• Poster presentation
  • P-I-0174

Revolutionizing translational research: large-scale targeted PRM proteomics assays enabled by a novel hybrid mass spectrometer

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  • New Technology: MS-based Proteomics

Abstract

Targeted mass spectrometry assay development for biomarker validation is no simple task. Although, the alure of finding a conclusive biomarker to indicate the presence of disease seems simple and straightforward in the discovery phase, the verification step is a painstakingly costly process. While traditional methods of analyzing target proteins defer to immunological or affinity-based assays, they suffer from lack of specificity and sensitivity. This is of critical concern for potential low abundant biomarkers in highly complex biomatrices. Here we present a simplified and streamlined approach moving from discovery to target verification by liquid chromatography coupled with a novel hybrid mass spectrometer instrument enabling targeted PRM assay for > 1000s of peptides associated with healthy and disease states and affected pathways. A quantitative targeted mass spectrometry (MS) method was created for > 1000s of peptides associated with proteins in disease related pathways, in less than 3 days by utilizing a gas-phase fractionation, Data Independent Acquisition (DIA) method to quickly map retention times and select charge state and optimal peptide transitions for targeted pathway protein assay. The targeted assays were then built by including optimal transitions in a PRM assay. The new instrument collects PRM data with 5x the throughput of previous generations of MS instruments, which provides the flexibility to create a high throughput (>60 SPD) quantitative targeted assay on a larger set of target compounds. To evaluate the assay"s quantitative performance, we spiked in the synthetically prepared disease biomarker associated SIL peptides by serial dilution into neat plasma digest processed. The depletion resin removed high abundant proteins like albumin and IgGs which constitute roughly 90 % of the plasma proteome, masking the identification of the low abundant proteins in a complex sample such as plasma. This was done in a 96-well plate format to demonstrate high-throughput capability of the workflow. The depleted samples were analyzed in triplicate to assess precision, accuracy and minimum Datapoints Per Peak (DPP) of the targeted assay. Early evaluation of the peptides demonstrated a % CV of < 20 % and a minimum of 10 DPP for roughly 70 % of the peptides in the quantitative PRM method. To further demonstrate the utility of this targeted assay we analyzed a mini cohort study that contained plasma samples of various high-prevalence disease with age- and ethnicity-matched normal plasma samples for protein/peptide biomarker detection and assessment. Novel Aspect: Novel Hybrid Mass Spectrometer Enables Streamlined Verification of Large-Scale Disease Biomarker Panel of more than 1000s targets.