Samuel, no single method will provide you with all answer on all possible modifications, and certainly not in "high throughput" (which leaves me puzzled - why are you looking for high throughput if you "only" want to characterize one protein?). You will probably need a combination of separations (2D, SDS-PAGE) combined with MS, Western (provided antibodies can be raised) and other techniques here - in other words, a good old full-blown biochemistry project. It will not be quick or high throughput.

Samuel, no single method will provide you with all answer on all possible modifications, and certainly not in "high throughput" (which leaves me puzzled - why are you looking for high throughput if you "only" want to characterize one protein?). You will probably need a combination of separations (2D, SDS-PAGE) combined with MS, Western (provided antibodies can be raised) and other techniques here - in other words, a good old full-blown biochemistry project. It will not be quick or high throughput.

Identification of the most protein modification derived from 1D or 2D gels can generally be done by MALDI-MS fingerprint analysis or by fragmentation spectra received by MALDI-PSD or ESI-MS/MS analysis. In combination with information from different databases lead to a first hint about protein-modification. Proteins separated by 1D or 2D-PAGE and blotted onto membranes can easily be stained or blotted with specific Phosph, Ubiq, Glyco and other modifications antibodies. Of course I agree with the previous comment that with a single method is not easy to identify all modifications at the same time but of course it is possible to identify a single modification kind in a single method.

Dear Samuel,

Mass spectrometry can address all of these PTMs, but each of them requires a unique sample preparation strategy so that is would be impossible to address them all with any single workflow. The only "high throughput" option were if you had antibodies against each of the specific PTMs you want to quantitate and could use an ELISA platform for analysis.

I say mass spectrometry is the way to go. One of the tools I use (and is developed by a colleague of mine) excels at this. It's called pride-asap (automatic spectrum annotation pipeline). I don't think there's anything out there that does a better job to be honest. You can find it at https://code.google.com/p/pride-asa-pipeline/ ! Keep me posted on what you think !

Detection of PTMs by mass spectrometry relies on alteration of the mass of tryptic peptides, which results from chemical modification occurring in the amino acid residue side chain. However, for the unequivocal assignment of a given modification site, tandem mass spectrometry (MS/MS) experiments are necessary to show that the mass shift detected in the precursor ion (the peptide from tryptic digestion) is also observed in the fragment ions carrying the modified amino acid residue.

An essential aspect of PTM identification is the need for high quality samples, i.e. high purity with low degradation and low abundance of contaminants, by the use of competitive separation techniques for intact proteins or peptides. As mentioned in the previous section, the very high dynamic range of protein concentrations in a cell poses serious difficulties for the identification of PTMs. Also it is difficult, if not impossible, to devise a strategy to allow the separation and analysis of all structurally diverse protein isoforms, because of the number of possible combinations of PTM locations within a protein with several PTMs. To understand the complexity of the problem, Thelen and Miernyka reported a virtual comparison of a liquid chromatography profile of a peptide containing zero to three PTMs [46], which highlighted the need to use a combination of strategies to improve the identification of PTMs. Currently, several approaches are used for identifying and quantifying proteins and PTMs, which have been conveniently reviewed in several high quality papers.

There are several strategies for PTM identification using mass spectrometry. These are essentially the same as those used for protein identification.

High-throughput proteomics allows the simultaneous identification of thousands of modified peptides. Of all the work-flow tasks, data processing, analysis and presentation are probably the most challenging aspects of proteomics. Unless targeted approaches are used, the characterization of PTMs using MS/MS data involves exhaustive searches of all potential combinations of mass shift for each identified peptide from a protein database. Several tools are most commonly used to perform this task, although the most used are Mascot (Matrixscience) , Sequest , Maxquant and Paragon.

I adjust my previous post and completely agree with Saeed, MS/MS - data is in fact needed for pride asap as well. We have open source tools called SearchGUI and PeptideShaker that can be used to explore the data. SearchGUI combines results of search engines (OMSSA and XTandem). It is usually followed by a peptideshaker run. This calculates a new ranked score based on the individual results. I recommend searchgui as it is very user friendly, contrasting to the other engines that are more often than not command line based.

So to find new, not annotated PTMs : pride asap can help

Find known PTM(s) in a sample : use searchgui

Process known PTM(s) and rank them : use peptideshaker (that uses searchgui's results)

https://code.google.com/p/pride-asa-pipeline/source/checkout

https://code.google.com/p/searchgui/

https://code.google.com/p/peptide-shaker/

Post-translational modifications are critical to the protein's function. Proteome differs from cell to cell and from time to time per se distinct genes are expressed in different cell types making it essential to identify even the basic set of proteins that are produced in a cell. Previous approach of mRNA analysis was found not to correlate with protein content as amount of protein produced for a given amount of mRNA depends on the gene it is transcribed from and on the physiological state of the cell. Besides differences caused due to translation from mRNA, many proteins undergo wide variety of chemical modifications after translation. Therefore, no single technique including high throughput can provide acceptable answer to possible modifications. Development of antibody specific to that modification can be a way out to study a particular protein. Also, there are antibodies specific to other modifications, which can be used to determine the set of proteins that have undergone the modification of interest. For glycosylation of proteins, certain lectins that bind sugars can be used. Another way is to subject a complex mixture of proteins to 2D-electrophoresis that allows small differences in a protein to be visualized by separating a modified protein from its unmodified form. Recent technique, PROTOMAP, which combines SDS-PAGE with shotgun proteomics enable detection of changes in gel-migration, such as those caused by proteolysis or post translational modification. Techniques such as ELISAs can be used for quantification of proteins. Now-a-days, matrix-assisted laser desorption/ionization (MALDI) coupled with ESI are being employed for rapid determination of proteins in particular mixtures. Also, fast parallel proteolysis (FASTpp) is also applied in proteomic assay, which enabled detection of specific proteins.

Thank you all. I'm trying to explore each of the suggestions you have given. I appreciate all your help. Thanks