Mass spectrometry protein preparation

Kussmann M, Nordhoff E, Rahbek-Nielsen H, Haebel S, Rossel-Larsen M, Jakobsen L, Gobom J, Mirgorodskaya E, Kroll-Kristensen A, Palm L, Roepstorff P (1997) Matrix-assisted laser desorption/ionization mass spectrometry sample preparation techniques designed for various peptides and protein analytes. J Mass Spectrom 32 593-601... 

At present identification and quantification of selenoamino acids in seafood relies solely on matching retention time of peaks with available standards. To unambiguously assign peaks, the use of HPLC coupled to a mass spectrometer is required, as for the identification of As species [158, 159]. However, at present the low concentrations of Se species, the low detection power, and the lack of knowledge about Se species does not make this possible [131, 135, 136, 163], Preparative scale isolation and purification of Se proteins as done for bacterial proteins is required to allow Se proteins and selenoamino acids to be characterized by mass spectrometry [164], Preparative gel and two-dimensional electrophoresis offers promise to obtain pure Se protein fractions. Selenium-containing... 

Li, F. et al., Drug-protein binding screening using tandem column affinity chromatography-tandem mass spectrometry, in preparation. 

Kussmann, M., Nordhoff, E., Rahbek-Nielsen, H., Haebel, S., Rossel-Larsen, M., Jakobsen, L., Gobom, J., Mirgorodskaya, E., Kroll-Kristensen, A., Pahn, L., and Roepstorff, R, Matrix-assisted laser desorption/ionization mass spectrometry sample preparation techniques designed for various peptide and protein analytes, Journal of Mass Spectrometry, 32, 593-601, 1997. 

Fig. 1. Schematic representation of protein identification by mass spectrometry and preparation of reference 2D maps.

Another means of moving beyond pure protein preparations to high-throughput characterization of proteomes is to enrich for phosphopeptides from complex mixtures by metal affinity chromatography (Andersson and Porath, 1986). Using this method, protein mixtures are proteolyzed to create peptides and phosphorylated peptides are enriched by metal affinity chromatography and subsequently identified by mass spectrometry. This method is limited, however, because in many cases phosphopeptides absorb poorly or nonphosphorylated peptides absorb nonspecifically to the metal affinity resins (Ahn and Resing, 2001). 

Vaidyanathan, S. Winder, C. L. Wade, S. C. Kell, D. B. Goodacre, R. Sample preparation in matrix-assisted laser desorption/ionization mass spectrometry of whole bacterial cells and the detection of high mass (>20kDa) proteins. Rapid Comm. Mass Spectrom. 2002,16,1276-1286. 

Wang, H., Hanash, S. (2005). Intact-protein based sample preparation strategies for proteome analysis in combination with mass spectrometry. Mass Spectrom. Rev. 24, 413 126. 

Nilsson CL et al. Identification of protein vaccine candidates from Helicobacter pylori using a preparative two-dimensional electrophoretic procedure and mass spectrometry. Anal Chem 2000 72 2148-2153. 

The ability to prepare monoliths within a mold of any shape was used by Lee et al. [128] who prepared monolithic ST-DVB microbeads within pulled fused silica needles and used them for the reversed-phase separation and on-line electrospray ionization mass spectrometry (ESI-MS) detection of proteins and peptides. As illustrated by Fig. 18, these monolithic microcolumns separated proteins far better than capillaries packed with commercial C18 silica or polymeric beads. 

Fion, N., Gellon, J. O., Jensen, H., and Girault, H. H. (2003). On-chip protein sample desalting and preparation for direct coupling with electrospray ionization mass spectrometry. ]. Chromatogr. A 1003, 11-19. 

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