General Technique
Identification of post translationally modified proteins. Post translational modification (PTM) of proteins plays a key role in the control of a wide range of biological functions and activities. Thus, development of technologies for the rapid and conclusive identification of PTMs is essential. We have made significant contributions to the field with development of technologies for the identification of sites of phosphorylation, glycosylation , and sumoylation.
1) Direct in vivo determination of individual phosphorylation sites in proteins is difficult, typically requiring purification to homogeneity of the phosphoprotein of interest. Our approach consists of three steps:
- selective phosphopeptide isolation from a peptide mixture via a series of chemical reactions;
- phosphopeptide analysis by µLC-MS/MS;
- identification of the phosphoprotein and the phosphorylated residue(s) by sequence database searching.
By incorporating stable isotope tags into this approach we can also detect quantitative changes in the phosphorylation state of proteins. (see quantitative proteomics technique).
2) The method for identifying N-linked glycosylation sites in proteins is based on the conjugation of glycoproteins to a solid support using hydrazide chemistry, stable isotope labeling of glycopeptides, and the specific release of formerly N-linked glycopeptides by peptide N-glycosidase F. The recovered peptides are then identified and quantified by MS/MS. This technology also is being used for reducing sample complexity before profiling peptide mixtures (see quantitative proteomics technique).
3) We have developed computational approaches to identify ubiquitin-like modifier (ULM) conjugates on proteins. Currently, we are focusing on the identification of sumoylated (SUMO) peptides. Standard MS peptide sequencing software tools are unable to identify many ULM conjugated peptides (including the SUMOs), which, due to the branched structure of the modified peptides, generate complex overlapping fragment ion spectra. In our experience, SUMOylated peptides mostly fragment on the SUMO chain. To address this problem, we have developed a software program named SUMmOn. While standard database search software treat modifications as indivisible units and use fragments from the target peptide only to assign a scan, SUMmOn computes and uses fragment ion series for both the modification and the modified peptide. This strategy has proven to be very successful in the identification of previously unknown SUMOylation sites using in-vitro SUMOylation of simple mixtures. We are now analyzing more complex mixtures.
Purpose/use/application of the technique:
Post translational modification (PTM) of proteins plays a key role in the control of a wide range of biological functions and activities. Thus, development of technologies for the rapid and conclusive identification of PTMs is essential. Modified proteins can be difficult to detect because they are often present in sub-stoichiometric amounts compared to the unmodified protein, and the chemical nature of the modification can hinder mass spectrometry analysis. By enriching the sample for the specifically modified form of the protein/peptide, the chances of identifying the modified peptide during mass spectrometric analysis are greatly improved (numbers 1 and 2 above). In addition, computational approaches are being developed that are tailored to the analysis of the unique MS/MS fragmentation patterns generated by post-translationally modified peptides (number 3 above). When combined with stable isotope labeling, the techniques can provide information about how the modification state changes when cells are exposed to different stimuli. This type of information greatly enhances our understanding of specific biological events.
Example(s) of projects at ISB that use this technique:
- Serum profiling - H. Zhang
- Prostate cancer metastasis- B. Lin
- Sumoylation- B. Raught, J.Aitchison
- T cell receptor activation-B. Wollscheid, A. Tao., R. Aebersold
Ongoing area of technology development:
We continue to make improvements in identification of phosphopeptides and sites of phosphorylation. We are developing a procedure that consists of the following steps:
- Peptide mixtures are methyl esterified to block carboxylates from further reaction in subsequent steps, and to introduce stable isotopes for quantitative analysis.
- Methylated peptides from two samples are combined, phosphate groups are activated using carbodiimide and imidazole, and reacted with excess polyamine to form phosphoramidate bonds. We are using a dendrimer as the solid support containing polyamine because it has a well characterized and uniform structure and has been used in other biological applications.
- Covalently bound phosphopeptides are eluted by acid hydrolysis of the phosphoramidate linkage and recovered for MS analysis.
This streamlined procedure represents a significant improvement over our previously published strategy, and in a preliminary study of dynamic changes of phosphorylation during T cell activation, we have identified and quantified 75 sites of tyrosine phosphorylation.
We are continuing to improve the sensitivity and specificity of the glycocapture technique.
Molecular genetic approaches are being developed to enhance detection of sumoylated and ubiquitylated peptides.
Representative publication(s):
Zhang, H., Li, X. J., Martin, D. B., and Aebersold, R. (2003). Identification and quantification of N-linked glycoproteins using hydrazide chemistry, stable isotope labeling and mass spectrometry. Nat Biotechnol 21, 660-6. Epub 2003 May 18.
Zhang, H., Yi, E. C., Li, X. J., Mallick, P., Kelly-Spratt, K. S., Masselon, C. D., Camp, D. G., 2nd, Smith, R. D., Kemp, C. J., and Aebersold, R. (2005). High throughput quantitative analysis of serum proteins using glycopeptide capture and liquid chromatography mass spectrometry. Mol Cell Proteomics 4, 144-55. Epub 2004 Dec 17.
Zhou, H., Watts, J. D., and Aebersold, R. (2001). A systematic approach to the analysis of protein phosphorylation. Nat Biotechnol 19, 375-8.
Pedrioli PG, Raught B, Zhang XD, Rogers R, Aitchison J, Matunis M, Aebersold R., Automated identification of SUMOylation sites using mass spectrometry and SUMmOn pattern recognition software. Nat Methods. 2006 Jul;3(7):533-9. PMID: 16791211 [PubMed - indexed for MEDLINE]
Nat Methods. 2005 Aug;2(8):591-8.
Comment in:
Nat Methods. 2005 Aug;2(8):579-80.
Quantitative phosphoproteome analysis using a dendrimer conjugation chemistry and tandem mass spectrometry.
Tao WA, Wollscheid B, O'Brien R, Eng JK, Li XJ, Bodenmiller B, Watts JD, Hood L, Aebersold R., The Bindley Bioscience Center and Department of Biochemistry, Purdue University, West Lafayette, Indiana 47907, USA.
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