Abstract
Biomedical research requires protein detection technology that is not only sensitive and quantitative, but that can reproducibly measure any set of proteins in a biological system in a high throughput manner. Here we report the development and application of a targeted proteomics platform termed index-ion triggered MS2 ion quantification (iMSTIQ) that allows reproducible and accurate peptide quantification in complex mixtures. The key feature of iMSTIQ is an approach called index-ion triggered analysis (ITA) that permits the reproducible acquisition of full MS2 spectra of targeted peptides independent of their ion intensities. Accurate quantification is achieved by comparing the relative intensities of multiple pairs of fragment ions derived from isobaric targeted peptides during MS2 analysis. Importantly, the method takes advantage of the favorable performance characteristics of the LTQ-Orbitrap, which include high mass accuracy, resolution, and throughput. As such it provides an attractive targeted proteomics tool to meet the demands of systems biology research and biomarker studies.
Highlights
From the ‡Institute for Systems Biology, Seattle, Washington, 98103; §Institute of Molecular Systems Biology, ETH (Swiss Federal Institute of Technology) and Faculty of Science, University of Zurich, Zurich, Switzerland; ¶Department of Genome Sciences, University of Washington, Seattle, Washington, 98195
We report a novel targeted quantitative proteomics platform termed Index-ion triggered MS2 ion quantification that alleviates many of the issues associated with current targeted methods. iMSTIQ involves the use of isotopically heavy “index” peptides to reproducibly trigger the acquisition of full MS2 spectra for the isotopically light target peptides, independently of the target peptides’ ion intensities
This labeling scheme generates isobaric pairs of reference and target sample peptides that in turn produce pairs of fragment ions separated by 4 Da/z units during MS2 analysis; b ions derived from the sample HL peptide appear in the spectrum at 4 Da/z units larger than the corresponding b ions from the reference LH peptide, and y ions derived from the reference LH peptide appear in the spectrum at 4 Da/z units larger than the corresponding y ions from the sample HL peptide
Summary
Index-ion Triggered MS2 Ion Quantification: A Novel Proteomics Approach for Reproducible Detection and Quantification of Targeted Proteins in Complex Mixtures*□S. Inclusion list methods can provide enhanced sensitivity and reproducibility, the gains are limited by the need to detect the precursor ion in question in an MS1 survey scan to trigger the generation of fragment ion spectra (MS2 or MS/MS) This can be problematic when the targeted peptides are of low abundance in biological samples with high complexity and dynamic range. Until recently, application of SRM has been limited by a prerequisite assay optimization process that typically involves selecting the most suitable transitions for each targeted peptide [18] Given these issues with the current targeted proteomics approaches, the development of a targeted quantitative proteomics platform that permits reproducible and accurate measurements in a high throughput manner, without the need for a priori selection of optimal transitions and rigorous retention time requirements, is highly desirable. We demonstrate the utility of the method by applying it to measure the temporal release of targeted inflammatory mediators from macrophages in response to lipopolysaccharide (LPS) stimulation
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