For pure samples e.g. purified protein/protein complex
- Identification of modification.
- Site localisation of post-translational modifications/synthetic modifications on proteins.
- Stoichiometry of modification e.g. number of phosphorylation sites on a protein.
For complex mixtures e.g. proteins extracted from cell line/tissue
- Identification of post-translational modifications on proteins e.g. phosphorylation, acetylation, methylation.
- Site localisation of modifications.
- If the modification of interest is present at sub-stoichiometric levels, we highly recommend enrichment of the modification prior to analysis. We routinely perform phosphopeptide enrichment. For other types of enrichment, please contact us.
Note: not all proteins in the proteome will be detected within a complex mixture. Protein detection depends on multiple factors such as protein abundance and the protein’s ability to be digested (refer to Protein Identification background information). To obtain information on a specific protein from within a complex mixture, purification/enrichment procedures are required prior to analysis.
For identification of protein modifications, we utilise the SolariX, Orbitrap Elite and Q-Exactive HF mass spectrometers. Both instruments are equipped with a nano-flow LC system with reverse phase column. We offer ETD, ECD, CID and HCD fragmentation.
How to prepare samples for analysis
For analysis of pure proteins/protein complexes:
We require a minimum of 20 µL sample at a concentration of 20 µM. Please provide details of the buffer composition that the sample is provided in. Desalting is required prior to mass spectrometry analysis and this service is provided in-house.
For analysis of complex mixtures e.g. cell lines/tissue:
For in-depth phosphoproteomics, sample preparation is typically performed by the customer and the digested proteins submitted directly for LC-MS/MS analysis either pre- or post-phosphoenrichment. Please refer to background information section in Protein/Peptide Identification for an idea of what this involves. Sample preparation that is tailored to your specific needs can be provided upon request. Please contact us to discuss your needs prior to sample submission.
LC-MS/MS analysis only: we require a minimum of 5 µg phosphoprotein per sample. Note: a desalting step must be performed prior to analysis. We highly recommend using a 4h gradient to maximise the number of phosphopeptides identified.
Phosphoenrichment and analysis: since typically <1 % of peptides detected are phosphorylated, we require a minimum of 200 µg total digested protein. We use either TiO2 or FeIMAC for the phosphoenrichment.
Note: PEG, glycerol and detergent are not amenable to mass spectrometry analysis and must be removed prior to sample submission. Analysis on lower sample volumes/concentrations can be attempted but results are not guaranteed.
To download the sample submission form click here.
Note: sample submission signifies agreement with our terms and Conditions of Service.