Comprehensive LESA Mass Spectrometry Imaging of Intact Proteins by Integration of Cylindrical FAIMS

The benefits of high field asymmetric waveform ion mobility spectrometry (FAIMS) for mass spectrometry imaging of intact proteins in thin tissue sections have been demonstrated previously. In those works, a planar FAIMS device coupled with a Thermo Elite mass spectrometer was employed. Here, we have evaluated a newly introduced cylindrical FAIMS device (the FAIMS Pro) coupled with a Thermo Fusion Lumos mass spectrometer for liquid extraction surface analysis mass spectrometry imaging of intact proteins in thin tissue sections from rat testes, kidney, and brain. The method makes use of multiple FAIMS compensation values at each location (pixel) of the imaging array. A total of 975 nonredundant protein species were detected in the testes imaging dataset, 981 in the kidney dataset, and 249 in the brain dataset. These numbers represent a 7-fold (brain) and over 10-fold (testes, kidney) improvement on the numbers of proteins previously detected in LESA FAIMS imaging, and a 10-fold to over 20-fold improvement on the numbers detected without FAIMS on this higher performance mass spectrometer, approaching the same order of magnitude as those obtained in top-down proteomics of cell lines. Nevertheless, high throughput identification within the LESA FAIMS imaging workflow remains a challenge

Crop Updates 2009 - Farming Systems

This session covers nineteen papers from different authors: Decision support technology 1. The use of high resolution imagery in broad acre cropping, Derk Bakker and Grey Poulish, Department of Agriculture and Food 2. Spraywise decisions – online spray applicatiors planning tool, Steve Lacy, Nufarm Australia Ltd 3. Testing for redlegged earthmite resistance in Western Australia, Svetlana Micic, Peter Mangano, Tony Dore and Alan Lord, Department of Agriculture and Food 4. Screening cereal, canola and pasture cultivars for Root Lesion Nematode (Pratylenchus neglectus), Vivien Vanstone, Helen Hunter and Sean Kelly,Department of Agriculture and Food Farming Systems Research 5. Lessons from five years of cropping systems research, WK Anderson, Department of Agriculture and Food 6. Facey Group rotations for profit: Five years on and where to next? Gary Lang and David McCarthy, Facey Group, Wickepin, WA Mixed Farming 7. Saline groundwater use by Lucerne and its biomass production in relation to groundwater salinity, Ruhi Ferdowsian, Ian Roseand Andrew Van Burgel, Department of Agriculture and Food 8. Autumn cleaning yellow serradella pastures with broad spectrum herbicides – a novel weed control strategy that exploits delayed germination, Dr David Ferris, Department of Agriculture and Food 9. Decimating weed seed banks within non-crop phases for the benefit of subsequent crops, Dr David Ferris, Department of Agriculture and Food 10. Making seasonal variability easier to deal with in a mixed farming enterprise! Rob Grima,Department of Agriculture and Food 11. How widely have new annual legume pastures been adopted in the low to medium rainfall zones of Western Australia? Natalie Hogg, Department of Agriculture and Food, John Davis, Institute for Sustainability and Technology Policy, Murdoch University 12. Economic evaluation of dual purpose cereal in the Central wheatbelt of Western Australia, Jarrad Martin, Pippa Michael and Robert Belford, School of Agriculture and Environment, CurtinUniversity of Technology, Muresk Campus 13. A system for improving the fit of annual pasture legumes under Western Australian farming systems, Kawsar P Salam1,2, Roy Murray-Prior1, David Bowran2and Moin U. Salam2, 1Curtin University of Technology; 2Department of Agriculture and Food 14. Perception versus reality: why we should measure our pasture, Tim Scanlon, Department of Agriculture and Food, Len Wade, Charles Sturt University, Megan Ryan, University of Western Australia Modelling 15. Potential impact of climate changes on the profitability of cropping systems in the medium and high rainfall areas of the northern wheatbelt, Megan Abrahams, Chad Reynolds, Caroline Peek, Dennis van Gool, Kari-Lee Falconer and Daniel Gardiner, Department of Agriculture and Food 16. Prediction of wheat grain yield using Yield Prophet®, Geoff Anderson and Siva Sivapalan, Department of Agriculture and Food 17. Using Yield Prophet® to determine the likely impacts of climate change on wheat production, Tim McClelland1, James Hunt1, Zvi Hochman2, Bill Long3, Dean Holzworth4, Anthony Whitbread5, Stephen van Rees1and Peter DeVoil6 1 Birchip Cropping Group, Birchip, Vic, 2Agricultural Production Systems Research Unit (APSRU), CSIRO Sustainable Ecosystems, Climate Adaptation Flagship, Qld, 3 AgConsulting, SA 4 Agricultural Production Systems Research Unit (APSRU), CSIRO Sustainable Ecosystems, Toowoomba Qld, 5 CSIRO Sustainable Ecosystems, SA, 6 Agricultural Production Systems Research Unit (APSRU), Department of Agriculture and Fisheries, Queensland 18. Simple methods to predict yield potential: Improvements to the French and Schultz formula to account for soil type and within-season rainfall, Yvette Oliver, Michael Robertson and Peter Stone, CSIRO Sustainable Ecosystems 19. Ability of various yield forecasting models to estimate soil water at the start of the growing season, Siva Sivapalan, Kari-Lee Falconer and Geoff Anderson, Department of Agriculture and Foo

