Identification of intact disulfide links within proteins

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IntroductionDisulfide-Bridges (also called cysteine-linkages) represent one of the most important structural element for protein folding and protein stabilization.
This posttranslational modification is acting also as a molecular switch for the regulation of the enzymatic activity in dependence of the redox state of the cell.
The detection of these disulfide bridges by mass spectrometry were difficult, often, biochemical methods has to be performed before.
The introduction of ETD fragmentation allowed the fragmentation of the crosslinked peptide specifically at the C-C bond, resulting in the release of the individually peptides. The sum of the masses of the single peptide has to fit to the mass of the selected precursor ion.
Up to now this calculation procedure was not fully automated. Several hundred or thousands of different spectra has to be inspected manually: A time consuming work...
At this point, our developed software screens unlimited numbers of spectra for the above described rules and illustrates the found hits.

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Electron Donator in the ETD fragmentation process

Workflow Overview
1.) Sample Preparation -> 2.) MS-Measurement -> 3.) Identify uncrosslinked peptids -> 4.) Prepare raw data -> 5.) Identify crosslinked peptids


Details
1.) Sample Preparation

  • Isolate your protein or protein complex
  • Avoid the use of any reducing agent like DTT, mercapto-ethanol or TCEP
  • Add iodacetamide (100mM) to inhibit reorientation of the C-C links
  • Digest your protein with an appropriate protease in solution or into SDS-gel.
    This is the most critical step, because none reduced proteins show some "resistance" to the action of proteases (because of their retaining 3D structure by the C-C). To overcome this effect, add some denaturating agents like acetonitrile. But take care,
    - that none of the agents interfere with the chromatography conditions (see below) !
    - that the action of the protease is not limited.


2.) MS-Measurement

  • Apply the sample to the LC-MS
    Attention: Crosslinked peptides are in the most cases larger than normal peptides. Be sure that all aggregates can pass through the column by choosing a HPLC material with an extended inner diameter like 300A particles.
  • For MS data, choose a mode with the best mass accuracy
  • For MSMS data choose ETD as fragmentation method (essential !!)
  • For MSMSMS data, choose CID fragmentation of the 4 most intensiv peaks of the ETD fragmentation.


3.) Identify uncrosslinked peptids/protein

  • First, perform a database search to identify the used protein using ETD parameters. Normally you will identify the cysteine free peptides.
  • If not the case, the digestion of the protein or its amount was not sufficient. Restart with sample preparation.......
  • If the protein identification was successful, note the accession number of the protein(s) in the used database


4.) Prepare raw data

  • Bruker Software
    • Create compounds and deconvolute them.
    • Export all compounds with MS, MSMS data and MS3 spectra as an xml file. This file can be used for search the cysteine crosslinkes.

  • Xcalibur Software
    • The raw data file can be used directly as data import.

  • other
    • Convert your raw data to an mzML file, which is suitable for data import.



5.) Start the Identification

Start >> the web form of the C-C analysing program and choose all the options in the form

Dependent of the amount of data in combination of the used parameters the program needs often a very long time. Limit for keep alive the connection is set to 8h. So, you have to be patient, because several millions possibilities for forming crosslinked peptides has to be calculated often.

Sometimes it is better, to start which low complex request (i.e. only 1 modification type)