Workflow

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Workflow for MS Full Service

Workflow


In gel digestion/peptide extraction/ZipTip clean-up

Please specify the protease for digestion in the MS service request form.  If not, trypsin will be used as the default protease.

All samples digested at the CGS will be desalted using ZipTip®

MALDI-TOF MS/MS analysis

For protein identification, each sample is first analyzed with MALDI-TOF MS to generate the PMF data (Scanning range: 900-4000 m/z). Then the five most abundant peptides (precursors) that are not on Exclusion List are selected for further fragmentation (MALDI-TOF/TOF) analysis to generate the full scan mass spectrum.

Manual MS/MS service is also available for users who want to look at specific precursors ions.  Please specify in the MS service request form.

Database searching / Results delivery

For protein identification by MALDI-TOF and MALDI-TOF/TOF MS analysis, data will be searched against NCBIprot and SwissProt database using our in house MASCOT searching engine.

Please specify the taxonomies for each sample (Taxonomy List).  Raw spectrum or specific parameter settings for database search is available on request during sample submission.  Late request will not be considered.  The search results in PDF format will be delivered to user via e-mail. 

MASCOT search analysis settings are as follow:
Fixed modification: carbamidomethyl (C); Variable modification: oxidation (M)
MSMS Fragment tolerance: 0.2 Da; Precursor tolerance: 75 ppm
Peptide charge: +1; Monoisotopic

NCBInr is obsolete and replaced by NCBIprot in late August 2016. NCBIprot contains all entries in NCBInr, only removing the gi number from the Fasta file. NCBInr is still available at our centre until Dec 2017.

MASCOT Score Interpretation

MOWSE is a sophisticated scoring algorithm for PMF data. It is developed from the observed distribution frequency of peptides in the source database. It is implemented in the algorithms MASCOT and it has been further transformed into a measure of absolute probability using a probability based scoring approach. For more information about the MASCOT scoring algorithm, please refer to Matrix Science Website http://www.matrixscience.com/help/scoring_help.html.