- Provides relative or absolute quantification of protein(s) in cases where no ELISA/LBA or other detection method/kit is available.
- Provides relative or absolute quantification of protein(s) in cases in cases where matrix interferences affect your current protein assay.
- Sample multiplexing to increase throughput via TMT’s.
- Can we developed for single or multiple (multiplexing) proteins per assay determination.
- Can we developed for proteins and site specific post translational modifications.
- Can provide equal LOD and LLOQ in matrix compared to ELISA in buffer.
- Can we performed under GCLP for clinical samples.
- Available for a few samples up to 1,000’s in single or multiple batches.
Targeted Mass Spectrometry Assay Development for Biomarker Validation
In addition to unbiased proteomic workflows for candidate biomarker discovery, we also offer services for developing targeted mass spectrometry assays to measure 1 – 50 target proteins in a single method. We have experience in developing assays with selected reaction monitoring (SRM) and parallel reaction monitoring (PRM). For biomarker candidate validation we recommend using PRM as it is generally simpler and faster to set up. Due to it being developed on an Orbitrap mass spectrometer, the high mass accuracy and use of all fragment ions means PRM is also very well suited for validation of modified peptides.
Whichever assay platform is chosen, the development process is broadly similar. Firstly we assess empirical data and on-line resources to select suitable proteotypic peptides. In the case of modified peptides, we have developed proprietary software to automate assessment of site-specific fragment ions required for accurate localisation.
Depending on the assay format chosen and the level of quantification required (relative or absolute) we will prepare reference materials and/or purchase isotope doped synthetic peptides to act as the quantitative reference. The prototype assay is then developed and characterised during which process a number of peptides may be excluded. The final assay format is then qualified using a synthetic matrix to determine the limits of detection, parallelism, accuracy and precision.
Finally, the qualified assay is used to analyse appropriately sized cohorts based on a power calculation using the known size effect from discovery experiments and the assay performance at the lower limit of quantification.
Routine Biomarker Assays
|SRM/PRM Targeted Assays||
Development of Validated Absolute Quantitative Assays for Multi-protein Biomarker Panels
Modern proteomic biomarker discovery generally produces a panel of 5 - 50 candidates that require further qualification in a larger cohort of samples. In this context the biomarkers are usually tryptic peptides and replication studies often use antibodies against the parent protein. Selected Reaction Monitoring (SRM) is a multiplexed, targeted mass spectrometry assay format for the routine measurement of biomarkers that is particularly suited to monitoring candidates that do not have validated commercially available antibodies especially post-translational modifications. Our SRM assay development service produces bespoke assay panels with defined performance characteristics that are suitable for routine use in large study cohorts. Our expert team will guide you in the best workflow for your biomarker candidates, including selection of proteotypic peptides and optimization of sample preparation where appropriate.
|The SRM Assay Development Process:|
|Phase 1||Selection of proteotypic peptides|
|Phase 2||Ordering AQUA peptides (if required)|
|Phase 3||SRM assay development and validation|
|Phase 4||Optimization of sample preparation (if required)|
|Phase 5||Biomarker qualification in small cohort|
|Phase 6||Biomarker validation in larger cohort|