2 D LC-MS/MS Mass Spectrometry

To achieve greater protein coverage of a complex sample it is recommended to use multi-dimensional separation.The sample is digested with trypsin. The digest is then loaded onto the first phase HPLC separation, this could be either a strong cation ion exchange (SCX) column, or a high pH (HpH) reverse phase column . The fractions from the chromatograph are collected and further separated in the second dimension 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.