Chemical degradation of 3H-labeled substance P in Tris buffer solution

Higa, Toshinobu; Desiderio, Dominic M.

doi:10.1016/0003-2697(88)90215-1

Cited by 11 publications

(23 citation statements)

References 12 publications

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“…At longer incubation times (e.g., 150 min in Figure 1), two new peaks in the mass spectra can be observed at m/z = 548 and 254. These peaks can be assigned to products associated with cleavage of the Pro 2 -Lys 3 The subP (3)(4)(5)(6)(7)(8)(9)(10)(11) product also contains a penultimate proline residue. As the system continues to incubate, (e.g., 2880 min.)…”

Section: Resultsmentioning

confidence: 99%

“…[cKP+H] + -the DKP product that forms by dissociation of subP (3)(4)(5)(6)(7)(8)(9)(10)(11) at the Pro 4 -Gln 5 peptide bond. The anticipated complementary fragment ion of this dissociation corresponding to Gln 5 -Met 11 -NH 2 [subP (5)(6)(7)(8)(9)(10)(11) ] is absent from all of the mass spectra. This is somewhat unsatisfying.…”

Section: Resultsmentioning

confidence: 99%

“…The peptide subP (≥95% purity) was obtained from Sigma Aldrich (St. Louis, MO, U.S.A.). The truncated subP (3)(4)(5)(6)(7)(8)(9)(10)(11) peptide, and several subP analogues involving a range of Pro→Ala substitutions, were synthesized in house, using standard FMOC solid-phase peptide synthesis carried out on an Applied Biosystems 433A Peptide Synthesizer (Applied Biosystems, Foster City, CA). [35] Each purified peptide was dissolved into pure ethanol solutions to concentrations of 500 µM and stored at −22 °C.…”

Section: Experimental Peptide Synthesis and Sample Preparationmentioning

confidence: 99%

“…These studies revealed that efficiency of ionizing and detecting cRP and cKP was low, relative to the larger peptides, as can be observed in the mass spectra presented below. For the kinetics plots shown below, in order to estimate the solution abundances of the different species that are present, we multiply the [subP (3)(4)(5)(6)(7)(8)(9)(10)(11) Methods for investigating proline configurations.…”

Section: Normalization Of Ms Peak Areas In Order To Determine Relativmentioning

confidence: 99%

“…Early studies of subP noted its instability, even when stored at low temperatures. [4,5] A number of processes may be responsible for the loss of function. At mM concentrations subP can form oligomers that are associated through hydrophobic residues near the C terminus.…”

Section: Introductionmentioning

confidence: 99%

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Substance P in Solution: Trans-to-Cis Configurational Changes of Penultimate Prolines Initiate Non-enzymatic Peptide Bond Cleavages

Conant

Fuller

El‐Baba

et al. 2019

J. Am. Soc. Mass Spectrom.

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We report ion mobility spectrometry and mass spectrometry studies of the non-enzymatic step-bystep degradation of substance P (subP), an eleven-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-NH 2 , in ethanol. At elevated solution temperatures (55 to 75 °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-NH 2 [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-NH 2 [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 trans→cis isomerization of the Arg 1-Pro 2 peptide bond. The subP (3-11) product has a long lifetime (t ½ ~ 30 hrs at 55 °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 trans→cis isomerization regulates the second bond cleavage event as well. From these data and our model mechanisms, we obtain transition state thermochemistry ranging from ΔH ‡ = 41 to 85 kJ•mol −1 and ΔS ‡ = −43 to −157 J•mol −1 •K −1 for each step in the reaction.

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