Protein mass spectrometric analysis

These columns have been used for separation of proteins of over 200 kDa MW in our experiments as shown by analysis using a ID gel. In addition, columns with larger particle sizes have been used to separate proteins of over 400 kDa (55-56). The NPS RP-HPLC method provides a liquid phase method for separating large intact proteins for further analysis. More specifically, it provides a means of separating proteins for interfacing to mass spectrometric analysis. 

Lee, S.W., Berger, S.J., Martinovic, S., Pasa-Tolic, L., Anderson, G.A., Shen, Y., Zhao, R., Smith, R.D. (2002). Direct mass spectrometric analysis of intact proteins of the yeast large ribosomal subunit using capillary LC/FTICR. Proc. Natl. Acad. Sci. USA 99, 5942-5947. 

Verma R et al. Proteasomal proteomics identification of nucleotide-sensitive pro-teasome-interacting proteins by mass spectrometric analysis of affinity-purified proteasomes. Mol Biol Cell 2000 11 3425-3439. 

Brancia, F.L., Oliver, S.G., and Gaskell, S.J. (2000) Improved matrix-assisted laser desorption/ionization mass spectrometric analysis of tryptic hydrolysates of proteins following guanidination of lysine-containing peptides. Rapid Comm. Mass Spectrom. 14, 2070-2073. 

Hansen, K.C., Schmitt-Ulms, G., Chalkley, R.J., Hirsch, J., Baldwin, M.A., and Burlingame, A.L. (2003) Mass spectrometric analysis of protein mixtures at low levels using cleavable 13C-isotope-coded affinity tag and multidimensional chromatography. Mol. Cell. Proteomics 2, 299-314. 

Veema, R., Chen, S., Feldman, R., Schieltz, D., Yates, J., Dohmen, J., and Deshaies, R. J. Proteasomal proteomics identification of nucleotide-sensitive proteasomal-interacting proteins by mass spectrometric analysis of affinity-purified proteasomes. Mol. Bid. Cell 2000, 71, 3425-3439. 

The analysis for proteins present in plasma or a cell extract is a challenging task due to their complexity and the great difference between protein concentrations present in the sample. Simple mixtures of intact proteins can be analyzed by infusion with electrospray ionization and more complex ones by matrix assisted laser desorption ionization. MALDI is more suited for complex mixtures because for each protein an [M+H]+ signal is observed while for ESI multiply charged ions are observed. Surface enhanced laser desorption (SEEDI) is a technique for the screening of protein biomarkers based on the mass spectrometric analysis of intact proteins [49]. However in most cases for sensitivity reasons mass spec-... 

Zu, X.L., BesanL P.G., Imhof, A. and Attwood, P.V. (2007) Mass spectrometric analysis of protein histidine phosphorylation. Amino Acids,... 

Figure 18.2 Redox proteomics and oxidatively modified proteins in the brain. Redox proteomics has the potential of detecting disease markers and identifying potential targets for drug therapy in neurodegenerative disorders. Redox proteomics involves the separation of brain proteins followed by detection, usually immunochemically, of oxidatively modified proteins, either from a two-dimensional western blot or from column eluents. Subsequent mass spectrometric analysis of tryptic digests and database searching leads to protein identification.

It was clear from this early work that mass spectrometric analysis would be the most appropriate technique to detect hit compounds for in situ Click chemistry applications. Although the DIOS-MS method was able to directly detect the low quantity of triazole product conversion in the presence of large amounts of protein and parent fragments, the sensitivity for this measurement was very low, with a poor signal-to-noise ratio. It was therefore both logical and desirable to optimize the sensitivity and selectivity of the MS... 

The idea that Gq activation leads to stimulation of EC formation is logical given that PLC is known to be activated by this class of G-proteins (Stemweiss et al. 1992). Activation of mGluRs has been shown to increase EC levels measured using mass spectrometric analysis of brain tissue (Jung et al. 2005), and activation of PLC and DAG lipase were implicated in this mGluR action. 

The purification of HCV14 2A protein after each column is summarized in Fig. 2. As seen in Fig. 2, the protein had a molecular weight of —16 kDa, matching its estimated molecular mass of 15,976 Da. The identity of this purified protein as HRV14 2A was further confirmed by mass spectrometric analysis (data not shown). Amino acid sequence analysis was also performed on the purified HRV14 2A protein. Its N-terminal amino acids were identified as GLGPRYGGIYTSN-, which was identical to the expected sequence. 

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