Abstract
Selected reaction monitoring mass spectrometry (SRM-MS) is playing an increasing role in quantitative proteomics and biomarker discovery studies as a method for high throughput candidate quantification and verification. Although SRM-MS offers advantages in sensitivity and quantification compared with other MS-based techniques, current SRM technologies are still challenged by detection and quantification of low abundance proteins (e.g. present at ∼10 ng/ml or lower levels in blood plasma). Here we report enhanced detection sensitivity and reproducibility for SRM-based targeted proteomics by coupling a nanospray ionization multicapillary inlet/dual electrodynamic ion funnel interface to a commercial triple quadrupole mass spectrometer. Because of the increased efficiency in ion transmission, significant enhancements in overall signal intensities and improved limits of detection were observed with the new interface compared with the original interface for SRM measurements of tryptic peptides from proteins spiked into non-depleted mouse plasma over a range of concentrations. Overall, average SRM peak intensities were increased by ∼70-fold. The average level of detection for peptides also improved by ∼10-fold with notably improved reproducibility of peptide measurements as indicated by the reduced coefficients of variance. The ability to detect proteins ranging from 40 to 80 ng/ml within mouse plasma was demonstrated for all spiked proteins without the application of front-end immunoaffinity depletion and fractionation. This significant improvement in detection sensitivity for low abundance proteins in complex matrices is expected to enhance a broad range of SRM-MS applications including targeted protein and metabolite validation.
Highlights
From the Biological Sciences Division and Environmental Molecular Sciences Laboratory, Pacific Northwest National Laboratory, Richland, Washington 99352
This study evaluated the improvements in selected reaction monitoring (SRM)-mass spectrometry (MS) measurements achievable based upon the increased signal levels obtained using a much more efficient electrospray ionization (ESI)-MS interface
Six standard peptides spiked into each sample at a level of 500 nM prior to LC-SRM analyses served as quality control (QC) peptides
Summary
Materials and Chemicals—The standard proteins bovine carbonic anhydrase, Escherichia coli -galactosidase, equine skeletal muscle myoglobin, chicken ovalbumin, and bovine cytochrome c and the standard peptides bradykinin fragment 1–7, kemptide, melittin, methionine enkephalin, renin substrate porcine, and [D-Ala2]-deltorphin II were purchased from Sigma. Sample Preparation—The initial mouse plasma protein concentration was ϳ40 mg/ml as determined by BCA protein assay (Pierce). Samples for SRM analysis were prepared by spiking different aliquots of the mixture of digested protein standards into a mouse plasma tryptic digest to desired concentrations (see Table I). The DC-only electric potential of the jet disrupter and conductance limit orifice electrode were independently controlled at 390 and 230 V for the high pressure ion funnel and 175 and 2 V for the low pressure ion funnel, respectively. The peak widths for each peptide were determined by averaging the data obtained using the two highest spiked concentrations (S8 and S9), and the elution time difference observed relative to the nearest QC marker peaks served as a peak integration window for the lower (S0 –S7) spike concentrations. Analyte peak identification at the lower concentrations was determined based on three main criteria. 1) The difference of elution time from the nearest QC peaks was within Ϯ10% of the average difference observed from the two highest concentrations. 2) Analyte peak widths were similar (i.e. within Ϯ10%) to the average of peak widths from the two top concentrations. 3) Analyte peak area decreased with decreasing analyte concentration levels
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
