Spectrometry-based Methods

ALIS An Affinity Selection-Mass Spectrometry System based on Continuous SEC 

The schematic shown in Fig. 3.1 describes an optimized, integrated SEC-RPC-MS-based affinity selection platform developed at NeoGenesis, dubbed the 

Protein and Bound Compounds Separated from Unbound Compounds in Mixture 

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Peptide mass fingeiprinting (PMF) is a mass spectrometry based method for protein identification. The protein is cleaved by an enzyme with high specificity (trypsin, Lys-C, Asp-N, etc.) or chemical (CNBr). The peptide mixture generated is analyzed by matrix-assisted laser desorp-tion/ionization (MALDI) or electrospray ionization (ESI)... 

Both plate reader and mass spectrometry based methods are commonly used for screening. The selection of a technique depends on instrument availability, throughput needs and the stage of compound advancement. For the characterization of compounds in drug development and clinical candidates, assays carried out using drug-like probes and analyzed by LC-MS/MS methods are considered the gold standard [117]. 

Dahlmann, J., Budakowski, W.R. and Luckas, B., Liquid chromatography-electrospray ionisation-mass spectrometry based method for the simultaneous determination of algal and cyanobacterial toxins in phytoplankton from marine waters and lakes followed by tentative structural elucidation of microcystins, /. Chromatogr., 994, 1-2, 45, 2003. 

Powell, K.D. Wales, T.E. Fitzgerald, M.C. Thermodynamic stability measurements on multimeric proteins using a new H/D exchange-and matrix-assisted laser desorption/ ionization (MALDl) mass spectrometry-based method. Protein Sci. 2002, 11, 841-851. 

Ma, L. Fitzgerald, M.C. A new H/D exchange- and mass spectrometry-based method for thermodynamic analysis of protein-DNA interactions. Chem. Biol. 2003, 10, 1205-1213. 

Mass spectrometry has found various applications in the chemistry of zinc-carbon bonds. The availability of a variety of ionization techniques has made possible the identification of different types of organozinc compounds—volatile, neutral, ionic, dimeric, polymetallic, solvent-containing etc. In the majority of the reported cases, molecular species have been observed. The experimental results demonstrated that Zn—C bonds are rather weak and easily cleaved upon or after ionization. It is obvious, however, that expanding mass spectrometry-based methods of analysis of zinc complexes will greatly benefit this field of chemistry, as well as facilitate applications of organozinc compounds to material sciences. 

D. R. Goodlett, Mass spectrometry-based methods for protein identification and phosphorylation site analysis, in ... 

Krylov, D.M. and Hurley, J.B. (2001). Identification of proximate regions in a complex of retinal guanylyl cyclase 1 and guanylyl cyclase activating protein-1 by a novel mass spectrometry based method. J Biol. Chem. 276 30648-30654. 

Krebs S, Medugorac I, Seichter D, Forster M. RNaseCut a MALDI mass spectrometry-based method for SNP discovery. Nucleic Acids Res 2003 31 e37. 

Loughrey C.S., Huddleston, M.J., Shou, W., Deshaies, R.J., Annan, R.S. and Carr, S. (2002) Mass spectrometry-based methods forphosphorylation site mapping of hyperphosphorylated proteins applied to Netl, a regulator of exit from mitosis in yeast. Mol. Cell. Proteomics 1, 186-196. 

These reactions are typically observed indirectly by measurement of offgassing signatures by mass spectrometry based methods, though interesting results have been obtained by NMR (26, 27). The effects of these reactions on the remaining, insoluble network structure are unfortunately more difficult to characterize. 

A Our lab is currently very involved in measuring the kinetic constants of possible inhibitors to various enzymes using mass spectrometry. Using a mass spectrometry-based method, we can measure and K-, of enzymes, substrates, and inhibitors... 

Figure 3. Example of a possible mass spectrometry-based method for the identification of protein markers used to differentiate between species when the genome sequence of one is unknown.

A consideration to bear in mind when using mass spectrometry for DNA analysis, either by SOMA or other methods, is that the presence of Na and K can result in the formation of adduct ions. The presence of adduct ions reduces both sensitivity and resolution and purification steps are therefore required to minimise their occurrence during sample preparation. Another concern of mass spectrometry-based methods is cost. However, once the mass spectrometer has been purchased ( US 100,000 to 300,000), the cost per sample is similar to that for other technologies. 

Purcell, A. W. and Gorman, J. J. 2004. Immunoproteomics Mass spectrometry-based methods to study the targets of the immune response. Mol. Cell. Proteomics 3 193-208. 

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