Discovery of Post-Translational Modifications (PTMs)
Many proteins, if not most, in archaea, prokaryotes and eukaryotes contain post-translational modifications (PTMs). PTMs increase the functions of proteins and define the structural plasticity of the proteome. The interplay between thousands of different molecules in cells or tissue maintains cellular integrity, morphology and regulates many important biological functions. Post-translational modifications include covalent modifications such as phosphorylation, glycosylation, ubiquitination, nitrosylation, methylation, acetylation, lipidation, proteolysis and many others.
Post-translational modifications are now considered as a mayor regulatory mechanism in eukaryotes and influence the metabolism of normal cell biology and pathogenesis. In proteomic studies, the discovery of post-translational modifications is achieved with the help of advanced mass spectrometry techniques. A multitude of mass spectrometry-based proteomic strategies has been investigated and reported in the proteomic literature.
How is a PTM discovery done?
A general PTM discovery experiment entails
1. The use of a quality control experiment or standard. For a nano-HPLC-MS/MS experiment, a standard of 125 fmol or greater is usually used. For phosphorylation studies, a phosphorylated peptide or protein or a mixture of different phosphorylated peptides or proteins may be used.
2. Next, a proteolytic digestion is performed. For example, on proteins in gel bands resulting from SDS-PAGE experiments. Trypsin is usually used for a first pass experiment, but other proteases or mixtures of proteases can also be used.
3. The use of nano-high performance liquid chromatography (HPLC) tandem mass spectrometry allows for the analysis of the digest. This analysis generates a dataset of fragment ions in the form of a mass list that can now be queried using search engines or bioinformatics software.
4. Next, a database search is performed using the peptide mass finger print data.
5. A typical search for PTMs involves the use of search engines such as X!tandem, SEQUEST, Mascot or other specialized PTM software.
6. However, to rule out false-positives a manual verification is often necessary.
Protein PTM Identifications (PTM ID)
Proteins PTM’s are identified by digesting proteins into peptides, analyzing peptides using tandem mass spectrometry (LC-MS/MS) followed by searching for modifications using X!tandem, SEQUEST, or other specialized PTM software. The observed post-translational modifications are usually verified by hand, and a likelihood score is produced. However, results can vary depending on the specific programs used.
In general, some modifications are easier to find than others. In addition, searches or scans for specific modifications or even unknown modifications using LC-MS/MS data can be performed. Usually, modifications can be found in the unimod database.
The chances of detecting the modification a researcher is interested in are completely dependent on the stability of the modification and how that modification affects peptide fragmentation in a mass spectrometer. The stoichiometry of the modification factures in as well, as does a number of other variables. However, the PTM discovery experiment may not always work the first time around and the analysis of some specific modifications may require specialized expertise and equipment.
International Journal of Proteomics
Journal of Proteomics
Journal of Proteome Research
Proteomics and Bioinformatics