MASSPEC and N-Terminal Sequencing about

 

Services offered

  • Intact Mass Analysis of proteins and peptides by ESI-MS
  • Automated Edman Degredation (N-Terminal Sequencing)
  • Sample clean-up (micro-desalting) and enrichment

Mass Spectrometry Analysis of Intact Proteins/Peptides

The intact protein or peptide will be  analysed on an AB Sciex Q-Trap 4000 mass spectrometer. Proteins are treated in a variety of ways depending on the sample and the MS method used.  After running the mass spectrometer on the intact sample the charged states are de-convoluted (reconstruted) and the result is reported as a mass.

Recommended for:

The accurate assignment of a protein/peptide mass

Advantages:

Highly accurate mass information

Disadvantages:

Generally larger amounts of sample are required
The protein must be pure (>90%)
The sample must be in solution
Not all proteins are amenable to MS analysis
Highly glycosylated, or variably glycosylated, proteins can be difficult to analyse

N-Terminal Sequencing

The microsequencing (Automated Edman degradation) is a method of sequencing amino acids in either a peptide or a protein. Phenylisothiocyanate is reacted with an uncharged N-terminal amino group, under mildly alkaline conditions, to form a cleavable derivative of N-terminal amino acid. Then, after consecutive transformations the more stable phenylthiohydantoin (PTH) amino acid derivative is formed. That PTH amino acid derivative can be identified by using chromatography or electrophoresis. The procedure can then be repeated again to identify the next amino acid. A major drawback to this technique is that the peptides being sequenced in such manner cannot have more than 50 to 60 residues.

Edman degradation is accomplished on a Shimadzu PPSQ 31A protein sequencer. The proteins may be provided in either solution or as a stained band on a PVDF membrane derived from an electroblot. At least 50 pmoles of protein are necessary to run 10-15 cycles. For extended number of cycles we recommend 100 pmoles of homogeneous protein. Sequencing of short peptides require 100-1000 pmoles.