Protein analyses offered include in-gel or in-solution protease digestion, chromatographic separation and tandem mass spectrometric analysis of the generated peptides, and interpretation of MS/MS data using software like Sequest, Mascot, and MaxQuant.
The facility also assists especially in applications for the isolation (enrichment strategies like lectin chromatography), detection and characterization of post-translationally modified peptides (e.g. phosphorylation, N- and O-linked glycosylation, oxidation, ubiquitination, cysteine modifications, acetylation/ methylation (+ di- and tri-methylation) and O-GlcNAc modification). Sites of modification are verified by manual inspection of the data.
Please consult facility staff for projects which implements specialized approaches related to proteomic PTM analysis. Also we offer service related to quantitative protein expression profiling (SILAC, N15 labeling, LFQ=label free quantification).
Post Translational Modifications (PTMs)
Protein post-translational modifications play a key role in many cellular processes such as cellular differentiation, protein degradation, signaling and regulatory processes, regulation of gene expression, and protein-protein interactions. Protein phosphorylation is the most commonly studied area of post-translational modification since it plays an important role in intracellular signal transduction and is involved in regulating cell cycle progression, differentiation, transformation, development, peptide hormone response, and adaptation. It has been estimated that one third of mammalian proteins may be phosphorylated and 50% may be glycosylated.
Other modifications our facility is investigating are palmitoylation, ubiquitination, and SUMOylation, along with other important modifications including methylation, dimethylation, acetylation, and C-modifications. These PTMs are determined using mainly the digested forms of the proteins by "bottom-up" analyses on the high mass accuracy and high resolution Thermo Scientific Q-Exactive mass spectrometer.
Small Molecules Quantitation
Small molecule analytical quantitation (drugs, pesticides, phytochemicals, etc.) can be used to measure the amount of a small molecule in complex protein and non-protein mixtures, such as plasma, urine, tablets, food and cell extracts. LC-MS/MS is performed using selected reaction monitoring (SRM) on a TSQ Vantage with samples ranging from clinical research to food and environmental analysis.
Metabolomics
Our lab has the capability to do targeted metabolomics and non-targeted metabolomics. For the targeted application, we use the TSQ Vantage in the MRM mode. For the non-targeted application, we use the Thermo LTQ Q-Exactive. Advanced bioinformatics analysis using the software SIEVE e.g. allows to identify patterns and potential biomarkers.