Services: Mapping of specified PTMs and sample requirements

Proteins are identified through identification of the peptides unique to a protein. Peptides (with or without PTM), in turn, are identified through fragmentation and determination of the corresponding fragment masses. A protein or protein mixture is digested by trypsin to generate tryptic peptides. Tryptic peptides are analyzed by mass spectrometry in line with reverse phase HPLC. Peptides are separated on an HPLC column and eluted peptides are analyzed in a mass spectrometer: peptide masses (m/z, mass per charge) are determined (MS spectrum), then, each analyzed peptide is fragmented and the corresponding fragment masses are determined (MS/MS spectrum). The MS/MS spectrum of each analyzed peptide is submitted to database search for identification. The peptide sequence and the residue bearing (if any) a specified PTM is identified by comparison of the peptide MS/MS spectrum to the MS/MS spectra generated by software for all tryptic peptides of all database proteins. A unique set of identified peptides (derived from the same parent protein) identifies the corresponding parent protein.

The following services are provided for mapping of specified PTMs:

Service Service Code Sample Requirements
Protein samples are trypsin digested by a customer and the digested samples (ready for LC/MS/MS analysis) are submitted to the core for LC/MS/MS analysis.
400
  • Total protein in a sample: ≥ 0.1 picomole/protein
  • Total protein concentration in a sample: ≥ 0.05 mg/ml
  • Sample should not contain detergents
Dissolved protein samples (in solution) are submitted to the core for both tryptic digestion and LC/MS/MS analysis.
401
  • Total protein in a sample: ≥ 0.1 picomole/protein
  • Total protein concentration in a sample: ≥ 0.05 mg/ml
  • Sample buffer: 50 mM ammonium bicarbonate NH4HCO3 or Tris-HCl, Ph 8.1-8.5
  • Samples should not contain detergents
Protein samples- gel bands/spots are submitted to the core for both in-gel tryptic digestion and LC/MS/MS analysis.
402
  • Gel thickness: 1mm (1mm spacer)
  • Total protein in a sample: a protein band or spot visible by coomassie or Sypro Ruby staining