J Proteome Res. 2013 Jul 5;12(7):3353-61. doi: 10.1021/pr400178v. Epub 2013 Jun 13. Targeted quantification of low ng/mL level proteins in human serum without immunoaffinity depletion. Shi T1, Sun X, Gao Y, Fillmore TL, Schepmoes AA, Zhao R, He J, Moore RJ, Kagan J, Rodland KD, Liu T, Liu AY, Smith RD, Tang K, Camp DG 2nd, Qian WJ. Abstract

We recently reported an antibody-free targeted protein quantification strategy, termed high-pressure, high-resolution separations with intelligent selection and multiplexing (PRISM), for achieving significantly enhanced sensitivity using selected reaction monitoring (SRM) mass spectrometry. Integrating PRISM with front-end IgY14 immunoaffinity depletion, sensitive detection of targeted proteins at 50-100 pg/mL levels in human blood plasma/serum was demonstrated. However, immunoaffinity depletion is often associated with undesired losses of target proteins of interest. Herein we report further evaluation of PRISM-SRM quantification of low-abundance serum proteins without immunoaffinity depletion. Limits of quantification (LOQ) at low ng/mL levels with a median coefficient of variation (CV) of ∼12% were achieved for proteins spiked into human female serum. PRISM-SRM provided >100-fold improvement in the LOQ when compared to conventional LC-SRM measurements. PRISM-SRM was then applied to measure several low-abundance endogenous serum proteins, including prostate-specific antigen (PSA), in clinical prostate cancer patient sera. PRISM-SRM enabled confident detection of all target endogenous serum proteins except the low pg/mL-level cardiac troponin T. A correlation coefficient >0.99 was observed for PSA between the results from PRISM-SRM and immunoassays. Our results demonstrate that PRISM-SRM can successfully quantify low ng/mL proteins in human plasma or serum without depletion. We anticipate broad applications for PRISM-SRM quantification of low-abundance proteins in candidate biomarker verification and systems biology studies.

TSQ Vantage  The large fraction of the eluent, at a fl ow rate of ∼ 3 μ L/min, was automatically dispensed every minute into a 96-well  plate  during ∼ 100  min  LC  run  using  the  Triversa NanoMate system (Advion BioSciences, Ithaca, NY).