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Applications of Various MS

Applications of Various MS

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07/12/2013

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SPECTROSCOPIC METHODS OF ANALYSIS—MASS SPECTROMETRY
Mike S. Lee
Milestone Development Services, Newtown, Pennsylvania
INTRODUCTION
The dramatically increased expenditures for both in-houseand outsourced pharmaceutical research and development(R&D) have led to a greater dependence on technology.New technologies are constantly introduced into drugdevelopment to address throughput issues and improvedevelopment cycles. The incorporation of new technol-ogies has resulted in fundamental change in the drugdevelopment paradigm. Recently, sample generating-based technologies such as high throughput biomolecularscreening and automated parallel synthesis have shiftedthe bottleneck to sample analysis-based technologies.The current focus on analytical techniques in thepharmaceutical industry emphasizes four primary figuresof merit: sensitivity; selectivity; speed; and highthroughput. Mass spectrometry (MS) provides each of these key attributes, and therefore, has been benchmarkedan effective solution for pharmaceutical analysis in eachstage of drug development (1). Perhaps more enablingthanthe MS-based technology itself is the diverse applicationsof MS in conjunction with sample preparation, chromato-graphic separation, and informatics. It is within thiscontext that MS has played an increasingly vital role in thepharmaceutical industry and has become the preferredanalytical method for trace-mixture analysis (Fig. 1
½
F1
).A variety of MS formats are widely accepted andapplied in the pharmaceutical industry. The specific MSapplication is often defined by the sample introductiontechnique. The pharmaceutical applications highlighted inthis article feature two types of sample introductiontechniques: dynamic and static. Dynamic sample intro-duction involves the use of high-performance liquidchromatography (HPLC) on-line with MS. The resultingliquid chromatography/mass spectrometry (LC/MS) for-mat provides unique and enabling capabilities forpharmaceutical analysis. The electrospray ionization(ESI) (2) and atmospheric pressure chemical ionization(APCI) (3) modes are the most widely used. Static sampleintroduction techniques primarily use matrix-assisted laserdesorption/ionization (MALDI) (4).The advances in MS instrumentation (5) and role of MSwithin the pharmaceutical industry (1) have been recentlyreviewed. This article will focus on MS technologies withregard to specific applications in drug development. Theintent of this article is to provide an overview of MSapplications and describe the significant integration of thistechnology into drug development. A detailed and in-depth overview of current MS technologies and appli-cations can be obtained from the recent proceedings of theAmerican Society for Mass Spectrometry Conference onMass Spectrometry and Allied Topics (www.asms.org)and the Association of Biomolecular Resource Facilities(www.abrf.org).
GENOMICS
Though the contributions of MS have been somewhatlimited in the field of genomics, there has been increasedparticipation and interest (6). Certainly, the worldwiderecognition received from the Human Genome Projectcreated a sense of urgency toward determining geneticvariation.Genomics refers to the study of genetic data to drawcorrelation between individual genetic inheritance andmedically or biologically important parameters. Forexample, these parameters may involve a patient’sresponse to a specific drug. Knowledge of the geneticbasis of individual drug response may provide under-standing of the observed variability in drug responsearising as a result of genetically determined differences indrug absorption, disposition, metabolism, or excretion (7).Furthermore, knowledge of genetic variation may beuseful during the target selection process when multipletargets are available within a specific disease state. Thus,the pharmaceutical industry has great interest indetermining the genetic variation in patient populations.Due to the existence of sequence variations, orpolymorphisms, no two human genomes are identical.Single nucleotide polymorphisms (SNPs) are the mostabundant genetic variation with an estimated frequency of 1 SNP per 500 basepairs. Since SNPs are so prevalent inthe genome, they can act as markers that are linked with aphenotype to provide a comprehensive measure of interaction with a specific drug. The validation of aENCL 100200012—8/10/2001——00012
Encyclopedia of Pharmaceutical Technology
Copyright
q
2002 by Marcel Dekker, Inc. All rights reserved.
2545
 
