Digests, enzymatic

A typical TIC chromatogram from an analysis of peptides resulting from enzymatic digest of myoglobin. The peaks represent individual peptides eluting from an LC column and being mass measured by a spectrometer coupled to it through a dynamic-FAB inlet/ion source. 

The crystal structure of myosin S-1 shows how the three subdomains (20K, 50K and 27K) of the myosin heavy chain (produced by further enzymatic digestion of... 

The sequence of a globular protein was confirmed by a combination of enzymatic digestion and HPLC with both Fourier-transform infrared spectroscopy (LC-FTIR spectroscopy) and mass spectrometry [11]. 

Such a process led to the full assignment of the sequence of the cytochrome c" protein shown in Figure 5.17, in which the polypeptide fragments generated by enzymatic digestion with endoproteinase Lys-C and Glu-C are also indicated. 

From a mass spectrometry perspective, these modifications, such as phosphorylation or glycosylation, manifest themselves as an increase in the molecular weight of both the parent protein and also of the polypeptides (produced by enzymatic digestion) containing the modification. 

Figure 5.65 LC-UV and LC-MS-MS (multiple-reaction monitoring (MRM)) traces from the analysis of a enzymatically digested solution of 100 p,g salmon testes DNA (for nomenclature, see text). Reprinted by permission of Elsevier Science from Comparison of negative- and positive-ion electrospray tandem mass spectrometry for the liquid chromatography-tandem mass spectrometry analysis of oxidized deoxynucleosides , by Hua, Y., Wainhaus, S. B., Yang, Y., Shen, L., Xiong, Y., Xu, X., Zhang, F., Bolton, J. L. and van Breemen, R. B., Journal of the American Society for Mass Spectrometry, Vol. 12, pp. 80-87, Copyright 2000 by the American Society for Mass Spectrometry.

The theory and application of this fluorescence method have been discussed in detail by LePecq and others (3,8). The assay requires that there is sufficient ionic strength to minimize ionic binding (e.g., O.IM sodium chloride), that the pH is 4-10, that no heavy metals are present, that the fluorescence is not enhanced on binding to other excipients (e.g., proteins) and that at least portions of the nucleic acids are not complexed. These requirements can usually he met when dealing with recombinant products in some cases the samples must he manipulated to create the appropriate conditions. In the intercalative method of dye binding, proteins rarely interfere with the assay, and procedures have been developed to remove the few interferences they may cause (e.g., the use of heparin or enzymatic digestion of the protein 9). 

Dizdaroglu, M., Hermes, W., Schulte-Frohlinde, D. and von Sonntag, C. (1978). Enzymatic digestion of DNA y-irradiated in aqueous solution. Separation of the digests by ion-exchange chromatography. Int. J. Radiat. Biol. 33, 563-569. 

Fig. 5.2 Chromatogram of the enzymatic digests of different types of insulin employed to demonstrate the suitability of the digestion procedure and the chromatographic system.

A 2D system coupled with a TOF-MS detector provides not only resolution for a large number of protein components, but also yields accurate intact molecular weight information (e.g., Opiteck et al., 1997 Liu et al., 2002 Millea et al., 2005). Moreover, by splitting the effluent just prior to the MS interface, a small portion can be diverted for MS analysis, whereas the bulk of the sample can be collected for subsequent analysis, following enzymatic digestion, to provide positive identification and characterization of the proteins present in the fraction. 

FIGURE 16.11 Multidimensional HPLC-CE separation of cytochrome c/myoglobin enzymatic digest presented in 2D format. For experimental details see text (reprinted with permission from Electrophoresis). 

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