Fungal Secretome Analysis via PepSAVI-MS: Identification of the Bioactive Peptide KP4 from<i>Ustilago maydis</i>

Kirkpatrick, Christine L.; Parsley, Nicole C.; Bartges, Tessa E.; Cooke, Madeline E.; Evans, Wilaysha S.; Heil, Lilian R.; Smith, Thomas J.; Hicks, Leslie M.

doi:10.1007/s13361-017-1880-z

Cited by 7 publications

(9 citation statements)

References 24 publications

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“…Overview PepSAVI-MS implements a multipronged approach for bioactive peptide discovery that utilizes selective extraction and fractionation of peptides from source material, bioactivity screening and mass spectrometry-based peptidomics for the identification of putative bioactive peptide targets. PepSAVI-MS was originally established for constitutively expressed peptides from botanicalsourced species and was recently validated for fungal secretomes (Kirkpatrick et al, 2018). Now, we extend this pipeline to capture secreted peptides from bacterial sources ( Fig.…”

Section: Resultsmentioning

confidence: 99%

“…PepSAVI‐MS is amenable to a variety of bioassay formats and can be modified to accommodate any target pathogen. High‐throughput microtitre‐based assays and agar diffusion‐based assays have been previously demonstrated with PepSAVI‐MS (Kirkpatrick et al ., , ). While many bacterial species bode well in high throughput 96‐well assays (as presented in the original implementation of the PepSAVI‐MS pipeline), this format is not amenable to some bacterial species that fail to grow to high density (e.g.…”

Section: Resultsmentioning

confidence: 99%

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Exploring bioactive peptides from bacterial secretomes using PepSAVI‐MS: identification and characterization of Bac‐21 from Enterococcus faecalis pPD1

Kirkpatrick

Parsley

Bartges

et al. 2018

Microbial Biotechnology

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SummaryAs current methods for antibiotic drug discovery are being outpaced by the rise of antimicrobial resistance, new methods and innovative technologies are necessary to replenish our dwindling arsenal of antimicrobial agents. To this end, we developed the PepSAVI‐MS pipeline to expedite the search for natural product bioactive peptides. Herein we demonstrate expansion of PepSAVI‐MS for the discovery of bacterial‐sourced bioactive peptides through identification of the bacteriocin Bac‐21 from Enterococcus faecalis pPD1. Minor pipeline modifications including implementation of bacteria‐infused agar diffusion assays and optional digestion of peptide libraries highlight the versatility and wide adaptability of the PepSAVI‐MS pipeline. Additionally, we have experimentally validated the primary protein sequence of the active, mature Bac‐21 peptide for the first time and have confirmed its identity with respect to primary sequence and post‐translational processing. Successful application of PepSAVI‐MS to bacterial secretomes as demonstrated herein establishes proof‐of‐principle for use in novel microbial bioactive peptide discovery.

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Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

Exploring bioactive peptides from bacterial secretomes using PepSAVI‐MS: identification and characterization of Bac‐21 from Enterococcus faecalis pPD1

Kirkpatrick

Parsley

Bartges

et al. 2018

Microbial Biotechnology

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“…Profiling of A. tricolor peptide library fractions 19-46 via LC-MS/MS revealed 5,868 unique features with masses, charge states, and retention times (1,(0)(1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15)000 Da,(2)(3)(4)(5)(6)(7)(8)(9)respectively) in the range of typical AMPs, highlighting the complex nature of the peptide library. The resulting list was filtered so that the maximum abundance of each feature was above 100 and detected in fractions 29-37, with <5% maximum abundance outside of the defined bioactive region (fractions [29][30][31][32][33][34][35][36][37]. This filtered list was modeled against the bioactivity profile using an elastic net penalized linear regression to identify the top 20 peptidyl features most likely contributing to bioactivity (Figure 2B).…”

