期刊
JOURNAL OF THE AMERICAN SOCIETY FOR MASS SPECTROMETRY
卷 30, 期 6, 页码 919-931出版社
SPRINGER
DOI: 10.1007/s13361-019-02159-w
关键词
Ion mobility spectrometry-mass spectrometry; Peptide conformation; Dissociation kinetics; Proline isomerization; Penultimate proline
资金
- National Institute of Health [R01 GM121751-03]
- Robert and Marjorie Mann Chair
- NSF [CHE-1707675]
- NIH [P41GM121751-01A1]
- Indiana University College of Arts and Sciences
We report ion mobility spectrometry and mass spectrometry studies of the non-enzymatic step-by-step degradation of substance P (subP), an 11-residue neuropeptide, with the sequence Arg(1)-Pro(2)-Lys(3)-Pro(4)-Gln(5)-Gln(6)-Phe(7)-Phe(8)-Gly(9)-Leu(10)-Met(11)-NH2, in ethanol. At elevated solution temperatures (55 to 75 degrees C), several reactions are observed, including a protonation event, i.e., [subP+2H](2+) + H+[subP+3H](3+), that appears to be regulated by a configurational change and two sequential bond cleavages (the Pro(2)-Lys(3) peptide bond is cleaved to form the smaller nonapeptide Lys(3)-Met(11)-NH2 [subP((3-11))], and subsequently, subP((3-11)) is cleaved at the Pro(4)-Gln(5) peptide bond to yield the heptapeptide Gln(5)-Met(11)-NH2 [subP((5-11))]). Each of the product peptides [subP((3-11)) and subP((5-11))] is accompanied by a complementary diketopiperazine (DKP): cyclo-Arg(1)-Pro(2) (cRP) for the first cleavage, and cyclo-Lys(3)-Pro(4) (cKP) for the second. Insight about the mechanism of degradation is obtained by comparing kinetics calculations of trial model mechanisms with experimental data. The best model of our experimental data indicates that the initial cleavage of subP is regulated by a conformational change, likely a transcis isomerization of the Arg(1)-Pro(2) peptide bond. The subP((3-11)) product has a long lifetime (t(1/2) 30h at 55 degrees C) and appears to transition through several structural intermediates prior to dissociation, suggesting that subP((3-11)) is initially formed with a Lys(3)-trans-Pro(4) peptide bond configuration and that slow transcis isomerization regulates the second bond cleavage event as well. From these data and our model mechanisms, we obtain transition state thermochemistry ranging from H-double dagger=41 to 85kJmol(-1) and S-double dagger=-43 to -157Jmol(-1)K(-1) for each step in the reaction.
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