Biomarkers
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12 Lewczuk, P et al (2010). Soluble amyloid precursor proteins in the cerebrospinal fluid as novel potential biomarkers of Alzheimer's disease: a multicenter study. Mol Psychiatry 15,138-145. 13 Richens, JL et al (2010). Quantitative validation and comparison of multiplex cytokine kits. J Biomol Screen 15, 562-568. 14 Chowdhury, F et al (2009). Validation and comparison of two multiplex technologies, Luminex and Mesoscale Discovery, for human cytokine profiling. J Immunol Methods 340, 55-64. 15 Lange, V et al (2008). Selected reaction monitoring for quantitative proteomics: a tutorial. Mol Syst Biol 4, 222. 16 Addona, TA et al (2009). Multi-site assessment of the precision and reproducibility of multiple reaction monitoring-based measurements of proteins in plasma. Nat Biotechnol 27, 633-641. 17 Hüttenhain, R et al (2009). Perspectives of targeted mass spectrometry for protein biomarker verification. Curr Opin Chem Biol 13, 518-525.
technique. As such, appropriate validation, not only of a target or a technology, but of a specific kit is required as the validity of a biomarker in a spe- cific disease state can be highly dependent on the antibody pair involved.
A powerful label-free technique which continues to be effectively applied to biomarker research is mass spectrometry (MS). In its various configura- tions – quadrupole MS/MS, SELDI-TOF or LC-MS – this technology operates independently of exoge- nous reagents to impart specificity or sensitive detection of the target. Furthermore, MS allows for the simultaneous measurement of multiple analytes making direct multiplex assays feasible. Historically, limitations in sensitivity and throughput as well as interference from abundant proteins in complex biological matrices have been significant issues in the adoption of MS for bio- marker screening and validation, however advances in instrumentation and analytical proto- cols continue to make the technique more accessi- ble to these applications. As comprehensively reviewed by Lange15,selected reaction monitoring (SRM) is a method commonly used for mass spec- trometric analysis. SRM delivers a unique frag- ment ion that can be monitored and quantified in the midst of complicated background matrices, and therefore allows for targeted detection of pre- viously identified proteolytic peptides. Using rigor- ous analytical protocols, the Clinical Proteomic Technology Assessment for Cancer Network has demonstrated that SRM allows for precise and reproducible measurement of spiked proteins in human plasma16. SRM techniques can allow for generic assay development that may be applicable to most protein biomarkers, increasing the feasibil- ity of MS as a time-saving approach over develop- ment of new antibody-based assays.
A major issue in adoption of MS techniques for
larger-scale biomarker identification and valida- tion efforts relates to sample preparation bottle- necks negatively impacting throughput. Automated systems have been developed to address this issue, such as the RapidFire ® system from Biocius Life Sciences Inc, which utilises small scale solid-phase extraction cartridges containing various chemistries that allow for sample prepara- tion in a wide range of applications. Using this sys- tem, increased analytical throughput of up to 20- fold has been achieved.
Although challenges remain with respect to sam- ple preparation and measurement of low-abun- dance proteins or small molecular-weight analytes in the presence of complex biological matrices such as blood plasma, many improvements continue to
22
Stéphane Parent is Principal Scientist and Dr Gregory Cosentino is R&D Director within Bio- discovery at PerkinElmer. They have been involved in the development of numerous AlphaLISA kits for the detection of biomarkers in biological samples.
Drug Discovery World Summer 2010
be described17 that make mass spectrometry an exciting technology with increasing utility in bio- marker research.
Conclusions
In this article we provide an overview regarding the application of biomarkers to current research and drug development activities. This area encompass- es a broad spectrum of end-uses, analytical strate- gies, and platform technologies – however com- mon requirements for appropriate use of biomark- ers include the need for their validation against the biology of interest, as well as the availability of sensitive and simple assays for their measurement. As new biomarkers are identified, technology improvements applied to gene expression profiling, quantitation of protein analytes, and imaging will continue to actively support this field for ongoing development into the future.
DDW
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