Optimized Analytical Methods
Determining the configurations of and modifying structures with known clinical utility constitute another effective strategy for generating new leads. Mass spectrometry (MS) and its adjunct methodologies have added growing precision and versatility to molecular research, delivering specific and quantitative results of immediate use to the pharmaceutical researcher.
To keep pace with the productivity requirements of metabolism studies in discovery, optimized analytical methods and efficient data interpretation tools are essential. Thus, pharmaceutical companies have increased their focus on early characterization of metabolites and identification of potential indicators of toxicity in drug discovery.
Scientists at Waters (Milford, MA), and their collaborators in the pharmaceutical industry, have applied Ultra Performance Liquid Chromatography (UPLC) coupled with quadrupole time-of-flight mass spectrometry (Q-TOF) to early metabolism studies.
Increasing throughput and information content for in vitro drug metabolism experiments by using UPLC coupled to Q-TOF/MS has produced an increase in metabolite peak resolution and spectral quality over high-performance liquid chromatography/mass spectrometry (HPLC/
MS), according to the company.
The extra resolution and sensitivity afforded by coupling UPLC with mass spectrometry reduces the risk of missing potentially toxic or reactive metabolites.
Applied Biosystems (Foster City, CA) couples its hybrid triple quadrupole/linear ion trap mass spectrometer, the Q TRAP LC/MS/MS System, into the BIOiTRAQ QT system to enable faster identification, characterization, and quantitation of biomarkers.
Sally Webb, proteomics marketing manager for the Americas at Applied Biosystems, says that this technology is a "hybrid system combining triple quadrupole sensitivity and specificity with ion trap technology, heightening confidence in biomarker identification and validation.
"In addition to biomarker ID and expression analysis with iTRAQ reagents, the system allows multiple reaction monitoring (MRM) workflows that provide specific and sensitive quantitation."
Webb adds that the Q TRAP system's MRM mode has been used successfully to "detect and quantitate biomarkers of interest in hypertension, such as angiotensin II in plasma. Since many drugs involve the inhibition of the conversion of angiotensin I to angiotensin II," this biomarker is of obvious interest to monitor vascular health.
Further applications are in "studying a family of Cytochrome P450 with an eye to predicting or monitoring an individual's reaction to a drug" based on that person's P450 profile.
As there is great genetic variability in one's endowment of these P450's, the company "has built a sensitive MRM assay that can analyze several isoforms responsible for the majority of drug metabolism in one experiment. This is unique in that the assay is looking directly at the proteins rather than indirectly by mRNA or enzyme substrate inhibition," continues Webb.
In effect, this approach constitutes the first protein assay of its kind in its research phase, according to Webb. Post-translational modifications, such as phosphorylation of peptides/proteins, are important in the regulation of many cellular processes including cell cycle, growth, apoptosis and signal transduction pathways.
"The Q TRAP system, because of its true hybrid nature, includes the highly specific precursor ion and neutral loss scans that target these modified peptides (triple quadrupole scans)," adds Webb.
"These, combined with the highest sensitivity ion trap MS/MS scans means you can target, detect, and quantitate phosphorylation of peptides/proteins in a single experiment."