The feeding tube of cyst nematodes: characterisation of protein exclusion

Plant parasitic nematodes comprise several groups; the most economically damaging of these are the sedentary endoparasites. Sedentary endoparasitic nematodes are obligate biotrophs and modify host root tissue, using a suite of effector proteins, to create a feeding site that is their sole source of nutrition. They feed by withdrawing host cell assimilate from the feeding site though a structure known as the feeding tube. The function, composition and molecular characteristics of feeding tubes are poorly characterised. It is hypothesised that the feeding tube facilitates uptake of host cell assimilate by acting as a molecular sieve. Several studies, using molecular mass as the sole indicator of protein size, have given contradictory results about the exclusion limits of the cyst nematode feeding tube. In this study we propose a method to predict protein size, based on protein database coordinates in silico. We tested the validity of these predictions using travelling wave ion mobility spectrometry--mass spectrometry, where predictions and measured values were within approximately 6%. We used the predictions, coupled with mass spectrometry, analytical ultracentrifugation and protein electrophoresis, to resolve previous conflicts and define the exclusion characteristics of the cyst nematode feeding tube. Heterogeneity was tested in the liquid, solid and gas phase to provide a comprehensive evaluation of three proteins of particular interest to feeding tube size exclusion, GFP, mRFP and Dual PI. The data and procedures described here could be applied to the design of plant expressed defence compounds intended for uptake into cyst nematodes. We also highlight the need to assess protein heterogeneity when creating novel fusion proteins

Deactivation of Cellulase at the Air-Liquid Interface Is the Main Cause of Incomplete Cellulose Conversion at Low Enzyme Loadings

Abstract Amphiphilic additives such as bovine serum albumin (BSA) and Tween have been used to improve cellulose hydrolysis by cellulases. However, there has been a lack of clarity to explain their mechanism of action in enzymatic hydrolysis of pure or low-lignin cellulosic substrates. In this work, a commercial Trichoderma reesei enzyme preparation and the amphiphilic additives BSA and Tween 20 were applied for hydrolysis of pure Avicel cellulose. The results showed that these additives only had large effects on cellulose conversion at low enzyme to substrate ratios when the reaction flasks were shaken. Furthermore, changes in the air-liquid interfacial area profoundly affected cellulose conversion, but surfactants reduced or prevented cellulase deactivation at the air-liquid interface. Not shaking the flasks or adding low amounts of surfactant resulted in near theoretical cellulose conversion at low enzyme loadings given enough reaction time. At low enzyme loadings, hydrolysis of cellulose in lignocellulosic biomass with low lignin content suffered from enhanced enzyme deactivation at the air-liquid interface

Analysis of recombinant glycoproteins by mass spectrometry

The advent of new technologies for analysis of biopolymers by mass spectrometry has revolutionised strategies for recombinant protein characterization. The principal recent developments have been matrix-assisted laser desorption/ionization and electrospray ionization mass spectrometry. Using these tools, accurate molecular mass determinations can now be obtained routinely - often using minute (picomole - femtomole) quantities of protein or protein fragments. These techniques have proved indispensible for detailed characterization of the post-translational modifications of recombinant proteins produced by eukaryotic systems. Glycosylation is arguably the most important and complex of these modifications and has prompted widespread use of these new techniques. In this mini-review article I describe recent advances in the use of mass spectrometry for analysis of recombinant glycoproteins

Ion mobility-mass correlation trend line separation of glycoprotein digests without deglycosylation

A high-throughput ion mobility mass spectrometer (IMMS) was used to rapidly separate and analyze peptides and glycopeptides derived from glycoproteins. Two glycoproteins, human α-1-acid glycoprotein and antithrombin III were digested with trypsin and subjected to electrospray traveling wave IMMS analysis. No deglycosylation steps were performed; samples were complex mixtures of peptides and glycopeptides. Peptides and glycosylated peptides with different charge states (up to 4 charges) were observed and fell on distinguishable trend lines in 2-D IMMS spectra in both positive and negative modes. The trend line separation patterns matched between both modes. Peptide sequence was identified based on the corresponding extracted mass spectra and collision induced dissociated (CID) experiments were performed for selected compounds to prove class identification. The signal-to-noise ratio of the glycopeptides was increased dramatically with ion mobility trend line separation compared to non-trend line separation, primarily due to selection of precursor ion subsets within specific mobility windows. In addition, isomeric mobility peaks were detected for specific glycopeptides. IMMS demonstrated unique capabilities and advantages for investigating and separating glycoprotein digests in this study and suggests a novel strategy for rapid glycoproteomics studies in the future