Towards practical time-of-flight secondary ion mass spectrometry lignocellulolytic enzyme assays

Robyn E Goacher,Alex Yi-Lin Tsai,Emma R Master

doi:10.1186/1754-6834-6-132

Abstract

BackgroundTime-of-Flight Secondary Ion Mass Spectrometry (ToF-SIMS) is a surface sensitive mass spectrometry technique with potential strengths as a method for detecting enzymatic activity on solid materials. In particular, ToF-SIMS has been applied to detect the enzymatic degradation of woody lignocellulose. Proof-of-principle experiments previously demonstrated the detection of both lignin-degrading and cellulose-degrading enzymes on solvent-extracted hardwood and softwood. However, these preliminary experiments suffered from low sample throughput and were restricted to samples which had been solvent-extracted in order to minimize the potential for mass interferences between low molecular weight extractive compounds and polymeric lignocellulose components.ResultsThe present work introduces a new, higher-throughput method for processing powdered wood samples for ToF-SIMS, meanwhile exploring likely sources of sample contamination. Multivariate analysis (MVA) including Principal Component Analysis (PCA) and Multivariate Curve Resolution (MCR) was regularly used to check for sample contamination as well as to detect extractives and enzyme activity. New data also demonstrates successful ToF-SIMS analysis of unextracted samples, placing an emphasis on identifying the low-mass secondary ion peaks related to extractives, revealing how extractives change previously established peak ratios used to describe enzyme activity, and elucidating peak intensity patterns for better detection of cellulase activity in the presence of extractives. The sensitivity of ToF-SIMS to a range of cellulase doses is also shown, along with preliminary experiments augmenting the cellulase cocktail with other proteins.ConclusionsThese new procedures increase the throughput of sample preparation for ToF-SIMS analysis of lignocellulose and expand the applications of the method to include unextracted lignocellulose. These are important steps towards the practical use of ToF-SIMS as a tool to screen for changes in plant composition, whether the transformation of the lignocellulose is achieved through enzyme application, plant mutagenesis, or other treatments.

Highlights

Time-of-Flight Secondary Ion Mass Spectrometry (ToF-SIMS) is a surface sensitive mass spectrometry technique with potential strengths as a method for detecting enzymatic activity on solid materials
The use of the 96-well filter plates for sample incubation and washing, combined with transferring washed biomass samples to tape for support in the ToF-SIMS, resulted in significant time and material savings compared to previous methods [1] (Table 1)
Several advances were made towards increasing the throughput and practicality of ToF-SIMS enzyme assays for lignocellulosic materials

Summary

Introduction

Time-of-Flight Secondary Ion Mass Spectrometry (ToF-SIMS) is a surface sensitive mass spectrometry technique with potential strengths as a method for detecting enzymatic activity on solid materials. Time-of-Flight Secondary Ion Mass Spectrometry (ToFSIMS) is a surface mass spectrometry technique with potential advantages for the study of lignocellulosic materials These advantages include direct analysis of complex solid substrates, the potential to monitor both lignocellulose degradation and modification, and the potential for fast enzyme assays due to the technique’s surface sensitivity [1]. We have identified 40 secondary ion peaks characteristic of lignin and polysaccharides in extracted pine [18] and have established good reproducibility of lignin and polysaccharide peak ratios across powdered wood samples [1] This peak list was further refined to exclude mass interferences from applied proteins, leading to ToF-SIMS assays to measure cellulase and laccase action on solvent-extracted spruce and aspen [1]

Methods

Results

Conclusion