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1.
bioRxiv ; 2024 May 10.
Article in English | MEDLINE | ID: mdl-38765996

ABSTRACT

Peptide phytohormones are decorated with post-translational modifications (PTMs) that are crucial for receptor recognition. Tyrosine sulfation on these hormones is essential for plant growth and development1. Measuring the occurrence and position of sulfotyrosine is, however, compromised by major technical challenges during isolation and detection2. We recently introduced a nanopore peptide sequencing method that sensitively detects PTMs at the single-molecule level3. By translocating PTM variants of the plant pentapeptide hormone phytosulfokine (PSK) through a nanopore, we here demonstrate accurate identification of sulfation and phosphorylation on the two tyrosine residues of PSK. Sulfation can be clearly detected and distinguished (>90%) from phosphorylation on the same residue. Moreover, the presence or absence of PTMs on the two close-by tyrosine residues can be accurately determined (>96% accuracy). Our findings demonstrate the extraordinary sensitivity of nanopore protein measurements, providing a new tool for identifying sulfation on peptide phytohormones and promising wider applications to identify protein PTMs.

2.
Anal Bioanal Chem ; 415(14): 2715-2726, 2023 Jun.
Article in English | MEDLINE | ID: mdl-37000211

ABSTRACT

Peptides are an important group of compounds contributing to the desired, as well as the undesired taste of a food product. Their taste impressions can include aspects of sweetness, bitterness, savoury, umami and many other impressions depending on the amino acids present as well as their sequence. Identification of short peptides in foods is challenging. We developed a method to assign identities to short peptides including homologous structures, i.e. peptides containing the same amino acids with a different sequence order, by accurate prediction of the retention times during reversed phase separation. To train the method, a large set of well-defined short peptides with systematic variations in the amino acid sequence was prepared by a novel synthesis strategy called 'swapped-sequence synthesis'. Additionally, several proteins were enzymatically digested to yield short peptides. Experimental retention times were determined after reversed phase separation and peptide MS2 data was acquired using a high-resolution mass spectrometer operated in data-dependent acquisition mode (DDA). A support vector regression model was trained using a combination of existing sequence-independent peptide descriptors and a newly derived set of selected amino acid index derived sequence-specific peptide (ASP) descriptors. The model was trained and validated using the experimental retention times of the 713 small food-relevant peptides prepared. Whilst selecting the most useful ASP descriptors for our model, special attention was given to predict the retention time differences between homologous peptide structures. Inclusion of ASP descriptors greatly improved the ability to accurately predict retention times, including retention time differences between 157 homologous peptide pairs. The final prediction model had a goodness-of-fit (Q2) of 0.94; moreover for 93% of the short peptides, the elution order was correctly predicted.


Subject(s)
Peptides , Tandem Mass Spectrometry , Chromatography, Liquid , Peptides/chemistry , Amino Acid Sequence , Amino Acids/chemistry , Chromatography, High Pressure Liquid
3.
Org Lett ; 25(11): 1907-1911, 2023 03 24.
Article in English | MEDLINE | ID: mdl-36917069

ABSTRACT

Post-translationally modified peptides are important regulating molecules for living organisms. Here, we report the stereoselective total synthesis of ß-1,2-linked l-arabinosylated Fmoc-protected hydroxyproline building blocks and their incorporation, together with sulfated tyrosine and hydroxyproline, into the plant peptide hormone PSY1. Clean glycopeptides were obtained by performing acetyl removal from the l-arabinose groups prior to deprotection of the neopentyl-protected sulfated tyrosine.