particular SNP represents an important stage forestablishing SNPs as a routine clinical diagnostic marker.The validation of SNPs via MALDI-TOF MS is emergingas a valuable genotyping tool (8,9). A schematic of aMALDI-TOF MS instrument is shown in Fig. 2
½
F2
.A recent study performed by Stroh et al. (10) comparedthe performance of MALDI-TOF MS with restrictionfragment length polymorphism (RFLP) and fluorescencepolarization (FP). The study involved the analysis of known mutations of the IL-1
b
gene. The procedureinvolved amplification of patient DNA samples usingstandard PCR techniques followed by a primer extensionstep where a separate post-PCR primer is hybridizeddirectly adjacent to the SNP site. The resulting MALDI-TOF MS data provided a direct confirmation of molecularweight for fast analysis of polymorphisms.Knowledge of the DNA sequence flanking the SNP siteallows for the optimum choice of post-PCR primer size,primer location, and dideoxy-nucleotide(s). Thus, theassay can be designed to extract complete informationabout the SNP regardless of its state. This emerging MS-based approach for SNP genotyping has potential to behighly automated without the requirement for fluorescenttags. Furthermore, “multiplexingcan be attained byselecting post-PCR primers of varying lengths to dedicatepredetermined regions of the mass spectrum to specificSNPs.
PROTEOMICS
The study of protein structure, function, quantity, andinteractions during maturation and progression of diseaseis referred to as proteomics. Analytical approaches that usea combination of two-dimensional (2-D) gel electrophor-esis for protein separation and MS analysis for proteinidentification followed by database searches is a widelypracticed proteomics strategy (11). The tryptic peptidesextracted from gels are analyzed by MALDI-TOF MS andmicrocolumn or capillary LC tandem mass spectrometry
Fig. 2
Schematic of a MALDI-TOF MS instrument. MALDI-TOF samples areprepared with a matrix that contains a small organic molecule capable of absorbingultraviolet light. A laser is used to desorb ions from the sample plate and the resultingions are forced into the flight tube by application of the acceleration voltage fromextraction grids. All ions leave the source with the same kinetic energy and traveldown the flight tube toward an ion reflector. Separation is based on mass with lighterions traveling faster than heavier ions. The ion reflector is used to correct for smallkinetic energy differences between ions of the same mass resulting in improvedresolution and mass accuracy. (Courtesy of Applied Biosystems, Framingham, MA.)
Fig. 1
Structure analysis matrix that illustrates pharmaceuticalanalysis preferences for four specific sample types: nontrace/-pure; nontrace/mixture; trace/pure; and trace/mixture. (Courtesyof Milestone Development Services, Newtown, PA.)
X-rayNMRMSUVIRHPLC/UVLC/MSLC/NMRTraceNon-TracePure Mixture
ENCL 100200012—8/10/2001——00012
Spectroscopic Methods of Analysis—Mass Spectrometry2546
 
(MS/MS) techniques. Typically, the MALDI-TOF MStechniques are used to quickly identify peptide fragmentsand confirm the presence of known proteins. Nano-scalecapillary LC/MS/MS techniques (using 50–100
m
mdiameter columns, operating at flow rates of 20–500nL/min) are used to further interrogate the complex proteinmixture at the low femtomole level. These techniquesrequire the use of a specialized ESI source shown in Fig. 3
½
F3
.The combination of MALDI-TOF MS and capillaryLC/MS/MS was recently described for the identification of disease state markers in human urine (12). In this study,urine proteins obtained from emphysema patients wereseparated on 2-D gels and selected spots were digestedwith trypsin and analyzed by MALDI-TOF. A databasesearch using Protein Prospector identified a potentialbiomarker for emphysema as human alpha-1-antitrypsin(A1AT). The corresponding MALDI spectrum containednine out of 18 peptides with masses that match theexpected tryptic digest fragments for A1AT.The same tryptic digest protein sample was analyzedby capillary LC/MS/MS using an ion trap massspectrometer followed by a database search withSEQUEST.
½
F4
Fig. 4 illustrates the components of an iontrap mass spectrometer. The highly automated data-dependent MS/MS analysis provided excellentsequence coverage for 11 tryptic peptides related toA1AT in a single LC/MS run. A tryptic peptide thatcorresponds to the A1AT sequence SVLGQLGITK observed at retention time (
) 30.5 min. was observedin the spectrum. The LC/MS data also providedsequence information on unmatched MALDI peaks.The need to detect lower concentration of protein andpeptide mixtures has resulted in the increased use of hybrid quadrupole/orthogonal TOF (QTOF) MS/MSinstruments (13) in conjunction with microcolumn LC.Fig. 5
½
F5
shows a schematic of a QTOF instrument. ThisLC/MS approach provides a resolution of ca. 0.1 massunits allowing for the analysis of complex product ionspectra (14,15). A recent publication by Chalmers andGaskell highlights the current challenges in proteomeanalysis with regard to MS instrumentation (16).
NATURAL PRODUCTS DEREPLICATION
Historically, an excellent source of novel lead drugcompounds is natural products. Natural product screeningactivities typically occur during drug discovery and
Fig. 3
Schematic of a nano-scale capillary ESI interface. This specialized LC/MSinterface, operating at flow rates from 20 to 500 nL/min and using 50 to 100
m
m IDcolumns, typically provides low femtomole sensitivity. Fully automated samplehandling and preparation procedures (i.e., desalting and preconcentration) combinedwith specialized devices for high separation and variable nL gradient flow ratesprovide unique capabilities for high-throughput analysis of proteins. (Courtesy of New Objective, Cambridge, MA.)
ENCL 100200012—8/10/2001——00012
Spectroscopic Methods of AnalysisMass Spectrometry 2547

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