Additionally this approach only detects proteins expressed at relatively high

Additionally this approach only detects proteins

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are between 10-200 kDa. Additionally, this approach only detects proteins expressed at relatively high levels and that have long half-lives [7,8]. Using a 40µg yeast lysate, the average protein abundance detected was 51,200 copies/cell with no proteins detected 2
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with abundances <1,000 copies/cell [8]. Since 1,500 spots were resolved on a 1.0 pH unit gel[8], several gels covering different pH ranges would be needed to resolve a whole cell lysate. Given these limitations this technology has limited potential for large scale proteome analysis[8]. 2D Fluorescence difference gel electrophoresis (DIGE) utilizes mass- and charge-matched, spectrally resolvable fluorescent dyes ( e.g., Cy3 and Cy5) to label two different protein samples in vitro prior to 2DE. Its main advantage over conventional 2DE is that both the control and experimental sample are run in the same polyacrylamide gel. These samples are then imaged separately but can be perfectly overlaid without "warping". This substantially raises the confidence with which protein changes between samples can be detected and quantified. Changes in relative level of protein expression may be detected that are as little as 1.2-fold for large volume spots[9]. Because detection is based on fluorescence, DIGE has a large dynamic range of about 10,000, which permits differential expression analysis of relatively low copy number proteins [9]. The limit of detection of DIGE for quantifying protein expression ratios is between 0.25 - 0.95 ng protein, which is similar to that for silver staining [9,10]. In a recent study [11], the relative level of expression of ~1,050 protein spots was compared in 250,000 laser dissected normal versus esophageal carcinoma cells. This analysis identified 58 spots that were up-regulated by >3-fold and 107 that were down- regulated by >3-fold in cancer cells. Mass Spectrometric Approaches to Protein Profiling Peptide/protein Disease Biomarker Discovery. Current approaches involve batch chromatography, matrix assisted laser desorption ionization mass spectrometry (MALDI-MS) and statistical analysis of large numbers of disease versus normal serum or other biological samples. Most recent studies have relied on surface enhanced laser desorption ionization time-of-flight mass spectrometry (SELDI-TOF-MS)[12,13]. The SELDI approach[13] involves using a gold coated chip with eight or sixteen 2 mm spots that are modified with chromatographic surfaces ( e.g. , anionic, cationic, hydrophobic, etc). After spotting a few microliters of serum, contaminants and salt are removed by washing with water, and the target dried by adding a MALDI matrix solution like - cyano-4-hydroxy-cinnamic acid. In a study by Petricoin et al [14] SELDI-MS analysis of serum from 50 control and 50 case samples from patients with ovarian cancer resulted in identifying 5 peptide biomarkers that ranged in size from 534 to 2,465 Da. The pattern formed by these markers was then used to correctly classify all 50 ovarian cancer samples in a masked set of serum samples from 116 patients who included 50 patients with ovarian cancer and 66 unaffected women. Similar promising results have been reported in studies of serum samples from breast and prostrate cancer patients. ( Li
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