A Viable New Strategy for the Discovery of Peptide Proteolytic Cleavage Products in Plant-Microbe Interactions

Abstract

Small peptides that are proteolytic cleavage products (PCPs) less than 100 amino acids are emerging as key signaling molecules that mediate cell-to-cell communication and biological processes that occur between and within plants, fungi, and bacteria. Yet, the discovery and characterization of these molecules is largely overlooked. Today, selective enrichment and subsequent characterization by mass spectrometry (MS)-based sequencing offers the greatest potential for their comprehensive characterization, however qualitative and quantitative performance metrics are rarely captured. Herein, we addressed this need by benchmarking the performance of an enrichment strategy optimized specifically for small PCPs using state-of-the-art de novo-assisted peptide sequencing. As a case study, we implemented this approach to identify PCPs from different root and foliar tissues of the hybrid poplar Populus X canescens 717-1B4 in interaction with the ectomycorrhizal basidiomycete Laccaria bicolor. In total, we identified 1660 and 2870 Populus and L. bicolor unique PCPs, respectively. Qualitative results supported the identification of well-known PCPs, like the mature form of the Photosystem II complex 5 kDa protein (~ 3 kDa). A total of 157 PCPs were determined to be significantly more abundant in root tips with established ectomycorrhiza when compared to root tips without established ectomycorrhiza and extramatrical mycelium of L. bicolor. These PCPs mapped to 64 Populus proteins and 69 L. bicolor proteins in our database, with several of them previously implicated in biologically relevant associations between plant and fungus.

Multi-omic characterization of bifunctional peroxidase 4-coumarate 3-hydroxylase knockdown in Brachypodium distachyon provides insights into lignin modification-associated pleiotropic effects

“…Peptides were analyzed by liquid chromatography and tandem mass spectrometry (LC-MS/MS) as previously described ( Villalobos Solis et al., 2020 ) using a Proxeon EASY-nLC 1200 liquid chromatography pump (Thermo Fisher Scientific) coupled to a Q Exactive Plus mass spectrometer (Thermo Fischer Scientific). Briefly, peptides were separated on an in-house-pulled nanospray emitter of 75 μm inner diameter packed with 30 cm of Kinetex C18 resin (1.7 μm, 100 Å, Phenomenex).…”

“…MS data were acquired with the Thermo Xcalibur software v4.27.19 using the top 10 data-dependent acquisition method. Other spectral data acquisition parameters have been reported before ( Villalobos Solis et al., 2020 ).…”

Advances and perspectives in discovery and functional analysis of small secreted proteins in plants

“…This may cause a problem for studying plant SSPs that contain a large portion of IDRs using proteomics approaches because MS has lower sensitivity than transcriptome sequencing. To increase the sensitivity of detecting SSPs in plants, it is necessary to enrich for IDRs containing proteins and low molecular weight proteins in protein extract using gel filters 95 or ultrafiltration devices 96 , 97 .…”

“…This classical approach for the discovery of novel natural products starts with an enrichment strategy to selectively isolate molecules of interest from highly complex crude extracts. For SSPs, common cellular extraction techniques use size exclusion ultrafiltration strategies, such as molecular weight cut-off spin column filters, to selectively enrich for low molecular weight protein fractions 96 , 97 . Other techniques include gel-based separations 49 , 95 , 133 , solvent extractions 89 , 134 , and size exclusion chromatography 134 , 135 .…”

Temporal dynamics of protein and post‐translational modification abundances in Populus leaf across a diurnal period

“…MS data was acquired with the Thermo Xcalibur software v4.27.19 using the topN method where N could be up to 10. Other spectral data acquisition parameters have been reported before [5].…”