Post-translational modification spectrometry

The Edman reaction enabled amino acid sequence analysis to be automated. Mass spectrometry provides a sensitive and versatile tool for determining primary strucmre and for the identification of post-translational modifications. 

Post-translational modification of proteins plays a critical role in cellular function. For, example protein phosphorylation events control the majority of the signal transduction pathways in eukaryotic cells. Therefore, an important goal of proteomics is the identification of post-translational modifications. Proteins can undergo a wide range of post-translational modifications such as phosphorylation, glycosylation, sulphonation, palmitoylation and ADP-ribosylation. These modifications can play an essential role in the function of the protein and mass spectrometry has been used to characterize such modifications. 

Roos, M., Soskic, V., Poznanovic, S., and Godovac-Zimmermaim, J. (1998). Post-translational modifications of endothelin receptor B from bovine lungs analyzed by mass spectrometry. J. Biol. Chem. 273, 924-931. 

Demirev, P. A. Lin, J. S. Pineda, F. J. Fenselau, C. Bioinformatics and mass spectrometry for microorganism identification Proteome-wide post-translational modifications and database search algorithms for characterization of intact H. Pylori. Anal. Chem. 2001, 73, 4566 573. 

Jensen, O.N. (2004). Modification-specific proteomics characterization of post-translational modifications by mass spectrometry. Curr. Opin. Chem. Biol. 8, 33 11. 

Strategy for Identification of Sites of In vivo Post-Translational Modifications of HMGAl Proteins by Mass Spectrometry... 

Freitas, M.A., Sklenar, A.R. and Parthun, M.R. (2004) Application of mass spectrometry to the identification and quantification of histone post-translational modifications. Journal of Cellular Biochemistry, 92, 691—700. 

Zhang, L., Freitas, MA., Wickham, J., Parthun, M.R., Klisovic, M.I., Marcucd, G. and Byrd, J.C. (2004) Differential expression of histone post-translational modifications in acute myeloid and chronic lymphocytic leukemia determined by high-pressure liquid chromatography and mass spectrometry. Journal of the American Society for Mass Spectrometry, 15, 77-86. 

Johnson, L., Mollah, S., Garcia, B. A., Muratore, T. F., Shabanowitz, J., Hunt, D. F., and Jacobsen, S. E. (2004). Mass spectrometry analysis of Arabidopsis histone H3 reveals distinct combinations of post-translational modifications. Nucleic Acids Res. 32 6511 -6518. 

Short polypeptides can be sequenced rapidly by fast atom bombardment mass spectrometry (FAB-MS). This technique not only provides the amino acid sequence of the peptide but also information on post-translational modifications. 

In order to assign the disulfide bonds of these molecules fast atom bombardment mass spectrometry (FABMS) which has been used not only to confirm amino acid sequence data but also to elucidate post-translational modifications of proteins, such as disulfide bonds, has been employed. For this purpose a sample of native Er-2, containing four methionines, was subjected to CNBr cleavage and without further fractionation directly... 

When working with purified enzymes, it can be useful to perform a close examination of their phosphorylation states and molecular masses. Mass spectrometry is often useful for this purpose. Post-translational modifications or sequence truncations can potentially alter the compound binding sites available and can also change the structure of potential inhibitory sites. For example, with protein kinases, phosphorylations distal from the ATP binding site can inactivate the kinase whereas phosphorylations near the ATP binding site can activate the catalytic activity. Often, practice does not permit control of such situations because the purified systems are often mixtures and cannot be controlled in the commonly used recombinant expression technologies. 

Another wide application of mass spectrometry is the detection and characterization of post-translational modifications such as myristoylation, phosphorylation, disulfide bridging, etc. The detection and localization of post-transla-tional modifications has been a rapidly developing area of mass spectrometry due to the functional importance of these modifications in biological systems. An example of this was recently shown for the case of the human rhinovirus HRV14 [10]. Electron density maps from crystallography data indicated a myristoylation of VP4. Mass analysis of VP4 also indicated a mass difference of 212 Da (consistent with myristoylation of VP4). Additional experiments with proteolytic digestion and tandem mass spectrometry were able to localize the modification to the N-terminus of VP4. 

MALDI quadrupole ion trap mass spectrometry has also been used to localize and identify the post-translational modifications on the Sindai virus [18]. The polymerase associated protein (P protein) from this virus is reported in the literature to be highly phosphorylated. In vitro studies have detected phosphorylation in different regions of the protein, while a single phosphorylation site was found in the in vivo studies. Mass spectral data, along with computer-aided analysis, enabled the identification and localization of two phosphorylation sites. 

The protein identification or sequence determination of a protein can be achieved using two different approaches top-down [22, 23] and bottom-up [24], A top-down experiment involves high-resolution measurement of an intact molecular weight and direct fragmentation of protein ions by tandem mass spectrometry (MS/MS) [25], This approach surveys an entire protein sequence with 100% coverage. Post-translational modifications such as glyco-... 

Tandem mass spectrometry coupled to electrospray ion source allowed not only to identify proteins but also to characterize post-translational modifications of a protein (phosphorylation, acetylation, methylation, glycosylation, etc.). Indeed, the presence of such modifications induces an increase of the peptide molecular masses compared to the calculated masses based on the theoretical sequence, which often directly identifies the type of modification. In addition, tandem mass spectrometry allows in general precise localization of the modification at specific residue of the peptide. Analysis of modifications allows to understand biological mechanisms because several processes are controlled and/or induced by such modifications (Mann and Jensen 2003). Being... 

Mass spectrometry is the method of choice for identification of proteins, quantitation of proteins expression at a given time but also for characterization of post-translational modifications. Indeed, allowing direct analysis of peptides and proteins, the MALDI and ES ionization modes have been revolutionary in the field of mass spectrometry applied to biology. 

Mass spectrometry Molecular weight (post-translational modifications)... 

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