Characterization of a Temperature-Controlled FAIMS System

High-field asymmetric waveform ion mobility spectrometry (FAIMS) focuses and separates gas-phase analyte ions from chemical background, offering substantial improvements in the detection of targeted species in biological matrices. Ion separations have been typically performed at atmospheric pressure and ambient temperature, although routine small molecule quantitation by LC-MS (and thus LC-FAIMS-MS) is generally performed at liquid flow rates (e.g., in excess of 200 μL/min) in which atmospheric pressure ionization sources (e.g., APCI and ESI) need to be run at elevated temperatures to enhance ion desolvation. Heat from the ionization source and/or the mass spectrometer capillary interface is shown to have a significant impact on the performance of a conventional FAIMS electrode set. This study introduces a new FAIMS system that uses gas heating/cooling to quickly reach temperature equilibrium independent of the external temperature conditions. A series of equations and balance plots, which look at the effect of temperature and other variables, on the normalized field strength (E/N), are introduced and used to explain experimental observations. Examples where the ion behavior deviates from the predicted behavior are presented and explanations based on clusters or changes in ion-neutral interactions are given. Consequences of the use of temperature control, and in particular advantages of using different temperature settings on the inner and outer electrodes, for the purpose of manipulating ion separation are described

Using gas modifiers to significantly improve sensitivity and selectivity in a cylindrical FAIMS device

(Graph Presented) Recent reports describing enhanced performance when using gas additives in a DMS device (planar electrodes) have indicated that comparable benefits are not attainable using FAIMS (cylindrical electrodes), owing to the non-homogeneous electric fields within the analyzer region. In this study, a FAIMS system (having cylindrical electrodes) was modified to allow for controlled delivery of gas additives. An experiment was carried out that illustrates the important distinction between gas modifiers present as unregulated contaminants and modifiers added in a controlled manner. The effect of contamination was simulated by adjusting the ESI needle position to promote incomplete desolvation, thereby permitting ESI solvent vapor into the FAIMS analyzer region, causing signal instability and irreproducible CV values. However, by actively controlling the delivery of the gas modifier, reproducible CV spectra were obtained. The effects of adding different gas modifiers were examined using 15 positive ions having mass-to-charge (m/z) values between 90 and 734. Significant improvements in peak capacity and increases in ion transmission were readily attained by adding acetonitrile vapor, even at trace levels ( 640.1%). Increases in signal intensity were greatest for the low m/z ions; for the six lowest molecular weight species, signal intensities increased by 3c10- to over 100-fold compared with using nitrogen without gas additives, resulting in equivalent or better signal intensities compared with ESI without FAIMS. These results confirm that analytical benefits derived from the addition of gas modifiers reported with a uniform electric field (DMS) also are observed using a non-homogenous electric field (FAIMS) in the analyser region.Peer reviewed: YesNRC publication: Ye

Deeper Protein Identification by Using FAIMS in Top-down Proteomics

Field Asymmetric Ion Mobility Spectrometry (FAIMS), when used in proteomics studies, provides superior selectivity, and enables more proteins to be identified by providing additional gas phase separation. Here, we tested the performance of cylindrical FAIMS for the identification and characterization of proteoforms by top-down mass spectrometry of heterogeneous protein mixtures. Combining FAIMS with chromatographic separation resulted in a 62% increase in protein identifications and an 8% increase in proteoform identifications as compared to samples analyzed without FAIMS. This increase was attributable, in part, to improved signal-to-noise for proteoforms with similar retention times. Additionally, our results show that the optimal compensation voltage of any given proteoform was correlated with the molecular weight of the analyte. Collectively these results suggest that the addition of FAIMS can enhance top-down proteomics in both discovery and targeted applications. </div

