023Mass Spectroscopy

in this lesson on protein structure from chapter 4 we like to look at mass spectroscopy this is a method of protein identification used to determine primary structure in a previous video lesson we saw that Edmunds sequencing was a chemical means of determined primary structure that is the sequence of amino acids within a peptide or protein in this case we're going to use more of a physical method as we'll see the molecule is first sprayed through a capillary tube and that enables it to form gas phase ions in the illustration at the bottom of our slide here here's our protein as this green sphere we're going to spray it through a capillary tube and that gives us these gas phase ions our first instrument ms1 sorts the ions so that only one emerges so we only have one selected that we're going to analyze each time the selected peptide is then sent through a collision chamber so that it's bombarded by an inert gas and that fragment sent in two different fragments of ions they tend to break at the peptide bonds and so each of these fragments differs by the number of amino acids present from the collision chamber these fragmented ions are then sent through our second instrument ms2 and that measures the charge to mass ratio of the pieces and we get a mass analysis so again we have different fragments corresponding to the presence of the different amino acid so each of these fragments is going to differ by a single amino acid and we'll see how that works to help us to determine the sequence it's important to keep in mind here this represents a physical breakage of the peptide and that allows us to detect any modifications to the protein remember with Edmonds sequencing we had to have a free amine terminus and it had to be deprotonated in order to carry out that analysis in this case the peptide can be modified in any way and because it's a physical breakage we can still analyze it what we get then from computer analysis is a printout that would look something like this at the bottom of the slide here the instrument measures the charge to mass ratio and we're going to determine the sequence by comparing the masses of increasingly larger fragments that differ by one amino acid so the difference in mass of successive Peaks identifies each of the residues let's see how that works in our illustration here the charge to mass ratio or mass is on our x-axis increasing from left to right so our smallest fragments are on the left and our largest are on the right on the y-axis we have the counts that is the prevalence of those fragments we're going to do this a number of times and we're going to get an averaging of the fragments that we see our first fragment our smallest one would correspond to a single amino acid we measure that mass and we find 71 point two and since we know the mass of the individual amino acids we know that corresponds to the amino acid alanine now we know that the first amino acid our peptide is Alan e the second-largest fragment would contain two amino acids we take the mass of that fragment subtract from it the mass of alanine that we already know seventy one point two and that difference is ninety seven that corresponds to the amino acid prolene now we know that the sequence of our peptide is alanine prolene and so in a similar way we can analyze each of the successively larger fragments to determine what's the next amino acid within the sequence so here we have two ways of determining the primary structure one a chemical method that's Edmond sequencing and the second a physical method mass spectroscopy so just keep in mind the differences between these two methods this concludes our studies in Chapter four we'll begin our studies in Chapter five with our next video lesson where we'll consider the prosthetic group that is at the core of oxygen binding proteins and we also want to consider how the group is bound within these proteins

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