2D LC ESI MS/MS Analysis for Protein Identification
The sample is digested with trypsin. The digest is then loaded onto a strong cation ion exchange (SCX) column. The fractions from the SCX chromatograph are collected and further separated using a reversed phase C18 column which is coupled with an ESI mass spectrometer. The ions entering the MS are continuously sampled and when a peptide is detected, the MS switches to MS/MS mode and automatically fragments the peptide. When the run is completed, software (e.g Mascot) is used to interrogate protein, DNA or EST databases and identify the protein(s).
Recommended for:
- Complex samples from body fluids, tissues or cells requiring fractionation prior to 1D nanoLC ESI MS/MS analysis
Advantages:
- No need to run 1D or 2D gels before the analysis
- High sensitivity
- Powerful separation, concentration and sample clean-up prior to MS/MS analysis
- Identify numerous proteins in a single sample
Disadvantages:
- Due to automated nature, some fragment spectra not ideal
- Relatively slow on a per sample basis
- Generate enormous amount of data for interpretation
For further details or advice on this APAF service please contact us at ms@proteome.org.au.