Molecular advances in microbial aminopeptidases

Nandan, Arya; Nampoothiri, K. Madhavan

doi:10.1016/j.biortech.2017.05.103

Cited by 23 publications

(11 citation statements)

References 78 publications

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“…DALDA and dmtDALDA are reported to be enzymatically stable, 35,36 but were observed to be degraded by plasma enzymes in the current study. Peptides are susceptible to enzymatic degradation at both termini by aminopeptidases and carboxypeptidases present in blood 37–40 . Thus, protection by specific structures at both termini of a peptide is essential for its stability.…”

Section: Discussionmentioning

confidence: 99%

Identification of exvivo catabolites of peptides with doping potential in equine plasma by HILIC‐HRMS

Guan

Fay

et al. 2020

Drug Testing and Analysis

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Bioactive peptides pose a great threat to sports integrity. The detection of these peptides is essential for enforcing their prohibition in sports. Identifying the catabolites of these peptides that are formed ex vivo in plasma may improve their detection. In the present study, the stability of 27 bioactive peptides with protection at both termini in equine plasma was examined under different incubation conditions, using HILIC coupled to HRMS. Of the 27 peptides, 13 were stable after incubation at 37°C for 72 hr, but the remaining 14 were less stable. Ex vivo catabolites of these 14 peptides were detected using their theoretical masses generated in silico, their appearance was monitored over the time course of incubation, and their identity was verified by their product ion spectra. Catabolites identified for chemotactic peptide, DALDA, dmtDALDA, deltorphins I and II, Hyp6‐dermorphin, Lys7‐dermorphin, and dermorphin analog are novel. A d‐amino acid residue at position 2 or 1 of a peptide or next to its C‐terminus protected the relevant terminal from degradation by exopeptidases, but such a residue at position 3 did not. A pGlu residue or N‐methylation at the N‐terminus of a peptide did not protect its N‐terminal. Ethylamide at the C‐terminus of a peptide provided the C‐terminal protection from attacks by carboxypeptidases. The C‐terminal Lys amide in DALDA, dmtDALDA, and Lys7‐dermorphin was susceptible to cleavage by plasma enzymes, which is the first report, to the authors’ knowledge. The results from the present study provide insights into the stability of peptides in plasma.

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Section: Discussionmentioning

confidence: 99%

Identification of exvivo catabolites of peptides with doping potential in equine plasma by HILIC‐HRMS

Guan

Fay

et al. 2020

Drug Testing and Analysis

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“…Aminopeptidases are a heterogeneous group of enzymes that catalyze the controlled hydrolysis of N-terminal residues from proteins or peptide substrates. Thus, they play pivotal roles in regulation of protein turnover in nearly all organisms [1][2][3][4]. Many but not all of these enzymes are zinc dependent.…”

Section: Introductionmentioning

confidence: 99%

Phosphonic Acid Analogs of Fluorophenylalanines as Inhibitors of Human and Porcine Aminopeptidases N: Validation of the Importance of the Substitution of the Aromatic Ring

et al. 2020

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A library of phosphonic acid analogs of phenylalanine substituted with fluorine, chlorine and trifluoromethyl moieties on the aromatic ring was synthesized and evaluated for inhibitory activity against human (hAPN) and porcine (pAPN) aminopeptidases. Fluorogenic screening indicated that these analogs are micromolar or submicromolar inhibitors, both enzymes being more active against hAPN. In order to better understand the mode of the action of the most active compounds, molecular modeling was used. It confirmed that aminophosphonic portion of the enzyme is bound nearly identically in the case of all the studied compounds, whereas the difference in activity results from the placement of aromatic side chain of an inhibitor. Interestingly, both enantiomers of the individual compounds are usually bound quite similarly.

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“…DALDA and dmtDALDA are known to be enzymatically stable, but were observed to be degraded by plasma enzymes at ambient temperature over 24 hours. Peptides are susceptible to enzymatic degradation at both termini by aminopeptidases and carboxypeptidases present in blood . Thus, protection by specific structures at both termini of a peptide is essential for its stability.…”

Section: Resultsmentioning

confidence: 99%

“…Peptides are susceptible to enzymatic degradation at both termini by aminopeptidases and carboxypeptidases present in blood. [39][40][41][42] (Table S4). This comparison reveals that the Lys residue at the C-terminus of DALDA is responsible for its instability in plasma − possibly from degradation by carboxypeptidase B.…”

Section: In Vitro Degradation Of Peptides In Equine Plasmamentioning

confidence: 99%

A comprehensive approach to detecting multitudinous bioactive peptides in equine plasma and urine using hydrophilic interaction liquid chromatography coupled to high resolution mass spectrometry

Guan

You

et al. 2019

Drug Testing and Analysis

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Bioactive peptides possess pharmacological effects and can be illicitly used in sports. To deter such misuse, an untargeted method using high resolution mass spectrometry (HRMS) has been developed for comprehensive detection of multitudinous exogenous peptides in equine plasma and urine. Forty‐four peptides were extracted using mixed‐mode solid‐phase extraction (SPE) from plasma and urine, separated with a hydrophilic interaction liquid chromatography (HILIC) column, and detected on an HRMS instrument. Ammonium formate as a mobile phase additive had effects on HILIC retention and charge state distribution of the peptides. The acetonitrile percentage in the reconstitution solution affected the solubility of peptide neat standards and peptides in plasma and urine extracts differently. The stability of the peptides in plasma at ambient temperature was assessed. The limit of detection (LOD) was 10–50 pg/mL for most of the peptides in plasma, and ≤ 500 pg/mL for the remaining. LOD was 100–400 pg/mL for the majority of the analytes in urine, and ≤ 4000 pg/mL for the others. The method was used successfully to analyze incurred plasma and urine samples from research horses administered dermorphin. Even in the absence of reference standards, dermorphin metabolites (aFGYPS‐NH2, YaFG, and YaF) were identified. These results demonstrate that data generated with this method can be retrospectively reviewed for peptides that are unknown at the time of sample analysis without requiring re‐analysis of the sample. This method provides a powerful novel tool for detection of numerous bioactive peptides and their metabolites in equine plasma and urine for doping control.

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Molecular advances in microbial aminopeptidases

Cited by 23 publications

References 78 publications

Identification of exvivo catabolites of peptides with doping potential in equine plasma by HILIC‐HRMS

Identification of exvivo catabolites of peptides with doping potential in equine plasma by HILIC‐HRMS

Phosphonic Acid Analogs of Fluorophenylalanines as Inhibitors of Human and Porcine Aminopeptidases N: Validation of the Importance of the Substitution of the Aromatic Ring

A comprehensive approach to detecting multitudinous bioactive peptides in equine plasma and urine using hydrophilic interaction liquid chromatography coupled to high resolution mass spectrometry

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