Improved label-free quantification of intact proteoforms using field asymmetric ion mobility spectrometry

The high-throughput quantification of intact proteoforms using a label-free approach is typically performed on proteins in the 0-30 kDa mass range extracted from whole cell or tissue lysates. Unfortunately, even when high-resolution separation of proteoforms is achieved by either high performance liquid chromatography or capillary electrophoresis, the number of proteoforms that can be identified and quantified is inevitably limited by the inherent sample complexity. Here we benchmark label-free quantification of proteoforms of Escherichia coli by applying gas-phase fractionation (GPF) via field asymmetric ion mobility spectrometry (FAIMS). Recent advances in Orbitrap instrumentation have enabled the acquisition of high-quality intact and fragmentation mass spectra without the need for averaging time-domain transients prior to Fourier transform. The resulting speed improvements allowed for the application of multiple FAIMS compensation voltages in the same liquid chromatography-tandem mass spectrometry experiment without increasing the overall data acquisition cycle. As a result, the application of FAIMS to label-free quantification based on intact mass spectra substantially increases the number of both identified and quantified proteoforms without penalizing quantification accuracy in comparison to traditional label-free experiments that do not adopt GPF

Comprehensive Single-Shot Proteomics with FAIMS on a Hybrid Orbitrap Mass Spectrometer

Liquid chromatography (LC) prefractionation is often implemented to increase proteomic coverage; however, while effective, this approach is laborious, requires considerable sample amount, and can be cumbersome. We describe how interfacing a recently described high-field asymmetric waveform ion mobility spectrometry (FAIMS) device between a nanoelectrospray ionization (nanoESI) emitter and an Orbitrap hybrid mass spectrometer (MS) enables the collection of single-shot proteomic data with comparable depth to that of conventional two-dimensional LC approaches. This next generation FAIMS device incorporates improved ion sampling at the ESI–FAIMS interface, increased electric field strength, and a helium-free ion transport gas. With fast internal compensation voltage (CV) stepping (25 ms/transition), multiple unique gas-phase fractions may be analyzed simultaneously over the course of an MS analysis. We have comprehensively demonstrated how this device performs for bottom-up proteomics experiments as well as characterized the effects of peptide charge state, mass loading, analysis time, and additional variables. We also offer recommendations for the number of CVs and which CVs to use for different lengths of experiments. Internal CV stepping experiments increase protein identifications from a single-shot experiment to >8000, from over 100 000 peptide identifications in as little as 5 h. In single-shot 4 h label-free quantitation (LFQ) experiments of a human cell line, we quantified 7818 proteins with FAIMS using intra-analysis CV switching compared to 6809 without FAIMS. Single-shot FAIMS results also compare favorably with LC fractionation experiments. A 6 h single-shot FAIMS experiment generates 8007 protein identifications, while four fractions analyzed for 1.5 h each produce 7776 protein identifications

