CFMP - Methods -> PTM Analysis

PTM analyses

We perform the analyses of various posttranslational modifications (PTMs), for example phosphorylation¹, SUMOylation, gykosylation^2,3, cystein redox state⁴ etc. Please contact us for details and to discuss your specific project. See also the exemplary citations at the end of the page.

Phosphorylations

We can perform large scale/global phosphorylation analysis from complex samples (Figure 1), which always involves a phosphopeptide enrichment step, to account for the low number of phosphorylated peptides compared to unphosphorylated ones (~1%). Moreover, to further reduce sample complexity, offline peptide fractionations (SCX, high-pH) can be employed.

Figure 1 - Workflow for large scale phosphoproteomic screening in complex samples

Beside discovery driven determinations of phosphorylation sites in a global manner, specific phosphosites can be monitored very sensitively using parent mass list-based approaches (Figure 2). Here the masses of phosphopeptides of interest are first calculated in-silico, to allow a subsequent targeted analysis within the mass spectrometer.

Figure 2 - Workflow for mapping specific phosphorylation sites using peptide parent mass lists

Glykosylations

We also perform glykosylation analyses following various strategies. For example, together with the Strahl group we established a protocol, which combined peptide glykosylation, lectin enrichment (ConA) and differential mannosidase treatment, to detect protein O-mannosylation in a large scale manner (see Figure 3 and Winterhalter et al²).

Examplary workflow for
differential O-Mannosylation
analysis

Figure 3 - Workflow of differential O-mannosylation analysis, including peptide deglycosylation, selective mannose peptide enrichment (lectin ConA), dimethyl labeling and differential mannosidase treatment. O-mannosylated peptides are identified by a characteristic shift in retention time, caused by the presence of the mannose group.

Cystein redox states

The analysis of cystein redox states is also in our portfolio. For this we apply differential cystein labeling techniques using common cystein-reactive reagents, like N-ethylmaleimide (NEM) or iodoacetamide (IAA). Differential labeling can either be performed with isotopologues of the same alkylation reagent (e.g. light vs heavy NEM --> see Figure 4), but alternatively also with two different reagents (1. NEM alkylation of free cysteines. 2. Reduction of oxidized cysteines 3. IAA alkylation of released cysteines). Finally, this then allows to determine differences in the oxidative state of cysteine sites in target proteins both sensitively and quantitatively.

Figure 4 - Diagramm depicting the principal workflow of an isotope-based differential cysteine redox state analysis.

References
1. Lin, T. C. et al. (2014) Cell-cycle dependent phosphorylation of yeast pericentrin regulates gamma-TuSC-mediated microtubule nucleation. Elife 3, e02208, doi:10.7554/eLife.02208 - Pubmed
2. Winterhalter, P. R., Lommel, M., Ruppert, T. & Strahl, S. (2013) O-glycosylation of the non-canonical T-cadherin from rabbit skeletal muscle by single mannose residues. FEBS letters 587, 3715-3721, doi:10.1016/j.febslet.2013.09.041 - Pubmed
3. Lommel, M. et al. (2013) Protein O-mannosylation is crucial for E-cadherin-mediated cell adhesion. Proc Natl Acad Sci U S A 110, 21024-21029, doi:10.1073/pnas.1316753110
4. Sobotta, M. C. et al. (2015) Peroxiredoxin-2 and STAT3 form a redox relay for H2O2 signaling. Nat Chem Biol 11, 64-70, doi:10.1038/nchembio.1695 - Pubmed