Abstract
Selected reaction monitoring (SRM)-MS is an emerging technology for high throughput targeted protein quantification and verification in biomarker discovery studies; however, the cost associated with the application of stable isotope-labeled synthetic peptides as internal standards can be prohibitive for screening a large number of candidate proteins as often required in the preverification phase of discovery studies. Herein we present a proof of concept study using an (18)O-labeled proteome reference as global internal standards (GIS) for SRM-based relative quantification. The (18)O-labeled proteome reference (or GIS) can be readily prepared and contains a heavy isotope ((18)O)-labeled internal standard for every possible tryptic peptide. Our results showed that the percentage of heavy isotope ((18)O) incorporation applying an improved protocol was >99.5% for most peptides investigated. The accuracy, reproducibility, and linear dynamic range of quantification were further assessed based on known ratios of standard proteins spiked into the labeled mouse plasma reference. Reliable quantification was observed with high reproducibility (i.e. coefficient of variance <10%) for analyte concentrations that were set at 100-fold higher or lower than those of the GIS based on the light ((16)O)/heavy ((18)O) peak area ratios. The utility of (18)O-labeled GIS was further illustrated by accurate relative quantification of 45 major human plasma proteins. Moreover, quantification of the concentrations of C-reactive protein and prostate-specific antigen was illustrated by coupling the GIS with standard additions of purified protein standards. Collectively, our results demonstrated that the use of (18)O-labeled proteome reference as GIS provides a convenient, low cost, and effective strategy for relative quantification of a large number of candidate proteins in biological or clinical samples using SRM.
Highlights
Selected reaction monitoring (SRM),1 known as multiple reaction monitoring, is a promising technology for reliable quantification of targeted analytes in complex biological and clinical applications [1,2,3,4,5,6,7,8,9,10]
Plasma Samples and Standard Proteins—Mouse plasma (ϳ40 mg/ml determined by BCA protein assay (Pierce)) obtained from Equitech-Bio, Inc. (Kerrville, TX), human female plasma (ϳ50 mg/ml) obtained from BioChemed Services (Winchester, VA), six nonhuman standard proteins obtained from Sigma-Aldrich, i.e. bovine carbonic anhydrase II, bovine -lactoglobulin, Escherichia coli -galactosidase, equine skeletal muscle myoglobin, chicken ovalbumin, and bovine cytochrome c, and two human standard proteins prostatespecific antigen (PSA) obtained from Sigma, and C-reactive protein (CRP) obtained from EMD Chemicals (Gibbstown, NJ) were used in this study
All tryptic peptides present in the individual samples should have their 18O-labeled counterparts as internal standards; the reference is considered as global internal standards (GIS)
Summary
18O-Labeled Proteome Reference as Global Internal Standards for Targeted Quantification by Selected Reaction Monitoring-Mass Spectrometry*□S. We present a proof of concept study using an 18O-labeled proteome reference as global internal standards (GIS) for SRM-based relative quantification. Our results demonstrated that the use of 18O-labeled proteome reference as GIS provides a convenient, low cost, and effective strategy for relative quantification of a large number of candidate proteins in biological or clinical samples using SRM. We investigated the concept of using an 18O-labeled proteome reference to generate global internal standards (GIS) as an alternative to stable isotope-labeled synthetic peptides for broad SRM-based relative quantification. Known ratios of standard proteins spiked into mouse plasma were used to evaluate the accuracy, reproducibility, and dynamic range of SRM-based quantification using the 18O-labeled GIS. Our results demonstrate that the 18O-labeled proteome reference affords sufficient quantification accuracy for SRM analysis of many protein targets in biological samples
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