Glycoproteins MALDI

Kim, Y. J. Freas, A. Fenselau, C. Analysis of viral glycoproteins by MALDI-TOF mass spectrometry. Anal. Chem. 2001, 73,1544—1548. 

Mokrzycki-Issartel, N., Bouchon, B., Farrat, S., Berland, P, Laparra, H Madelmont, J.-C., and Theisen, M. (2003). A transient tobacco expression system coupled to MALDI-TOF-MS allows validation of the impact of differential targeting on structure and activity of a recombinant therapentic glycoprotein produced in plants. FEBS Lett. 552 170-176. 

Tire MALDI method is especially useful for complex mixtures of peptides and can be utilized in peptide sequencing. The technique is also appropriate for studying mixtures of glycoproteins. Negative-ion MALDI can be applied to oligonucleotide mixtures. Further improvements in resolution in both MALDI... 

G. Lochnit and R. Geyer, An optimized protocol for nano-LC-MALDI-TOF-MS coupling for the analysis of proteolytic digests of glycoproteins, Biomed. Chromatogr., 18 (2004) 841-848. 

The MALDI spectrum of a mixture of four oligosaccharides derived from the cleavage of a glycoprotein. Reproduced from Finnigan MAT documentation, with permission. 

With bovine fetuin as a model system, the use of PMP labeling in the routine rp-HPLC profile analysis of glycoproteins has been evaluated. Comparable profiles for oligosaccharides can be obtained regardless of whether they are released by automated chemical or manual enzymatic treatments. The PMP-oligosaccharide samples recovered fi om HPLC are suitable for MALDI TOP and LC ESI mass spectrometric analysis. The presence of all three N-linked and one of the 0-linked oligosaccharides was confirmed. 

Figure 11.25 Mass spectrometric sequencing of oligosaccharides. Carbohydrate-cleaving enzymes were used to release and specifically cleave the oligosaccharide component of the glycoprotein fetuin from bovine serum. Parts A and B show the masses obtained with MALDI-TOF spectrometry as well as the corresponding structures of the oligosaccharide-digestion products (using the same scheme as that in Figure 11.18) ...

The degree of glycosylation of the glycopolypeptides was estimated via H NMR, MALDI-TOF, and periodate assay (Glycoprotein Carbohydrate Estimation Kit (Pierce, Rockford, IL). H NMR spectra were acquired on a Bruker DRX-400 NMR spectrometer under standard quantitative conditions at 25 °C, and the standard protocols for the periodate assay were as described by the manufacturer. Comparison of sample solutions to a calibration curve of proteins of known carbohydrate content, combined with quantitative amino acid analysis of the samples, permitted estimation of the degree of substitution of the glycopolypeptides. 

The first two protocols presented in this work focus on high-throughput gel-based techniques. Chapter 1 describes 2-D DICE, a method used to examine the entire proteome within discreet pi fractions, whereas Chapter 2 details several complementary techniques for the specific analysis of glycoproteins found in tissue samples. The next chapters. Chapters 3 and 4, focus on SELDI-MS and MALDI-TOF, two techniques that make use of mass spectrometry for the characterization and identification of proteins. These techniques may be used alone or in combination with other proteomic methods, such gel-based assays, where mass spectrometry is useful for additional characterization and identification of proteins. 

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