Mass spectrometry and sequencing

Joubert-Caron R et al. Protein analysis by mass spectrometry and sequence database searching a proteomic approach to identify human lymphoblastoid cell line proteins. Electrophoresis 2000 21 2566— 2575. 

As for all synthetic products to be tested in biological systems, a careful analytical characterization of peptide libraries is crucial in order to confirm their identity and establish their quality. Compared to individual peptides, however, the analysis of peptide libraries is complicated due to the fact that the peptides are either bound to a solid support or arranged in highly complex mixtures. This poses certain restrictions on which analytical methods can be used to characterize combinatorial libraries. For example, analytical methods that are based on the separation of product components, such as high performance liquid chromatography (HPLC) and capillary electrophoresis (CE), are only of limited use for the analysis of peptide libraries, in particular of those made up of complex nnixtures (>100 peptides per mixture). The analytical methods beneficially applicable to peptide libraries include amino acid analysis, mass spectrometry, and sequencing. 

CHARACTERIZATION OF A RECOMBINANT HEPATITIS E PROTEIN VACCINE CANDIDATE BY MASS SPECTROMETRY AND SEQUENCING TECHNIQUES... 

Table I Summary of mass spectrometry and sequencing data from the unmodified and...

Joubert-Caron, R., Le Caer, J.P., Montandon, F., Poirier, F., Pontet, M., Imam, N., Feuillard, J., Bladier, D., Rossier, J., and Caron, M., 2000, Protein analysis by mass spectrometry and sequence database searching a proteomic approach to identify human lymphoblastoid cell line proteins [In Process Citation]. Electrophoresis 21 2566-2575. 

The structural information won from HPLC or lectin columns can and should be conflrmed by independent methods. These include NMR, mass spectrometry, and sequencing of purifled oligosaccharide chains with exoglycosidases. 

E Peptide Sequencing Using Mass Spectrometry and Sequence Databases... 

Nesvizhskii, A.I. (2007) Protein identification by tandem mass spectrometry and sequence database searching. Methods in Molecular Biology, 367,87-119. 

Figure 5.11. Generic approaches to identify interacting proteins within complexes. The complex is isolated from cells by affinity purification using a tag sequence attached to a protein known to be in the complex. Alternatively, the complex can be immunprecipitated with an antibody to one of the proteins in the complex. The proteins are resolved by polyacrylamide gel electrophoresis, proteolyzed, and the mass of the resulting peptides is determined by mass spectrometry. Alternatively, the proteins can be proteolyzed and the resulting peptides resolved by liquid chromatography. The peptide masses are then determined by mass spectrometry and used for database searching to identify the component proteins.

The HPLC-MS/MS assay was also successfully applied to the measurement of UV-induced dimeric pyrimidine photoproducts [123, 124]. The latter lesions were released from DNA as modified dinucleoside monophosphates due to resistance of the intra-dimer phosphodiester group to the exonuclease activity during the hydrolysis step [125, 126]. The hydrolyzed photoproducts exhibit mass spectrometry and chromatographic features that allow simultaneous quantification of the three main classes of photolesions, namely cyclobutane dimers, (6-4) photoproducts, and Dewar valence isomers, for each of the four possible bipyrimidine sequences. It may be added that these analyses are coupled to UV detection of normal nucleosides in order to correct for the amount of DNA in the sample and obtain a precise ratio of oxidized bases or dimeric photoproducts to normal nucleosides. 

Christie, J.F., Dunbar, B. and Kennedy, M.W. (1993) The ABA-1 allergen of the nematode Ascaris suum. epitope stability, mass spectrometry, and N-terminal sequence comparison with its homologue in Toxocara canis. Clinical and Experimental Immunology 92, 125-132. 

Stahl, N., Baldwin, M. A., Teplow, D. B., Hood, L., Gibson, B. W., Burlingame, A. L., and Prusiner, S. B. (1993). Structural studies of the scrapie prion protein using mass spectrometry and amino acid sequencing. Biochemistry 32, 1991-2002. 

Fig. 7. Protein identification with electrospray tandem mass spectrometry and a triple quadrupole mass spectrometer. Fragment spectra of several peptides are generated during one investigation. From the fragment spectra short sequence stretches can be read. Together with their mass location in the peptide of the measured mass, they can be used to specifically identify a protein in the database. Because the protein identification depends only on one peptide, several proteins can be identified from one sample.

Ermer, for example, utilized LCQ to monitor impurity profiles of various batches of ramorelix used in toxicological studies, clinical stndies and scale-up. Ramorelix is a synthetic glycosylated decapeptide with monoisotopic of 1530.7. The toxicological batch served as the benchmark against which all other batches were compared. Molecnlar weights of impurities were determined by ESI mass spectrometry, and nsed in conjunction with UV peak area % to gauge impurities in batches nsed in clinical trials. These impurity profiles were compared to those of batches used in the toxicologically qualified batch. Eour impurities were detected with the same value. They were believed to be diastereoisomers of ramorelix, i.e., a peptide sequence with one of the amino acids in the opposite enantiomeric form. 

Alban PS, Popham DL, Rippere KE, Krieg NR. 1998. Identification of a gene for a rubrerythrin/nigerythrin-like protein from Sprillum volutans by using amino acid sequence data from mass spectrometry and NH2-terminal seqnencing. J Appl Microbiol 85 875-82. 

Mass spectrometry and high-resolution NMR spectroscopy, applicable to small samples of carbohydrate, yield essential information about sequence, configuration at anomeric and other carbons, and positions of glycosidic bonds. 

Structural studies of glycosphingolipids involves determination of the structure of the oligosaccharide chain and of the lipid moiety. For the oligosaccharide chain, it is necessary to determine the composition, molar ratio, and sequence of the monosaccharides, their pyranose or furanose nature, and the position of glycosidic bonds and their configuration for the lipid moiety, the composition of the fatty acids and sphingosine bases must be determined. Used for these purposes are the classical, chemical methods, conventionally accepted in the chemistry of carbohydrates and lipids and based on the degradation of compounds, enzymic, and physicochemical methods, primarily mass spectrometry and n.m.r. spectroscopy. 

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