Analysis of Post-Translational Modifications

Post-translationally modified proteins, such as glycoproteins and phosphopro-teins, are prone to decomposition during the sample preparation and/or MALDl process, and also in the mass analyzer. Labile molecules should be analyzed using the DHB matrix [21] or the THAP or DHAP matrices [44] using the linear TOF-MS mode. Post-translahonally modified pephdes should be analyzed in both linear 

TOF mode and reflector TOF mode for the observation of labile modifications [45] (differences between linear and reflectron TOFs in the detection of metastable ions are detailed in Chapter 2). It may also be useful to try negative- and positive-ion modes, as certain species ionize more efficiently in the negative-ion mode [46]. An alternative to the MALDl-MS/MS analysis of modified peptides is electron-transfer dissociation (ETD)-based ESl-MS/MS, where the modification is retained during peptide ion fragmentation, making the assignment of the modification site more straightforward. 

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Mann, M., Jensen, O.L. (2003). Proteomic analysis of post-translational modifications. Nature Biotech. 21, 255. 

MS-Based Analysis of Post-Translational Modifications (PTMs)... 

Proteomics includes a variety of technologies that include differential protein display on gels, protein chips, quantitation of protein amoimts, analysis of post-translational modifications, characterization of protein complexes and networks and bioinformatics. All this information in combination with genome and phenotype studies will ultimately yield a comprehensive picture of a cellular or tissue proteome (Wasinger and Corthals 2002). 

In conclusion, because ECD and ETD induce fragmentation along the peptide backbone that is essentially sequence independent and preserves post-translational modifications, this method is an effective alternative dissociation method for peptides and proteins, especially for the analysis of post-translational modifications. 

In comparison with the bottom-up approach, the top-down approach for de novo protein sequencing is faster and can be applied on proteins in mixture. However, for analysis of post-translational modifications, these two approaches are complementary and a combination of them is interesting. The bottom-up approach allows detection of low-stoichiometric modifications even if a high-stoichiometric modification is missed. Furthermore, enzymatic digestion leads to the loss of all connectivity information from the original protein species. On the other hand, the top-down approach allows observation of the global pattern of... 

M. Mann and O. N. Jensen, Proteomic analysis of post-translational modifications, Nat. Biotechnol., 21 (2003) 225-261. 

Mann, M. and Jensen, O.N. (2003) Proteomic analysis of post-translational modification. Nat. Biotechnol. 27,255-261. 

Here, we will describe a range of applications of MALDI-MS, from the concepts of in-depth analysis of purified proteins to applications of MALDI-MS in a broader, proteomics-based research where proteins are identified, characterized, and quantified. In addihon, issues of sample preparation, protein characterization and identification strategies and bioinformatic tools for data interpretation wiU be discussed. The concepts of peptide fragmentation, sequencing and derivatization, analysis of post-translational modifications and the clinical apphcations of MALDI-MS are also briefly outlined. 

Yan, B., Vallier-Douglass, J., Brady, L., Steen, S., Han, M. and Pace, D., et al. Analysis of post-translational modifications in recombinant monoclonal antibody IgCl by reversed-phase liquid chromatography/mass spectrometry. /. Chromatogr. 1164 153-161, 2007. 

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