An alternative approach to peptide mass fingerprinting involves tandem mass spectrometry (MS/MS). QIT instruments also have a relatively limited charge capacity and consequently have a limited dynamic range. Both types of instrument are also suitable for MS/MS applications (described below) but tend to be limited to mass accuracies of 50 to 100 ppm, and low sensitivity due to the scanning nature of the design. Among the other mass spectrometer designs that have been employed for protein analysis are triple-stage quadrupole (TSQ) instruments and quadrupole ion trap (QIT) devices. Additionally, peptide mass fingerprinting cannot be used reliably for quantitative analysis because of ion suppression problems associated with the MALDI ionization process. 69(23): 4741-50 (1997)), the technique's limited accuracy leads to ambiguities when it is applied to complex mixtures, and the likelihood of false positive assignments rises to unacceptable levels. Although peptide mass fingerprinting has been shown to facilitate the identification of multiple proteins in simple mixtures (Jensen et al., Anal. Those skilled in the art have coined the name Peptide Mass Fingerprinting (“PMF”) for this method of identifying proteins. 22(6): 338-45 (1993) Henzel et al., Identification of 2-D Gel Proteins at the femtomole level by Molecular Mass Searching of Peptide Fragments in a Protein Sequence Database, Techniques in Protein Chemistry V, John Crabb ed., (1994) and Pappin et al., Peptide Mass Fingerprinting using MALDI-TOF mass spectrometry, Current Biology 3:327-332 (1993)). The generated list of peptide ion mass values is then presented to any of a number of previously described search-engines for protein identification (see Mann et al., Use of mass spectrometric molecular weight information to identify proteins in sequence databases, Biological Mass Spectr. The MALDI instrument characteristically generates a mass spectrum of singly charged peptide ions with a mass accuracy of 20 to 50 ppm. In single stage mass spectrometry the instrument of choice has been a Matrix Assisted Laser Desorption Ionization (MALDI) Time of Flight Mass Spectrometer (TOF-MS or MALDI-TOF). The utility of both single stage and tandem mass spectrometry for the identification of cellular proteins using protein and nucleotide databases is well documented. The discrepancy can be attributed to a variety of factors such as poor experimental reproducibility across various transcriptional profiling platforms, effects of cellular compartmentalization, translation efficiency, post-translational modifications, and protein degradation systems. Varshavsky, The N-end rule: functions, mysteries, uses, Proc. Morris, Post-transcriptional gene regulation, Wiley-Liss Inc (1997) and A. Gygi et al., Correlation between protein and mRNA abundance in yeast, Mol. However, research in this field has demonstrated that there is often a poor correlation between measured mRNA levels and levels of the actual protein encoded by the mRNA.
The field has expanded considerably with the development of DNA arrays (e.g., the GeneChip™ arrays marketed by Affymetrix, Santa Clara, Calif.). Gene expression analysis techniques measure changes in mRNA levels and relate these changes to a cellular response characteristic to a given stimulus.
Elucidating changes in expression profiles should allow for a greater understanding of many biochemical processes on a macroscopic level. Internal stimuli include genetic variations and disease states, while external stimuli include changes in environmental conditions (e.g., temperature, pH, osmolality, etc.) or chemical concentrations (e.g., drugs, hormones, toxins, etc.) Understanding how gene expression profiles vary dynamically as conditions fluctuate can provide valuable insight into the identification and development of novel therapeutic targets, treatments and disease progression/regression markers (biomarkers).
Roth et al., Finding DNA regulatory motifs within unaligned noncoding sequences clustered by whole-genome mRNA Quantitation, Nat. DeRisi et al., Exploring the metabolic and genetic control of gene expression on a genomic scale, Science 278:680-686 (1997) F. Technology platforms such as transcriptional profiling or gene expression analysis are making it possible to better understand cellular physiology and to develop correlations between gene expression (mRNA) and cellular responses to internal and environmental stimuli. The growing importance of genomic and proteomic information in biotechnology and pharmaceutical research and development has stimulated the development of many innovative technologies.
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