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Mass Spectrometry Unit

Samples submission: Guidelines for Proteomics sample submission/prep

PROTEIN PHOSPHORYLATION DETECTION/LOCALIZATION:

Phospho site localization depending on amount of phosphopreotein you supply - i.e., a visible coomassie band = above 10 nanograms of phosphorylated proteins. Samples submitted for mass spectrometry analysis can come from either:

• SDS gels
• liquid solutions

PROTEIN PHOSPHO-ANALYSIS FROM GEL:

1. Reduce contamination from keratin & other proteins. Here's how:
   Tips to avoid keratin contamination in gel and sample preparation - Feb 08 [.ppt, 3.7 Mb]
2. Use MS-compatible staining procedures: Coloidal coomassie stains rather than Silver (Ag). If you can not visualize bands with Coomasie try to scale up your isolation to increase the amount of protein. Ag stain, with its lower detection limit may not contain sufficient protein for MS detection. Ag ions (a) inhibit trypsin digestion by binding at the active site of trypsin and chymotrypsin. (b) in the MS process, they induce ion suppression & background interference. Sypro Ruby gives better mass spectrometry results and is easier to use.

If you do want to use silver stain make sure to use the MS Facility Silver stain protocol.

• Colloidal coomassie stain protocol
• Silver stain protocol
• Sypro Ruby protocol

PROTEIN PHOSPHO-ANALYSIS FROM LIQUIDS:

Reduce contamination from keratin & other proteins. Contamination, primarily in the form of proteins present on the fingers and hair of investigators as well as dust in the lab should be avoided. Some general recommendations:

• always wear gloves when handling samples
• the buffer should contain no urea, no detergents
• see Protein precipitation protocols for cleaning samples

References/Usefull literature:
Phospho enrichment done in our MS Facility: Heegaard, H.H. et al. (2007) Mol.Cell. Proteomics, 6(10) pg.1778