Quantitative targeted proteomics
Custom and curated targeted assays that provide protein/peptide/isoform specific proteome characterization. Accurate and precise quantification, with additional sensitivity, greater throughput, and reduced sample requirements compared to discovery workflow.
APPLICATIONS
- Target engagment (indirect/direct)
- Isoform characterization
- Protein complex stoichiometry
- Protein expression profiling
- Post-translational modifications
WORKFLOW OVERVIEW
- One-time assay design
- Identify/select peptides
- Curate peptides
- Generate custom assay
- Source isotopically heavy standards
- Validate assay
- Subsequent sample analysis
- High-throughput lysis & sample preparation
- Apply custom targeted assay for precise and accurate quantification
SERVICES PROVIDED
Services
In brief:
- Project consultation / design
- Custom targeted assay design
- Sample preparation
- Typically request minimally processed cells, tissues, biofluids, IPs, etc.
- LC-MS data acquisition on an appropriate instrument
- Data analysis
Deliverables
Our goal is to provide interpretable proteomics data and we’ve curated our deliverables to enable routine analysis, without special software. Deliverables include:
- QC data
- Protein annotations, gene symbols, quantitaive values
- Upon request:
- RAW mass spec. files
- Written document containing a detailed description of materials and methods for publications
PROJECT SPOTLIGHT: Covalent Inhibitor Target Engagement
A protein target of interest (purified, or in matrix (tissue, xenograft, cultured cells, etc)) is treated with a small molecular covalent inhibitor, and the compound covalently binds the target at a specific cysteine. Total protein is isolated and digested into peptides with a protease. Isotopically heavy synthetic internal standard peptides are spiked into the digested sample. Internal standard peptides and their endogenous counterparts are identified, sequenced, and quantified by LC-MS. The amount of drug bound to the target (TE) is characterized by dose-responsive disappearance of cysteine-containing peptide(s) of interest compared to stable control peptides.
A Limit of Quantification (LoQ) assay is completed for each targeted assay to ensure sensitivity to the target of interest.
KRAS G12C signal normalized to SFEDIHHR to control for KRAS abundance. A robust treatment-induced reduction in G12C peptide signal is observed, indicative of covalent engagement of sotorasib.