for highly sensitive, large scale targeted protein quantification in a single analysis.
abstract: 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.
authors.affiliation: Biological Sciences Division, Pacific Northwest National Laboratory , Richland, Washington 99352, United States.
authors.forename: Tujin
authors.initials: T
authors.lastname: Shi
authors.forename: Thomas L
authors.initials: TL
authors.lastname: Fillmore
authors.forename: Yuqian
authors.initials: Y
authors.lastname: Gao
authors.forename: Rui
authors.initials: R
authors.lastname: Zhao
authors.forename: Jintang
authors.initials: J
authors.lastname: He
authors.forename: Athena A
authors.initials: AA
authors.lastname: Schepmoes
authors.forename: Carrie D
authors.initials: CD
authors.lastname: Nicora
authors.forename: Chaochao
authors.initials: C
authors.lastname: Wu
authors.forename: Justin L
authors.initials: JL
authors.lastname: Chambers
authors.forename: Ronald J
authors.initials: RJ
authors.lastname: Moore
authors.forename: Jacob
authors.initials: J
authors.lastname: Kagan
authors.forename: Sudhir
authors.initials: S
authors.lastname: Srivastava
authors.forename: Alvin Y
authors.initials: AY
authors.lastname: Liu
authors.forename: Karin D
authors.initials: KD
authors.lastname: Rodland
authors.forename: Tao
authors.initials: T
authors.lastname: Liu
authors.forename: David G
authors.initials: DG
authors.lastname: Camp
authors.forename: Richard D
authors.initials: RD
authors.lastname: Smith
authors.forename: Wei-Jun
authors.initials: WJ
authors.lastname: Qian
mesh_terms: D002853:Chromatography, Liquid; D004797:Enzyme-Linked Immunosorbent Assay; D005260:Female; D006801:Humans; D017430:Prostate-Specific Antigen / Q000097:blood* / Q000302:isolation & purification; D012680:Sensitivity and Specificity; D053719:Tandem Mass Spectrometry
chemical_list: D017430:Prostate-Specific Antigen