Crop Updates 2009 - Farming Systems

This session covers nineteen papers from different authors: Decision support technology 1. The use of high resolution imagery in broad acre cropping, Derk Bakker and Grey Poulish, Department of Agriculture and Food 2. Spraywise decisions – online spray applicatiors planning tool, Steve Lacy, Nufarm Australia Ltd 3. Testing for redlegged earthmite resistance in Western Australia, Svetlana Micic, Peter Mangano, Tony Dore and Alan Lord, Department of Agriculture and Food 4. Screening cereal, canola and pasture cultivars for Root Lesion Nematode (Pratylenchus neglectus), Vivien Vanstone, Helen Hunter and Sean Kelly,Department of Agriculture and Food Farming Systems Research 5. Lessons from five years of cropping systems research, WK Anderson, Department of Agriculture and Food 6. Facey Group rotations for profit: Five years on and where to next? Gary Lang and David McCarthy, Facey Group, Wickepin, WA Mixed Farming 7. Saline groundwater use by Lucerne and its biomass production in relation to groundwater salinity, Ruhi Ferdowsian, Ian Roseand Andrew Van Burgel, Department of Agriculture and Food 8. Autumn cleaning yellow serradella pastures with broad spectrum herbicides – a novel weed control strategy that exploits delayed germination, Dr David Ferris, Department of Agriculture and Food 9. Decimating weed seed banks within non-crop phases for the benefit of subsequent crops, Dr David Ferris, Department of Agriculture and Food 10. Making seasonal variability easier to deal with in a mixed farming enterprise! Rob Grima,Department of Agriculture and Food 11. How widely have new annual legume pastures been adopted in the low to medium rainfall zones of Western Australia? Natalie Hogg, Department of Agriculture and Food, John Davis, Institute for Sustainability and Technology Policy, Murdoch University 12. Economic evaluation of dual purpose cereal in the Central wheatbelt of Western Australia, Jarrad Martin, Pippa Michael and Robert Belford, School of Agriculture and Environment, CurtinUniversity of Technology, Muresk Campus 13. A system for improving the fit of annual pasture legumes under Western Australian farming systems, Kawsar P Salam1,2, Roy Murray-Prior1, David Bowran2and Moin U. Salam2, 1Curtin University of Technology; 2Department of Agriculture and Food 14. Perception versus reality: why we should measure our pasture, Tim Scanlon, Department of Agriculture and Food, Len Wade, Charles Sturt University, Megan Ryan, University of Western Australia Modelling 15. Potential impact of climate changes on the profitability of cropping systems in the medium and high rainfall areas of the northern wheatbelt, Megan Abrahams, Chad Reynolds, Caroline Peek, Dennis van Gool, Kari-Lee Falconer and Daniel Gardiner, Department of Agriculture and Food 16. Prediction of wheat grain yield using Yield Prophet®, Geoff Anderson and Siva Sivapalan, Department of Agriculture and Food 17. Using Yield Prophet® to determine the likely impacts of climate change on wheat production, Tim McClelland1, James Hunt1, Zvi Hochman2, Bill Long3, Dean Holzworth4, Anthony Whitbread5, Stephen van Rees1and Peter DeVoil6 1 Birchip Cropping Group, Birchip, Vic, 2Agricultural Production Systems Research Unit (APSRU), CSIRO Sustainable Ecosystems, Climate Adaptation Flagship, Qld, 3 AgConsulting, SA 4 Agricultural Production Systems Research Unit (APSRU), CSIRO Sustainable Ecosystems, Toowoomba Qld, 5 CSIRO Sustainable Ecosystems, SA, 6 Agricultural Production Systems Research Unit (APSRU), Department of Agriculture and Fisheries, Queensland 18. Simple methods to predict yield potential: Improvements to the French and Schultz formula to account for soil type and within-season rainfall, Yvette Oliver, Michael Robertson and Peter Stone, CSIRO Sustainable Ecosystems 19. Ability of various yield forecasting models to estimate soil water at the start of the growing season, Siva Sivapalan, Kari-Lee Falconer and Geoff Anderson, Department of Agriculture and Foo

Metal Complexes of Bridging Neutral Radical Ligands: pymDTDA and pymDSDA

Metal complexes of the 4-(2′-pyrimidyl)-1,2,3,5-dithiadiazolyl (pymDTDA) neutral radical ligand and its selenium analogue (pymDSDA) are presented. The following series of metal ions has been studied using M(hfac)2 as the coordination fragment of choice (hfac = 1,1,1,5,5,5 hexafluoroacetylacetonato): MnII, CoII, NiII, and ZnII. The binuclear cobalt and nickel complexes of pymDTDA both exhibit ferromagnetic (FM) coupling between the unpaired electrons on the ligand and the metal ion, while the binuclear zinc complex of pymDTDA is presented as a comparative example incorporating a diamagnetic metal ion. The binuclear manganese complex of pymDTDA, reported in a preliminary communication, is compared to the pymDSDA analogue, and new insight into the magnetic behavior reveals that intermolecular magnetic coupling, mediated by chalcogen− oxygen contacts, gives rise to a significant increase in the χT product at low temperature. Surprisingly, the binuclear nickel complex of pymDSDA forms dimers in the solid state, as do the mononuclear complexes of cobalt and nickel with pymDTDA. In addition, mixed mononuclear/binuclear complexes of Mn- and Zn(pymDTDA) have been identified