Multiplex substrate profiling by mass spectrometry for proteases

Rohweder, Peter; Jiang, Zhenze; Hurysz, Brianna; O’Donoghue, Anthony J.; Craik, Charles S.

doi:10.1016/bs.mie.2022.09.009

Cited by 8 publications

(6 citation statements)

References 84 publications

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“…To avoid that potential issue, these two sequences were selected; since they were already demonstrated to be competent substrates; the top 30 observed cleavages are presented in File S1 . This is similar to what has been done previously with the MSP-MS platform where the top cleaved peptides were used to generate model substrates; this strategy has been shown to provide optimal substrates for other proteases ( 32 , 33 ). Compared to one replicate of LapX ΔC S319A as a negative control, neither top hit was observed in the catalytically inactive serine to alanine variant ( File S1 ).…”

Section: Resultsmentioning

confidence: 58%

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Proteases influence colony aggregation behavior in Vibrio cholerae

Detomasi,

Batka,

Valastyan

et al. 2023

Journal of Biological Chemistry

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Section: Resultsmentioning

confidence: 58%

“…To determine LapX substrate specificity, LapX FL and LapX ΔC were subjected to MSP-MS ( 32 , 33 ). We reasoned that analysis of LapX ΔC could provide information concerning a role for the C-terminal domain in substrate preference, as this domain is unannotated and its function, if any, is unknown.…”

Section: Resultsmentioning

confidence: 99%

Proteases influence colony aggregation behavior in Vibrio cholerae

Detomasi,

Batka,

Valastyan

et al. 2023

Journal of Biological Chemistry

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“…This approach, termed multiplex substate profiling by mass spectrometry, has been used to generate substrate specificity profiles for more than 80 different proteases. 50 RgpB cleaved 83 out of 2964 peptide bonds after 10 min incubation with the peptide library. We analyzed the sequence of the amino acids surrounding the cleavage site and found that RgpB had a strong preference for cleaving between R and G with the sequence FLVRG (Supporting Information, section 2.4).…”

Section: Resultsmentioning

confidence: 99%

“…Cleavage at any one of the 2964 peptide bonds within this library can be detected and quantified by liquid chromatography–tandem mass spectrometry. This approach, termed multiplex substate profiling by mass spectrometry, has been used to generate substrate specificity profiles for more than 80 different proteases . RgpB cleaved 83 out of 2964 peptide bonds after 10 min incubation with the peptide library.…”

Section: Results and Discussionmentioning

confidence: 99%

A Protease-Responsive Polymer/Peptide Conjugate and Reversible Assembly of Silver Clusters for the Detection of Porphyromonas gingivalis Enzymatic Activity

Retout,

Amer,

Yim

et al. 2023

ACS Nano

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We report the reversible aggregation of silver nanoparticle (AgNP) assemblies using the combination of a cationic arginine-based peptide and sulfur-capped polyethylene glycol (PEG). The formation and dissociation of the aggregates were studied by optical methods and electron microscopy. The dissociation of silver clusters depends on the peptide sequence and PEG size. A molecular weight of 1 kDa for PEG was optimal for the dissociation. The most important feature of this dissociation method is that it can operate in complex biofluids such as plasma, saliva, bile, urine, cell media, or even seawater without a significant decrease in performance. Moreover, the peptide–particle assemblies are highly stable and do not degrade (or express of loss of signal upon dissociation) when dried and resolubilized, frozen and thawed, or left in daylight for a month. Importantly, the dissociation capacity of PEG can be reduced via the conjugation of a peptide-cleavable substrate. The dissociation capacity is restored in the presence of an enzyme. Based on these findings, we designed a PEG-peptide hybrid molecule specific to the Porphyromonas gingivalis protease RgpB. Our motivation was that this bacterium is a key pathogen in periodontitis, and RgpB activity has been correlated with chronic diseases including Alzheimer’s disease. The RgpB limit of detection was 100 pM RgpB in vitro. This system was used to measure RgpB in gingival crevicular fluid (GCF) samples with a detection rate of 40% with 0% false negatives versus PCR for P. gingivalis (n = 37). The combination of PEG-peptide and nanoparticles dissociation method allows the development of convenient protease sensing that can operate independently of the media composition.

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“…In conclusion, the revealed temperature tolerance and catalytic properties of globupain render it as a promising protease in diverse industrial and biotechnology sectors. Further studies focused on indepth knowledge of the substrate specificity (O'Donoghue et al, 2012, Rohweder et al, 2022, effects of protease inhibitors and resistance to organic solvents and chemical denaturants may provide a deeper understanding of the applicability of globupain.…”

Section: Discussionmentioning

confidence: 99%

Activation mechanism and activity of globupain, a thermostable C11 protease from the Arctic Mid-Ocean Ridge hydrothermal system

Røyseth

Hurysz

Kaczorowska

et al. 2023

Preprint

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Deep-sea hydrothermal vent systems with prevailing extreme thermal conditions for life offer unique habitats to source heat tolearant enzymes with potential new enzymatic properties. Here, we present the novel C11 protease globupain, prospected from a metagenome-assembled genome of uncultivated Archaeoglobales sampled from the Soria Moria hydrothermal vent system located on the Arctic Mid-Ocean Ridges. By sequence comparisons against the MEROPS-MPRO database, globupain showed highest sequence identity to C11-like proteases present in human gut and intestinal bacteria,. Successful recombinant expression in Escherichia coli of the active zymogen and 13 mutant substitution variants allowed assesment of residues involved in maturation and activity of the enzyme. For activation, globupain required the addition of DTT and Ca2+. When activated, the 52 kDa proenzyme was processed at Lys137 and Lys144 into a 12 kDa light- and 32 kDa heavy chain heterodimer. A structurally conserved His132/Cys185 catalytic dyad was responsible for the proteolytic activity, and the enzyme demonstrated the ability to activate in-trans. Globupain exhibited caseinolytic activity and showed a strong preference for arginine in the P1 position, with Boc-QAR-aminomethylcoumarin (AMC) as the best substrate out of a total of 17 fluorogenic AMC substrates tested. Globupain was thermostable (Tm activated enzyme = 94.51 ± 0.09°C) with optimal activity at 75 °C and pH 7.1. By characterizing globupain, our knowledge of the catalytic properties and activation mechanisms of temperature tolerant marine C11 proteases have been expanded. The unique combination of features such as elevated thermostability, activity at relatively low pH values, and ability to operate under high reducing conditions makes globupain a potential intriguing candidate for use in diverse industrial and biotechnology sectors.

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Multiplex substrate profiling by mass spectrometry for proteases

Cited by 8 publications

References 84 publications

Proteases influence colony aggregation behavior in Vibrio cholerae

Proteases influence colony aggregation behavior in Vibrio cholerae

A Protease-Responsive Polymer/Peptide Conjugate and Reversible Assembly of Silver Clusters for the Detection of Porphyromonas gingivalis Enzymatic Activity

Activation mechanism and activity of globupain, a thermostable C11 protease from the Arctic Mid-Ocean Ridge hydrothermal system

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