You will prepare and submit a term paper on Mass Spectrometry. Your paper should be a minimum of 1250 words in length. The first cluster has a charge state of 2 while the second (m/z=574.3) has +1. The peptide at m/z 574.3 is present in greater amounts. while the peptide at m/z 461.4 could have a more folded conformation (due to higher charge state).
These ions which are at the lowest range of the mass spectrum represent the immonium ions of certain amino acids. Diagnostic amino acids have specific m/z. 72 is the m/z value for the immonium ion of valine. (molecular mass of valine residues is 99.1 Da. the difference (99-72) of ~27 Daltons represents the removal of 1 carbon (M=12) and 1 oxgyen (M=16) and addition of H+(M=1.)
The ions at m/z 175.12 represent the y1” ions or the C terminal of the protein. This is arginine based on the difference in mass of the” fragment (175.12) and 19 (1 oxygen and 3 H which are found in the C terminal end ) to get 156.12, the mass of the arginine residue.
c. After digestion, the peptide fragments that are produced have basic amino-terminal residues, which make them ideal for positive ionization mass spectrometry analysis because they are easily protonated (to produce MH+).
iii. The Table shows that aldolase mutant K238A reacted with DHAP because the final mass differed from that of the calculated and measured masses by 155.2 and 152.8 Daltons respectively. The changes in mass mutant K236A is not enough to indicate a reaction with DHAP.
iv. K236 is the enzyme that is responsible for the aldolase activity because when it was mutated, it lost its ability to form the Schiff base. which is necessary for cleaving F1,6BP to glyceraldehyde-3-phosphate and DHAP ( (Mathews & Van Holde, 1996). K238 is not in the active site because mutation did not affect its ability to react with DHAP.
The gene is then expressed and the translated protein is reacted with DHAP to see if there is Schiff base formation. Tandem mass spectrometry is performed on the mutant and wild type peptides after reacting with DHAP. The non-reactive mutant (with the active site changed) will not have DHAP moiety in its mass spectra compared to the wild type and the mutant that has the non-active lysine residue.