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Home arrow Health arrow Analysis of Protein Post-Translational Modifications by Mass Spectrometry
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Quantitative Measurements

Quantitative aspects of MALDI spectra have been discussed in Section 3.8.1.3, but quantitation has also been achieved with a number of other ionization techniques. Recent reviews summarize quantitative methods for glycoproteins [337, 338]. A number of applications have incorporated stable isotopes into reagents used to derivatize glycans. Comparing mixed spectra of glycans from control and test samples where differential derivatization has been performed with normal and isotopically labeled reagents allows, for example, changes in glyco- sylation in disease states to be monitored. Thus, Kang et al. [339] have used CH3 and C2H3 derivatization to monitor changes in breast cancer by MALDI-TOF MS. Atwood et al. [340] have used a mixture of 13CH3I and 12C2H1H2I for deri- vatization. Although these derivatives have the same nominal mass, derivatized glycans could be separated at 30,000 resolution with an FT-ICR instrument.

Other isotope-labeled derivatives have involved derivatization at the reducing terminus and have included 13C6-labeled 2-AA [341] and aniline [342] and 2H4-labeled pyridine [343]. Hahne has produced two reagents (Figure 3.10) with various stable isotope labels. Of these, the aminooxy reagent (B), which formed an oxime with the glycan, showed the greatest labeling efficiency. Four 13C and one 15N labels were incorporated in the positions shown. Thus, all three labeled derivatives introduced a five unit increase in mass and experiments were conducted using labeled and unlabeled reagents as mentioned earlier. However, on fragmentation, the fragment containing the dimethylpyridine group exhibited a different mass according to which reagent had been used. Consequently, the glycan profile from glycans derivatized with any of the three labeled derivatives looked the same, but, if mixed, the glycans labeled with each specific reagent could be identified by the mass of the fragment.

 
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