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).
Complex samples from body fluids, tissues or cells requiring fractionation prior to 1D nanoLC ESI MS/MS analysis
No need to run 1D or 2D gels before the analysis
Powerful separation, concentration and sample clean-up prior to MS/MS analysis
Identify numerous proteins in a single sample
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 firstname.lastname@example.org.