蛋白质组学-QUANTP~1.pptVIP

2. Though charge-matched, the covalently modified proteins generated by DIGE have slightly altered protein migration properties relative to the bulk of the unlabeled material, because of the additional mass of the dyes. Disadvantages Righetti G, et al. Mass Spectrom Rev, 2002, 21, 287-302 Disadvantages 3. Post-translational modifications that generate altered Cy2/Cy3/Cy5 labeling efficiencies, position/sequence-dependent differential quenching effects, differential solubility effects for labeled protein, and different exposure times required to image individual cyanine dyes, all limit accurate determination of subtle changes in intensity ratios. Fluoresence cross-talk between excitation/emission band-pass filters often causes Cy5-labeled material to emit when illuminating at the optimal Cy3 excitation wavelength, further complicating the quantitation of small changes in protein abundance. Righetti G, et al. Mass Spectrom Rev, 2002, 21, 287-302 Disadvantages 4. One cannot simply run a DIGE gel and cut out the spots for direct MS analysis. The real centroid of the spot will not be aligned with the fluorescent spot. The vast majority of the spots will be present in too low an amount to be directly amenable to MS analysis There is no way to predict where the covalent fluorescent label will be attached, so that peptide identification might be problematic. After the gel has been removed from the special scanner for fluorescence, the spots will no longer be visible, and cutting them out will simply be impossible. Righetti G, et al. Mass Spectrom Rev, 2002, 21, 287-302 Absolute Quantitation Barnidge DR, et al. Anal Chem 2003, 75, 445-51 ITRAQ Question 1.目前定量方法存在的问题 2.对上述问题一种或几种解决方法. Acid-labile isotope codedextractants (ALICE) Wang JH, et al. Anal Chem, 2002,74,4969-79 Wang JH, et al. Anal Chem, 2002,74,4969-79 Solution(2) 1. The size of the ICAT label (~500 Da) is a large modification that remains on each peptide throughout the MS analysis. This can complicate