Subject(s)
Glycopeptides , Tyrosine , Glycopeptides/chemistry , Tyrosine/chemistry , Hydroxyproline , Sulfates/chemistry , Peptides/chemistry
4.
ACS Chem Biol ; 17(8): 2054-2064, 2022 08 19.
Article in English | MEDLINE | ID: mdl-35867905

ABSTRACT

We studied the mechanistic and biological origins of anti-inflammatory poly-unsaturated fatty acid-derived N-acylethanolamines using synthetic bifunctional chemical probes of docosahexaenoyl ethanolamide (DHEA) and arachidonoyl ethanolamide (AEA) in RAW264.7 macrophages stimulated with 1.0 µg mL-1 lipopolysaccharide. Using a photoreactive diazirine, probes were covalently attached to their target proteins, which were further studied by introducing a fluorescent probe or biotin-based affinity purification. Fluorescence confocal microscopy showed DHEA and AEA probes localized in cytosol, specifically in structures that point toward the endoplasmic reticulum and in membrane vesicles. Affinity purification followed by proteomic analysis revealed peroxiredoxin-1 (Prdx1) as the most significant binding interactor of both DHEA and AEA probes. In addition, Prdx4, endosomal related proteins, small GTPase signaling proteins, and prostaglandin synthase 2 (Ptgs2, also known as cyclooxygenase 2 or COX-2) were identified. Lastly, confocal fluorescence microscopy revealed the colocalization of Ptgs2 and Rac1 with DHEA and AEA probes. These data identified new molecular targets suggesting that DHEA and AEA may be involved in reactive oxidation species regulation, cell migration, cytoskeletal remodeling, and endosomal trafficking and support endocytosis as an uptake mechanism.


Subject(s)
Lipopolysaccharides , Monomeric GTP-Binding Proteins , Animals , Cyclooxygenase 2/metabolism , Dehydroepiandrosterone/metabolism , Lipopolysaccharides/metabolism , Lipopolysaccharides/pharmacology , Macrophages/metabolism , Mice , Monomeric GTP-Binding Proteins/metabolism , Peroxiredoxins , Proteomics , RAW 264.7 Cells
5.
Chem Commun (Camb) ; 53(93): 12528-12531, 2017 Nov 21.
Article in English | MEDLINE | ID: mdl-29116266

ABSTRACT

Activity-based protein profiling has emerged as a powerful tool for visualizing glycosidases in complex biological samples. Several configurational cyclophellitol isomers have been shown to display high selectivity as probes for glycosidases processing substrates featuring the same configuration. Here, a set of deoxygenated cyclophellitols are presented which enable inter-class profiling of ß-glucosidases and ß-galactosidases.

6.
J Med Chem ; 60(15): 6704-6720, 2017 08 10.
Article in English | MEDLINE | ID: mdl-28704052

ABSTRACT

We report the synthesis and biological evaluation of a series of 7-aryl-1,2,4-triazolo[4,3-a]pyridines with mGlu2 positive allosteric modulator (PAM) activity and affinity. Besides traditional in vitro parameters of potency and affinity, kinetic parameters kon, koff and residence time (RT) were determined. The PAMs showed various kinetic profiles; kon values ranged over 2 orders of magnitude, whereas RT values were within a 10-fold range. Association rate constant kon was linearly correlated to affinity. Evaluation of a short, medium, and long RT compound in a label-free assay indicated a correlation between RT and functional effect. The effects of long RT compound 9 on sleep-wake states indicated long RT was translated into sustained inhibition of rapid eye movement (REM) in vivo. These results show that affinity-only driven selection would have resulted in mGlu2 PAMs with high values for kon but not necessarily optimized RT, which is key to predicting optimal efficacy in vivo.


Subject(s)
Excitatory Amino Acid Agonists/pharmacology , Pyridines/pharmacology , Receptors, Metabotropic Glutamate/agonists , Triazoles/pharmacology , Allosteric Regulation , Animals , CHO Cells , Cricetulus , Excitatory Amino Acid Agonists/chemical synthesis , Kinetics , Piperidines/pharmacology , Pyridines/chemical synthesis , Rats, Sprague-Dawley , Sleep, REM/drug effects , Structure-Activity Relationship , Triazoles/chemical synthesis , Tritium
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