| 1D NanoLC ESI MS/MS analysis for Protein Identification |
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The sample is digested with trypsin and loaded onto a Capillary LC system coupled to an MS/MS instrument. The peptides are separated using a reverse phase C18 column and directly eluted into the MS. 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). Where the protein of interest is not in the database, de-novo sequencing and Blast searching can be used to identify peptides similar to those in known proteins.
Recommended for:
Advantages:
- High confidence identification of proteins
- Powerful separation, concentration and sample clean-up prior to MS/MS analysis
- Ability to identify numerous proteins in a single run
- Can be coupled to other chromatography steps upstream
Disadvantages:
- Due to automated nature, some fragment spectra not ideal
- Relatively slow on a per sample basis when compared to MALDI-TOF or MALDI-TOF/TOF MS
For further details or advice on this APAF service please contact us at ms@proteome.org.au. | |
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