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Biochemical Applications

Rapid protein identification using capillary LC/MS/MS and database searching

Traditional methods of protein identification generally require the isolation of individual proteins by two-dimensional gel electro- phoresis. The combination of capillary LC/MS/MS with intelligent, data-dependent acquisition and probability-based database searching makes it possible to rapidly identify as many as 100 proteins in a single analysis.

In this example, a capillary LC and ion trap mass spectrometer were used to acquire data from a mixture of five tryptically digested proteins at a concentration of 1 pmol/µl each (Figure ). Using intelligent, automated data- dependent acquisition, a full scan product ion (MS/MS) spectrum was acquire from the most abundant relevant ion in each mass scan throughout the entire run. All MS and MS/MS data were acquire from a single analysis.

Protein identification was accomplished using MASCOT software that correlated the uninterpreted MS/MS data with sequences in a database. Figure  demonstrates the excellent match between the observed MS/MS spectrum from the most abundant ion (m/z 807.2) in the chromatographic peak at 17.55 minutes and the theoretical y-ion series predicted for a tryptic peptide from human apolipoprotein, one of the proteins in the sample mixture.


[ Michael Linscheid.  Westmorel, D.G]

Clinical Applications

Metabolite Identification
Metabolite identification is central to many of the activities in preclinical development. A more complete characterization of pharmacokinetic properties is per- formed in animals (typically, rats and dogs) during this stage. The knowledge of the biotransformation pathways of the lead candidate to its metabolites is used to indicate the magnitude and duration of activity. Metabolite identification is critical to many of these activities, and plays an important role in establishing the dose and toxicity levels. The identification of metabolite structures with LC/ MS and LC/MS/MS techniques are an effective approach due to their ability to analyze trace mixtures from complex samples of urine, bile, and plasma. The key to structure identification approaches is based on the fact that metabolites generally retain most of the core structure of the parent drug (Perchalski et al., 1982). Therefore, the parent drug and its corresponding metabolites would be expected to undergo similar fragmentations and to produce mass spectra that indicate major substructures.

Impurity Identification
Synthetic impurities are of particular concern during process research and safety evaluation activities. Often, impurities are the result of synthetic by-products or starting materials of the scale-up process. Impurities provide a comprehensive indicator of the chemical process and are diagnostic of overall quality. The resulting information is used by process chemists to guide process optimization. Knowledge of the identity and relative amount of impurities is used to diagnose process reactions so that changes in reagents and reaction conditions lead to better yields and higher quality material. Although it is often difficult to assign an exact time period for the completion of chemical process research activities, it is usually the rate-determining step for preclinical develop- ment activities. With an increasing number of novel lead candidates that enter into preclinical development, considerable resources are needed to identify impurities. LC/MS-based approaches provide integrated sample clean-up and structure analysis procedures for the rapid analysis of impurities. This advantage was demonstrated during the preclinical development of TAXOL.

LC/MS played an important role for the identification of impurities con- tained in extracts and process intermediates from Taxus brevifolia and T. baccata biomass. Because drugs derived from natural sources often have a very diverse set of structural analogs, it is important to determine which analogs are carried through the purification process and ultimately appear as impurities. This task presents a unique challenge during the preclinical stage of drug development due to the highly complex nature of the samples.

The spectrum contains abundant molecular ions atm/z 854, 871, and 912, which correspond to [M H], [M NH]4, and [M NH4CHCN]3, respectively. This distinct molecular ion pattern is used to determine the molecular weight of the resulting impuritie.[Lee, M.S, Kerns, E.H]

High-sensitivity detection of trimipramine and thioridazine
For most compounds, MS is more sensitive than other LC detectors. Trimipramine is a tricyclic antidepressant with sedative properties. Thioridazine is a tranquilizer. Figure  shows these compounds in a urine extract at a level that could not be detected by UV. To get the maximum sensitivity from a single-quadrupole mass spectrometer, the analysis was done by selected ion monitoring.

Food ApplicationsIdentification of aflatoxins in food
Aflatoxins are toxic metabolites produced in foods by certain fungi. Figure  shows the total ion chromatogram from a mixture of four aflatoxins. Even though they are structurally very similar, each aflatoxin can be uniquely identified by its mass spectrum.



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