MadSci Network: Molecular Biology

Re: How ,do bilogists determine the amino-acid sequence of a protein molecule?

Date: Fri Sep 22 00:53:02 2006
Posted By: Sebastien De Landtsheer, Undergraduate, Immunology, Laboratoire National de Santé
Area of science: Molecular Biology
ID: 1158789277.Mb


These days, the amino-acid sequence of a protein is inferred by the nucleic
acid sequence. Sequencing a DNA is indeed far more easier than sequencing a
protein. Once you know the DNA sequence, usually there is no problem
knowing the protein squence.
For the technique concerning DNA sequencing, I can advise you to read here

The genetic code is univocal knowing the organism you work with :

To answer your question, there are indeed ways to know directly the
sequence of a protein. First the protein is analysed interms of weight and
composition. This is done by running the protein in acrylamide gels :

Then there are two techniques : 
1) the chemical (and old) one, which is, to my knowledge, never used
anymore these times : it is called the Edman reaction : with a certain
chemical, you digest (remove) the last amino-acid of a chain, which is then
identified (liquid chromatography). Then you repurify your protein and you
do the reaction again, and this provides you the nature af the second
amino-acid. This is pure organic chemistry :

2) Mass spectrometry : here you have two possibilities (in terms of
ionisation) : MALDI and electrospray. The protein is first digested with
different endoproteases (trypsin for exemple), then the peptides are
ionised, selected and fragmented. The filiation of the fragments provides
you the sequence. For the technical explanation (which are very difficult)
about mass spectrometry I advise you this site :

Hope this helps

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