Re: how are proteins sequenced?

How are proteins sequenced?
The short answer is the most proteins are sequenced by locating the gene for that protein and then sequencing the DNA. This is because DNA sequencing is cheaper and easier than chemically sequencing the protein.

However, I think that what you were most likely asking about is sequencing the protein by chemical means. As you may know proteins are composed of amino acids that are linked together by peptide bonds. A peptide bond is formed between the final carbon of the one amino acid and the beginning nitrogen of the next amino acid. Therefore all proteins begin with a free nitrogen and end with a free carbon. The basic strategy of protein sequencing is to use a chemical that binds to either the free nitrogen (N-terminal sequencing) or the free carbon (C-terminal sequencing) at neutral pH. At acidic pH, the chemical attacks the peptide bond between the amino acid residue that it has bonded with and the next amino acid in the chain and actually breaks that bond. The modified amino acid is then separated from protein and identified using high pressure liquid chromatography.

There are actually machines that do most of the work. Machines using N-terminal sequencing are most common. The specific chemistry that is used to cut up the protein is called Edman chemistry and you can find out the specifics of the reaction in any undergraduate level biochemistry book or an organic chemistry book.

N-terminal sequencing normally requires that the protein be cut up into peices with proteases. Proteases are proteins that cut up other proteins. Optimally, you want to cut your protein into pieces of about 30 amino acids, because that is the size that most automated sequencing machines can handle. Sometimes the first residue of a protein is modified and there isn't a free nitrogen to start the sequencing process with, in that case you can either use C-terminal sequencing or Mass Spectrometry.

Mass Spectometry is a technique that breaks the protein up into fragments that can be identified by their mass and charge. You compare the pattern of fragments optained from mass spectrometry with a database of sequences and hope that your sequence matches one in the database. For that reason, the smaller the peice of protein that use mass spectrometry on, the more likely you are to deduce its sequence.

Why can't you sequence protein using chromatography?
Its easy to identify individual amino acids using chromatography because they have very different chemical properties. While the chemical properties of a protein depend on the entire sequence, you can't deduce the sequence of a protein from its chemical properties because there are many different sequences that would give you the same chemical property. By chemical properties, I am referring to things like mass, total charge, stickiness (hydrophobicity) which are commonly used to separate proteins on various chromatographic columns. Chromatography is also used to purify the peices of protein once you have cut it up with proteases so that you have a very pure sample of each peice for doing the chemical sequencing.

Clare