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FAQs - Characterization of Purified Biomolecules
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[+] How should I provide my sample for mass determination and/or structural analysis?
For this type of analysis, your samples should fulfil these criteria:
- minimal volume: above 5 µl
- minimal recommended concentration: 1-5 pmol/µl (optimal concentration: 10-500 pmol/µl)
- if possible, dissolved in volatile solvents (free of non-volatile buffers, salts, detergents, glycerol, etc.)
- salt and buffer containing samples may be submitted (we will desalt them on ZipTips prior to the analysis)
[+] What will FGCZ provide as results of mass determination experiment (MALDI/ESI)?
The resulting mass spectra will be delivered in .pdf format or as an excel spreadsheet. For ESI data, a list of deconvoluted neutral masses
M will be provided, too. Raw data will be delivered upon request (as .txt file).
No, gel bands cannot be processed for intact mass determination analyses
[+] Which samples can I submit for the identification of disulfide bonds?
The analysis of disulfide bonds can be effectively performed only on purified or low-complexity protein samples.
- Digestion with multiple enzymes may be required.
- recommended amount: 10 µg (minimal recommended amount: 1-2 µg)
All details can be found
here
[+] What will FGCZ provide as a result of the disulfide bonds analysis?
In most cases, the analysis will be performed using LC-MS methods, as described
here. The data will be analysed using the software
Byonics (Protein Metrics).
The report will include .pdf result file depicting MS and MS/MS data of the linked disulfide peptides and, if available, Byonics result file.
[+] Can you identify the N- and/or C-Terminal protein sequence(s)?
The N- and C-Terminal protein identification depends on their protein physicochemical properties.
- The simple analysis of intact proteins via MALDI-ISD (in-source fragmentation) may be successful for purified proteins.
- for N-Terminal analysis, alternative methods based on N-Terminal labelling are implemented (e.g. dimethyl labelling)
- for the large-scale identification of N-Termini (e.g. for identification of protease substrates) we do offer the approach TAILS