Research On The Role Of Acidic Amino Acid On Peptide Fragmentation In Mass Spectrometry

With the unremitting efforts of scientists, the Human Genome Project proposed in the early1990s has been basically completed. However the post-genomics era is arriving, and the study of the proteome is a very important content in it. To carry out this important research, you need to determine the amino acid sequence composition, structure and function of each protein.With the rapid development of biological mass spectrometry in recent years, it has become an indispensable identification techniques of the determination of protein’s amino acid sequence. If using MS to measure the related protein sequence, protein was required to be enzymatic with protease (for example, the trypsin can hydrolyze Lys-or Arg-C-terminal peptide bond) into a peptides, and then these peptides were identified and analyzed. Although scientists have established a peptide mass spectrometry database, in the realistic determination and analysis, often a strong fragmentation peak or unconventional ion is encountered. The role of acidic amino acids in the cleavage of peptide mass spectrometry has not been elucidated. Therefore, categoring the peptides to study the cleavage of a certain class of peptide can provide a scientific basis and theoretical support to further improve the peptide mass spectrometry database, to broaden the application of mass spectrometry in peptide and proteomics research.In this paper, we selected the peptides containing acidic amino acids, and then studied the role of acidic amino acids in the peptide cleavage respectively by oxidation and changing the type or location of the amino acid. And we also study peptide cleavage differences around the modification of the N-terminal acetylation and C-terminal amidation of the peptides containing acidic amino acid. In the mass spectrometry experiments, the differences in peptide cleavage around the peptide acid change and endpoint modification were evaluated respectively through the mass spectrometry efficiency curve and the MS/MS spectra; in the quantum chemical calculations, the charge distribution on the main-chain amide bond atoms was also studied in the peptide acid change and endpoint modification; associating experimental results and the charge distribution of theoretical calculations to explore the role of acidic amino acids in the peptide mass spectrometry cracking process. The dissertation consists of the following three parts.In the first part, With easily oxidized amino acids-cysteine peptide as the object, the changes of peptide cleavage efficiency before and after oxidation were explored by mass spectrometry and quantum chemical calculations. It was shown in the mass spectrometry tests that, species plus oxygen cleavage more easily than the original peptides after cysteine oxidation, and with the degree of oxidation becoming deeper (that is, the acidic more strong), the peptides cracked more likely. This was also confirmed by the charge distribution results obtained by quantum chemical calculations. For the peptide RGDC, with the deepening of the oxidation degree, the Qo/QN value of amide bond3which was the main breakpoint has been increased, this was also to say the proton attraction of the nitrogen atom on the amide bond become stronger than the oxygen atoms and it contribute to the occurrence of cracking reactions; for GRCG and its derivatives, with the deepening of the degree of oxidation, the Qh/Q(o+N) values of amide bonds have been reduced, suggesting that protons can flow more easily to the amide bonds, and it will promote the cleavage reaction.In the second part, Mass spectrometry experiments and quantum chemistry calculation methods were utilized to study the changes in the peptide cleavage with the change of amino acids in the peptide specific location (sample peptide is LAXSV, X=D, E, C) or the change of the same amino acid position in peptides (the amino acid which is studied here is glutamate, respectively ELASV, LEASV, LAESV, LASEV and LASVE). It has been shown in the mass spectrometry tests that peptides becomes more difficult cracking with the change of amino acids by aspartic acidâ†'glutamic acidâ†'cysteine in the specific location; and peptides became harder to crack with the glutamic acid position from the N-terminal to the C-terminal by degrees. And also in the charge distribution, with the change of amino acids by aspartic acidâ†'glutamic acidâ†'cysteine in the specific location, the QN values of amide bonds have been increased, and this indicates that the proton flow more difficult to the nitrogen atom on the amide bonds; with the glutamic acid position from the N-terminal to the C- terminal the Qo/QN values of amide bonds becomes smaller, and it can be shown that compared with the oxygen atom, proton flow more difficult to the nitrogen atoms of amide bond. Both of these cases will make the peptide becomes more difficult to fragment.In the third part, the changes in peptide cleavage under the modification of peptides endpoint-N-terminal acetylation or C-terminal amide were discussed by a platform of mass spectrometry experiments and quantum chemical calculations. The peptides studied in this part were GRCG, and LAXSV (X=D, E, C) before and after the N-terminal acetylation and C-terminal amidation.It was indicated in studies that the peptide crack more easily with the N-terminal acetylation; however the cleavage of the peptide has no change or become more difficult after the C-terminal amidation. The above conclusion has also been strongly support by the charge distribution changes in the amide bond. For the peptide with N-terminal acetylation the QN values of amide bonds have increased, indicating that the nitrogen atom attract protons easier, and promoting peptide cleavage. For peptides with C-terminal amidation the QN values of amide bonds have no change or decreased, indicating that the ability of the nitrogen atom to attract protons did not change or became more difficult, so that the peptide cleavage has no change or become harder to cracking.The research results show that under oxidizing conditions, the increase of the peptide acidity can make proton flow more easily to the amide bond or its nitrogen atom so as to promote the cleavage of the peptide. With the direct change of the amino acids, the acidity enhancement makes the nitrogen atom in amide bond more reject protons, and this will make the peptide more difficult to crack. N-terminal acetylation can promote the cleavage of the peptide, but the C-terminal amidation contrast.