Protein Identification by 1D nLC-MS-MS

Reverse Phase Chromatography

There are two services available for the identification of proteins from either gel bands or solutions. The Standard service uses a 4000 QTRAP for analysis and the Enhanced service uses a LTQ Orbitrap for analysis. The difference between the services therefore relates to the capabilities of the ms systems and their ability to identify proteins in increasingly complex mixtures..

For single SDS-PAGE gel bands of reasonable intensity by colloidal coomassie then our Standard service using our 4000 QTRAP can be used. For gel bands of high molecular weight or low intensity then the Enhanced service would be more suitable. Also for complex samples such as gel sections or solution samples then our Enhanced service using our LTQ Orbitrap system is again recommended..

Sample Process:

1D band/2D spot samples are processed in a laminar flow cabinet, using our optimised in-gel digestion protocol, in order to minimise dust contamination (i.e. keratin). The digests are then extracted prior to analysis by 1D nLC-MS/MS either using our Standard service (4000 QTRAP) or Enhanced service (LTQ Orbitrap). The resultant data is then processed using the Mascot search engine and a Mascot report produced that can be emailed to the user or provided as hardcopy.

Protein samples in solution can be precipitated with TCA then re-suspended in Urea, reductively alkylated and then digested with trypsin in solution prior to analysis by 1D nLC-MS/MS. The digests can then be analysed using either service (Standard or Enhanced) prior to Mascot database searching. The resultant Mascot report can be emailed to the user or provided as hardcopy.