Translation phosphorylation characterization

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. 

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. 

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... 

Ovalbumin, a member of the albumins. Ovalbumin is a glycoprotein (hen ovalbumin Mr 44.5 kDa 385 aa) characterized by four sites of post-translational modifications. Beside the acetylated N-terminus, the carbohydrate moiety is located at Asn , and the two phosphorylated serines at residues 68 and 344. Ovalbumin is a non-inhibitory member of the serine protease inhibitor family of the serpins. It has been classified as ovalbumin A1-A3, depending on the number of phosphorylated serine residues. Ovalbumin comprises 60% of the total protein amount of egg-white [A. D. Nisbet et al., Eur. J. Biochem. 1981, 115, 335 J. A. Huntington, P. E. Stein, J. Chromate. B 2001, 756, 189]. 

FT-ICR-based top-down proteomics has been applied to protein discovery in Methanococcus jannaschii [134], Shewanella oneidensis [135], and human HeLa cells [136]. Examples of applications of the top-down approach for targeted protein characterization include the analysis of post-translationally modified histones [137,138], determination of variations in the sequence of P-thymosin [139], and phosphorylation analysis of cardiac troponin I [140]. 

Small RNAs may also be involved in regulating the translation of mRNA in eukaryotic cells. Of the stimulatory RNAs, the best characterized is a small RNA of about 160 nucleotides, which accumulates in cells after infection with adenovirus. This virus-associated RNA, VA-RNAi, which is required to maintain general protein synthesis, acts by inhibiting the phosphorylation of the alpha subunit of initiation factor eIF-2. 

In a scenario that occurs often in a protein biochemistry lab setting, a researcher has isolated a protein of unknown function, or he/she has overexpressed a protein in Escherichia coli and wishes to characterize it. A common practice today is to submit a small amount of the protein to a core mass spectrometry laboratory for a molecular weight measurement. Using either ESI or MALDl, a molecular weight with a precision and accuracy of 0.05% or better can be measured. This, of course, depends heavily on the purity of the protein sample, the relative size of the protein, the presence or absence of post-translational modifications (PTM) (e.g., phosphorylation, glycosylation, etc.), the resolution of the mass analyzer, and so on. Primary structure information (i.e., amino acid... 

---