Precursor Ion Scanning
This service makes use of state of the art nanoLC technology developed by Dionex/LC Packings and linear ion trap technology developed by Applied Biosystems/Sciex.
The service uses a technique called parent ion scanning (more commonly known as Parents of -79) to detect phosphopeptides prior to generation of tandem MS data that is then used to determine the site of phosphorylation. During collisionally induced dissociation (CID) of the phosphopeptides in negative ion mode the phosphate is released as a PO3- ion (-79) which is then detected by the system.
This reporter ion is then used to identify the originating peptide whose mass is then used to calculate its respective charge states in positive ion mode for tandem MS analysis. E.g. if a phosphopeptide is detected as a [M-2H] ion then the system will calculate the [M+2H], [M+3H] and [M+4H] ions for analysis by tandem MS in positive ion mode.
A summary report is then generated from a comparison of the tandem MS data and the known protein sequence to assign the sites of phosphorylation.
The protein of interest must have good intensity by colloidal coomassie in order to proceed. 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 digested samples are then extracted and used in a 3 stage analysis using our state of the art nLC system (Dionex/LC Packings) coupled to our 4000 QTRAP (Applied Biosystems/Sciex). Stages are as follows: Identification of protein(s) by nLC-MS-MS; Parents of -79 (PO3-) analysis to detect phosphorylated peptides; Data interpretation.
Mascot reports of protein identification(s) and identification of phosphorylation site(s) are provided electronically or as hardcopy.