Section: Pepsavi-msmentioning

confidence: 99%

PepSAVI-MS Reveals a Proline-rich Antimicrobial Peptide in Amaranthus tricolor

et al. 2019

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Traditional medicinal plants are a rich source of antimicrobials; however, the bioactive peptide constituents of most ethnobotanical species remain largely unexplored. Herein, PepSAVI-MS, a mass spectrometry-based peptidomics pipeline, was implemented for antimicrobial peptide (AMP) discovery in the medicinal plant Amaranthus tricolor. This investigation revealed a novel 1.7 kDa AMP with strong activity against Escherichia coli ATCC 25922, deemed Atr-AMP1. Initial efforts to determine the sequence of Atr-AMP1 utilized chemical derivatization and enzymatic digestion to provide information about specific residues and post-translational modifications. EThcD (electron-transfer/higher-energy collision dissociation) produced extensive backbone fragmentation and facilitated de novo sequencing, the results of which were consistent with orthogonal characterization experiments. Additionally, multistage HCD (higher-energy collisional dissociation) facilitated discrimination between isobaric leucine and isoleucine. These results revealed a positively charged proline-rich peptide present in a heterogeneous population of multiple peptidoforms, possessing several post-translational modifications including a disulfide bond, methionine oxidation, and proline hydroxylation. Additional bioactivity screening of a simplified fraction containing Atr-AMP1 revealed activity against Staphylococcus aureus LAC, demonstrating activity against both a Gram-negative and a Gram-positive bacterial species unlike many known short chain proline-rich antimicrobial peptides.

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“…To address this gap, PepSAVI-MS was developed to identify low abundance bioactive peptides in complex natural product extracts (Kirkpatrick et al, 2017 , 2018a , b ; Moyer et al, 2019 ; Parsley et al, 2019 ). PepSAVI-MS is a top-down peptidomics approach that leverages modern mass spectrometry and relies on traditional bioassays for bioactivity characterization (e.g., 96-well plate or disk-diffusion).…”

Section: Introductionmentioning

confidence: 99%

Implementation of Microfluidics for Antimicrobial Susceptibility Assays: Issues and Optimization Requirements

Parsley

Smythers

Hicks

2020

Front. Cell. Infect. Microbiol.

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Despite the continuous emergence of multi-drug resistant pathogens, the number of new antimicrobials reaching the market is critically low. Natural product peptides are a rich source of bioactive compounds, and advances in mass spectrometry have achieved unprecedented capabilities for the discovery and characterization of novel molecular species. However, traditional bioactivity assay formats hinder the discovery and biochemical characterization of natural product antimicrobial peptides (AMPs), necessitating large sample quantities and significant optimization of experimental parameters to achieve accurate/consistent activity measurements. Microfluidic devices offer a promising alternative to bulk assay systems. Herein, a microfluidics-based bioassay was compared to the traditional 96-well plate format in respective commercially-available hardware. Bioactivity in each assay type was compared using a Viola inconspicua peptide library screened against E. coli ATCC 25922. Brightfield microcopy was used to determine bioactivity in microfluidic channels while both common optical and fluorescence-based measurements of cell viability were critically assessed in plate-based assays. Exhibiting some variation in optical density and fluorescence-based measurements, all plate-based assays conferred bioactivity in late eluting V. inconspicua library fractions. However, significant differences in the bioactivity profiles of plate-based and microfluidic assays were found, and may be derived from the materials comprising each assay device or the growth/assay conditions utilized in each format. While new technologies are necessary to overcome the limitations of traditional bioactivity assays, we demonstrate that off-the-shelf implementation of microfluidic devices is non-trivial and significant method development/optimization is required before conventional use can be realized for sensitive and rapid detection of AMPs in natural product matrices.

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Fungal Secretome Analysis via PepSAVI-MS: Identification of the Bioactive Peptide KP4 fromUstilago maydis

Cited by 7 publications

References 24 publications

Exploring bioactive peptides from bacterial secretomes using PepSAVI‐MS: identification and characterization of Bac‐21 from Enterococcus faecalis pPD1

Exploring bioactive peptides from bacterial secretomes using PepSAVI‐MS: identification and characterization of Bac‐21 from Enterococcus faecalis pPD1

PepSAVI-MS Reveals a Proline-rich Antimicrobial Peptide in Amaranthus tricolor

Implementation of Microfluidics for Antimicrobial Susceptibility Assays: Issues and Optimization Requirements

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