Transforming the speed, cost and ease of assay development
Biomarker validation
Modern proteomics methods can produce a large number of candidate biomarkers that then need validating before taking into clinical qualification studies. Historically this required production of monoclonal antibody pairs for each target, development of immunoassays such as ELISA and testing one biomarker at a time requiring large volumes of sample material. Typically such efforts cost several $100,000’s and could take years. In many cases it was not even possible to develop adequate monoclonals and so a semi-quantitative method such as Western blotting with polyclonal antibodies had to be used.
Marker Multiplexing
More recently new technologies have been introduced that allow the testing of multiple biomarkers in a single assay. Doing this with immunoassays is difficult and the level of cross-talk between individual assays within the multiplex are difficult to measure and often lead to significant variation in results between labs and over time.
In the past five years a new method for the measurement of between 1 to over 100 candidate biomarkers using isotope dilution mass spectrometry has been developed. Selective Reaction Monitoring (SRM) takes advantage of the mass spectrometer’s ability to select a target peptide, fragment it and then quantitatively detect one or more specific fragments (Fig 1). The combination of parent and daughter ion masses, called a transition, is unique and provides considerable benefits in both sensitivity and specificity of mass spectrometry for protein biomarkers. Perhaps most significant is the fact that such SRM assays typically require only a few microliters of plasma and can precisely quantitate large numbers of biomarkers with no interference between determinations.
Figure 1 – Principle of SRM
In the triple quadrupole instrument Q1 is set to allow a single parent ion (orange circle) to enter the collision cell at Q2. The selected ions are then fragmented to produce daughter ions. As the daughter ions exit Q2 only the daughter ion of a set mass (green triangle) can pass through Q3 and hit the detector. All other ions are deflected away.
SRM can be run in a semi-quantitative mode. To make it fully quantitative a known amount of each target peptide can be introduced into the sample in a form that is heavier than the natural peptide. This is achieved by introducing heavy isotopes of carbon, nitrogen or oxygen into the reference peptides during synthesis or by chemical tagging using isotopic TMT reagents. Then the amount of naturally occurring peptide can be determined based on the ratio of its MS ion intensity compared to that of the known concentration of heavy isotope peptide standard.
PS Biomarker Services™ Assay Development module can produce and validate SRM assays using any of the established methods such as AQUA or QCONCAT. However, we predominantly utilise the combination of isotopic peptide labelling with TMT and SRM to enable a seamless switch from TMT-based discovery into biomarker validation. This means we can use Orbitrap Velos data for selection of parent and daughter ion transitions and often the same samples for method development and assay validation. Recently we demonstrated the ability to develop a TMT-SRM assay for eight candidate biomarkers of skin sensitivity to chemical allergens in just four weeks from discovery. Because the method uses regular synthetic peptides and small amounts of TMT it is relatively inexpensive compared to the cost of antibody production and considerably quicker to implement.
For some low-abundance biomarkers there may be a need to perform enrichment prior to measurement by SRM. Typically this can be achieved using an immunoprecipitation (IP) step prior to SRM. Proteome Sciences has considerable experience of developing IP-SRM methods using commercially available antibodies to the candidate biomarker. Unlike ELISA development however only a single antibody is required and it is not necessary to have absolute specificity as cross-reacting proteins will be differentiated by the SRM component of the assay. Typically development of IP-SRMonly requires an additional four weeks if antibodies are available.
The number of commercially available ELISA’s has dramatically increased over recent years. However, many groups are finding the sensitivity and particularly specificity of many of these assays in human body fluids is poor. Furthermore, limitations on sample availability preclude the use of many such assays. There are some limits to SRM sensitivity in complex samples such as human plasma but in many cases the limitations of immunoassays are readily addressed by adopting TMT-SRM saving wasted time and costs. Our expert scientists can help you select the optimum biomarker validation strategy and undertake development of TMT-SRM and regular SRM assays and/or the validation of various immunoassay options.
