How do scientists determine the kinds and numbers of amino acids in a protein?

Question

How do scientists determine the kinds and numbers of amino acids in a protein?

Answer 1

This is a general question, so here is a general answer.

In the old days they needed to figure out what the stuff ways.  It was a lot of biochemistry to determine that proteins were made up of specific kinds of amino acids, just a limited repertoire.  Edman degradation can be used to do this.

Then of course is the whole story of how the DNA code was broken and the DNA-RNA-protein dogma was understood.  The bottom line for your question is that there are three DNA bases that specify each amino acid.  It is a degenerate code, so sometimes more than one group of three will specify the same amino acid.  But, once the hard part was done, then it turns out that you just need the DNA sequence to figure out the kinds and numbers of amino acids in a protein.  In Eukaryotes, things with a nucleus, you sometimes have to worry about where the coding regions begin and end.  The coding regions are called exons, and then there are spacer regions of variable size called introns which separate the exons.

Once you have the sequence, then there are ways to figure out where the proteins start and end.  All (actually it's mostly all but there are exceptions), proteins start with a methionine codon, which is ATG.  And there are three stop codons.  Starting with that initial group of three bases you just group the rest in threes and go until you hit a stop codon.  Again, there are exceptions, but almost all proteins are coded for in this way.

...and the most amazing thing of all, to me at least, is that this is a universal feature of life on our planet.  From viruses to bacteria and lions, tigers and bears, we all use this same system!

Answer 2

First, the protein is completely hydrolyzed to amino acids by heating in concentrated HCl.  Individual amino acids are then separated from each other by chromatography and quantitated.  Standards are used to determine which amino acids migrate how quickly.  Based on the molecular weight of the protein and the ratios of amino acids to each other, the number of each amino acid is calculated.

Answer 3

Today the easiest method, because so many genes (and complete genomes) have been sequenced, it to to predict the peptide sequence based on the DNA sequence.

This method is not 100% accurate. Computer programs can predict the open reading frame of genes, however they are not always correct. Many genes are alternatively spliced, so you can get several different proteins from the same gene (usually tissue specific). Finally, protein prediction does not account for post-translational modifications (such as cleavage) which will change the composition of the final product.

Answer 4

The protein is hydrolyzed by acid and the amino acid content is then analyzed by a machine called amino acid analyzer. That gives you the ratio of the amino acids in the protein. The molecular weight of the protein can be determined by gel electrophoresis. When you have all the results, you can calculate how many each of the amino acids are present.

Answer 5

they use specific enzymes to break down the protein