Discovery to Validation
In the biomarker discovery pipeline, how researchers transition from discovery to verification, while maintaining high throughput, presents a challenge.
One possible solution: multiple reaction monitoring (MRM), a sensitive, quantitative mass spectrometry (MS) tool coupled with global internal standards that bypass synthetic protein standards, an approach under development from Christie L. Hunter, Ph.D., senior staff scientist at Applied Biosystems (www.appliedbiosystems.com).
In MRM, based on triple quadrupole MS platforms, the first analyzer transmits the peptide ion of interest, the peptide ion is fragmented in a collision cell, then the second analyzer transmits only one of the characteristic sequence ions of interest to the detector.
MRM experiments can be quantitative, but one of today’s challenges in peptide MRM analysis is how to maintain this quantitative accuracy across many patient samples and push the throughput envelope. Good internal standards enable this.
Typically, researchers create internal standards by synthesizing the peptide of interest with a stable isotope labeled amino acids. However, this can become prohibitively expensive in protein biomarker verification when internal standards for 50–100 potential proteins are required. Translation: potentially hundreds of synthetic stable isotoped labeled peptides will have to be made.
Dr. Hunter now has developed “more generic, more global internal standards using chemistry rather than making synthetic peptides.”
In the proof of principal experiment, Hunter focused on profiling proteins from 20 sets of twins. Digested protein from all 40 samples was pooled and labeled with a heavy label to create a global internal standard. The label under development is a derivative of Applied Biosystems’ iTRAQ reagents for MRM, called mTRAQ reagents, a pair of nonisotopic amine labeling reagents.
Light-labeled individual samples were spiked with pooled heavy labeled patient sample, and the light and heavy peptides from putative biomarkers were monitored by MRM, providing normalized peptide and protein ratios for every patient sample. MRMs can provide high quantitative reproducibility with coefficient of variance of <10 percent and with Dr. Hunter’s generic internal controls, coefficient of variances can often squeeze under five percent.
Dr. Hunter said that after this verification and prioritizing of discovered biomarkers, the next step could include absolute quantitation. “Throughput is definitely a challenge that we’re all facing in terms of how many samples and proteins can be monitored during verification and we are working to maximize the numbers of MRMs in a single run.” (There are now 600 MRMs in a single run, but Dr. Hunter hopes to double that number.)