Introduction to Peptide Sequencing

Introduction to Peptide Sequencing


I’d like to talk a little bit about peptide
sequencing. Let’s say we’ve got a peptide. This one has five amino acids making it up,
so it’s a pentapeptide and what we can do is we can figure out what the structure of
the peptide is if we can order these or find the order of these amino acids in the peptide. That’s called primary structure. Now what you might want to do is say, okay,
why don’t we just go ahead and hydrolyze everything. That is we just break apart these peptide
bonds and we can do that if we use very strong acid and heat. We can break them up into their component
amino acids, but unfortunately if we do that, why we can figure out what the amino acids
are by ion exchange chromatography, we’ll never know what the order was in the original
peptide. So, what we’re going to do is look at some
ways that we can manage that and we can do that using first of all the Sanger reagent. What the Sanger reagent will do is it will
react with the end, the amino end of the peptide, which is the first amino acid in the chain
and it will break that amino acid off the chain and then you’re left with this kind
of compound, which will actually fluoresce at a unique frequency depending upon the amino
acid that’s connected to this grouping. So, what that allows you to do is actually,
we’re looking at the fluorescence frequency, will tell you what is connected at this point,
which will tell you what was on the end of the peptide. That will tell you what was first in the peptide
chain. So, the Sanger reagent will allow us to do
that. Cyanogen bromide is a chemical treatment that
allows you to cut an amino acid peptide chain specifically after methionine. So, if you reacted this peptide with methionine,
it would break after methionine leaving this peptide plus valine. Just to give you a very brief look at the
reaction itself, you can see it’s a chemical process that specifically interacts with the
methionine side chain. That’s what the side chain looks like here. And then it breaks the peptide apart at methionine
but just after the methionine specifically. Right, there’s two others I want to talk
about and those are chymotrypsin and trypsin and these are enzymes and they cut specifically
after certain amino acids in the structure. Chymotrypsin will cut after Phe, Trp, and
Tyr. So, it will be phenylalanine, tryptophan,
and tyrosine, specifically after those. Just to give you a very brief explanation
on what these do as enzymes. If you’ve got an enzyme here, it’s going
to take some sort of substrate and the substrates in this case involve these particular amino
acids for chymotrypsin and phenylalanine, tryptophan, and a tyrosine and you get this
activated complex. The thing though is that the enzyme has a
specific shape in this what’s called the active site and it will only take certain
things at certain shapes. I want to show you, I want to show you what
this looks like with the, with regard to the amino acids. And let’s look at phenylalanine, tyrosine,
and tryptophan and I want you to see that the side chains are fairly similar in structure. So, those are the ones that will specifically
fit into this hole in the enzyme. So, then what happens is that the enzyme will
cause this to break apart and then you get the enzyme back in its original form plus
products from the reaction and the products from this particular process will be the peptide
chains that are broken apart. So, this will cut specifically after these. So, there’s a Tyr, it will cut after that. You’ll be left with these two peptide chains
as products. Now trypsin is specifically used to cut after
Arg and Lys. So, arginine and lysine. Let me show you what that looks like and arginine
and lysine are down here and you can see that those two side chains look fairly similar
as well and that works pretty much in the same way, except now these are specific for
arginine and lysine residues after the end and then will cut after those in the chain. So, here’s an Arg here, so it will cut after
the Arg and you’ll be left with these two, these two peptides right here. Alright, so that’s what the, that’s what
all that stuff will tell you and we can use this information to actually sequence a longer
peptide and I’ll show you an example of that in a minute.

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