Tandem post-translational modifications

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. 

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... 

Perhaps a more challenging question is how we can discover new PTM reactions. In principle, there are analytic tools that can be used to research this topic. One such tool is top-down FT-MS, which determines the molecular weight of the whole protein with high accuracy. By comparing the obtained tandem MS (MS/MS) result with the expected MS/MS result, post-translational modifications can be identified (150). Crystallography can also discover new PTM, if a protein expressed in the proper host can be crystallized. Some rare modifications or protein side chains were discovered this way (151). However, the success of using these methods would require that... 

With large proteins, the determination of the primary sequence and post-translational modifications is most efficiently done after digestion with tiypsin or another protease to generate smaller peptides. In this case, the peptides are first separated by HPLC, most commonly RP-HPLC, and the column eluant is directed into the MS. In this hyphenated method, known as LC-MS or liquid chromatography-tandem mass spectroscopy (LC-MS/MS), the individual peptides are analyzed, allowing the identification of post-translational modification sites. In some cases, there are potentially multiple sites in a single peptide that may be modified. 

Jebanathirajah JA, Pittman JL, Thomson BA, Budnik BA, Kaur P, Rape M, Kirschner M, Costello CE, O Connor PB. Characterization of a new qOq-FTICR mass spectrometer for post-translational modification analysis and top-down tandem mass spectrometry of whole proteins. J Am Soc Mass Spectrom 2005 16 1985-1999. 

Jung, H.R. Liu, W. Jensen, O.N. Determination of post-translational modifications of Histone H3 using chromatography, ion mobility tandem mass spectrometry and customized bioinformatics tools. Proc. 56th ASMS Conference on Mass Spectrometry and Allied Topics, Denver, CO, June 1-5, 2008, MP 394. 

Kjeldsen, R, Giessing, A.M., Ingrell, C.R., Jensen, O.N. (2007) Peptide sequencing and characterization of post-translational modifications by enhanced ion-chaiging and liquid chromatography electron-transfer dissociation tandem mass spectrometry. Anal Chem, 79 (24), 9243-9252. 

Tandem mass spectrometry (MS/MS) is very useful for the amino acid sequencing of peptides, and has been used widely in both protein biochemistry and pro-teomics to identify proteins, to deduce the sequence of a peptide, and to detect and locate post-translational modifications. Until around a decade ago, the concept of amino acid sequencing by MS-technologjes was synonymous with ESI-MS/MS, but today MALDI-MS/MS techniques are implemented in high-performance instruments such that the quality of MALDI tandem mass spectra is comparable with that of ESI-MS/MS spectra. Currently, MALDI tandem mass spectrometers exist in a number of geometries, including TOF-TOF, Q-TOF, ion trap and orbitrap analyzers that each provide unique analytical features for the sequencing of peptides and proteins by MS/MS (details of the instrumentation for different types of MS/MS are provided in Chapter 2). 

Wallace A., Boyce J., and Balland A. (2002), Structural Characterization of Man-nose-6-phosphate-containing glycans by tandem mass spectrometry, IBC s Conference on The Impact of Post-Translational and Chemical Modifications on Protein Therapeutics, San Diego, CA. 

Mass spectrometry not only allows the precise determination of the molecular mass of peptides and proteins but also the determination of their sequences, especially when used with tandem mass techniques. Indeed, fragmentation of peptides and proteins gives sequence information that can be used for protein identification, de novo sequencing, and identification and localization of post-translational or other covalent modifications [ 17-24]. 

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