ChipSoftX is an entirely new operating software for the TriVersa NanoMate automated nanoelectrospray source. Besides improvement in program compatibility with Windows and integration of existing software features, it also provides access to the new Developers Kit – a platform for customized method development with direct access to robot controls allowing entirely novel analysis workflows such as LESAPLUS.
Native mass spectrometry seeks to probe noncovalent protein interactions in terms of protein quaternary structure, protein–protein and protein–ligand complexes. The ultimate goal is to link the understanding of protein interactions to the protein environment by visualizing the spatial distribution of noncovalent protein interactions within tissue. Previously, we have shown that noncovalently bound protein complexes can be directly probed via liquid extraction surface analysis from dried blood spot samples, where hemoglobin is highly abundant. Here, we show that the intact hemoglobin complex can be sampled directly from thin tissue sections of mouse liver and correlated to a visible vascular feature, paving the way for native mass spectrometry imaging.
R.L. Griffiths and H.J. Cooper Anal. Chem., 2016, 88 (1), pp 606–609
Analyst, 2015, Accepted Manuscript DOI: 10.1039/C5AN00933B
Liquid extraction surface analysis (LESA) is a surface sampling technique that allows electrospray mass spectrometry analysis of a wide range of analytes directly from biological substrates. Here, we present LESA mass spectrometry coupled with high field asymmetric waveform ion mobility spectrometry (FAIMS) for the analysis of dried blood spots on filter paper. Incorporation of FAIMS in the workflow enables gas-phase separation of lipid and protein molecular classes, enabling analysis of both haemoglobin and a range of lipid (phosphatidylcholine or phosphatidylethanolamine, and sphingomyelin species) from a single extraction sample. The work has implications for multiplexed clinical assays of multiple analytes.
Young, LM, Saunders, JC, Mahood, RA, Revill, CH, Foster, RJ, Tu, LH, Raleigh, DP, Radford, SE, Ashcroft, AE
Nature Chemistry7,73–81doi:10.1038/nchem.2129
The search for therapeutic agents that bind specifically to precursor protein conformations and inhibit amyloid assembly is an important challenge. Identifying such inhibitors is difficult because many protein precursors of aggregation are partially folded or intrinsically disordered, which rules out structure-based design. Furthermore, inhibitors can act by a variety of mechanisms, including specific or nonspecific binding, as well as colloidal inhibition. Here we report a high-throughput method based on ion mobility spectrometry–mass spectrometry (IMS–MS) that is capable of rapidly detecting small molecules that bind to amyloid precursors, identifying the interacting protein species and defining the mode of inhibition. Using this method we have classified a variety of small molecules that are potential inhibitors of human islet amyloid polypeptide (hIAPP) aggregation or amyloid-beta 1-40 aggregation as specific, nonspecific, colloidal or non-interacting. We also demonstrate the ability of IMS–MS to screen for inhibitory small molecules in a 96-well plate format and use this to discover a new inhibitor of hIAPP amyloid assembly.
Randall EC, Bunch J, Cooper HJ; Anal Chem. 2014 Nov 4;86(21):10504-10. doi: 10.1021/ac503349d. Epub 2014 Oct 23
Top-down identification of proteins by liquid extraction surface analysis (LESA) mass spectrometry has previously been reported for tissue sections and dried blood spot samples. Here, we present a modified “contact” LESA method for top-down analysis of proteins directly from living bacterial colonies grown in Petri dishes,without any sample pretreatment. It was possible to identify a number of proteins by use of collision-induced dissociation tandem mass spectrometry followed by searches of the data against an E. coli protein database. The proteins identified suggest that the method may provide insight into the bacterial response to environmental conditions. Moreover, the results show that the “contact” LESA approach results in a smaller sampling area than typical LESA, which may have implications for spatial profiling.
Martin NJ, Griffiths RL, Edwards RL, Cooper HJ. J Am Soc Mass Spectrom. 2015 May 20. [Epub ahead of print]
Liquid extraction surface analysis (LESA) mass spectrometry is a promising tool for the analysis of intact proteins from biological substrates. Here, we demonstrate native LESA mass spectrometry of noncovalent protein complexes of myoglobin and hemoglobin from a range of surfaces. Holomyoglobin, in which apomyoglobin is noncovalently bound to the prosthetic heme group, was observed following LESA mass spectrometry of myoglobin dried onto glass and polyvinylidene fluoride surfaces. Tetrameric hemoglobin [(αβ)2 4H] was observed following LESA mass spectrometry of hemoglobin dried onto glass and polyvinylidene fluoride (PVDF) surfaces, and from dried blood spots (DBS) on filter paper. Heme-bound dimers and monomers were also observed. The ‘contact’ LESA approach was particularly suitable for the analysis of hemoglobin tetramers from DBS.
Our research focuses on in situ analysis of intact proteins from biological substrates. We combine ambient surface techniques and ion mobility spectrometry with high resolution mass spectrometry.
We are particularly interested in native ambient mass spectrometry, in which folded proteins, protein assemblies and protein complexes are sampled directly from thin tissue sections. Native ambient mass spectrometry, such as liquid extraction surface analysis (LESA), is integrated with mass spectrometry imaging to provide simultaneous spatial and structural information.
We also apply LESA for the analysis of intact but unfolded proteins from a range of substrates including living microbial colonies growing on agar and other solid substrates, dried blood spots and tissue sections. The combination of LESA, ion mobility spectrometry and mass spectrometry enables the detection of hundreds of proteins.
Initially, we purchased the TriVersa® NanoMate® for direct infusion and coupling to LC and we still use the equipment for that purpose. Advion Interchim Scientific’s chip technology has revolutionized nanospray as far as ease of use. The ESI Chip™ is robust and bypasses any problems with non-uniformity. It allows us to move simply to the next nozzle if there is an issue with spray. The spray sensing capability is very clever and necessary for our overnight runs. More recently, we have used the LESA capability of the TriVersa NanoMate® for our in situ analyses of proteins in tissue, dried blood spots and microbial colonies.
I would recommend the TriVersa® NanoMate® to anyone with a mass spectrometer who uses nanoelectrospray.
Liquid Extraction Surface Analysis Mass Spectrometry of ESKAPE Pathogens
Havlikova et al. J Am Soc Mass Spec. 2021
Top-down LESA MS/MS was used for protein identification in four ESKAPE pathogens as well as E. faecalis V583 and a clinical isolate of A. baumannii.
A: From drug-sized molecules to fragments, we analyze a little bit of everything. My primary research focuses around discovering the structure of proteins including enzyme structure and function in the development of antibiotics.
In addition, we have a collaboration with UCB, a global pharma focused on central nervous system and immunology disorders, involving library screening.
Q: Why did you incorporate the TriVersa NanoMate® into your laboratory?
A: We have a NanoMate 100 and a TriVersa NanoMate®. It is reasonably well-known that you need nanoESI for noncovalent interactions – ligand screening, but it was essential to my research to speed up and to automate the process. I had watched too many demonstrations that involved pulled capillaries, and I knew it would drive us crazy. A second instrument was absolutely vital to meet the needs of our collaboration with UCB.
Q: What benefits have you experienced with the TriVersa NanoMate®?
A: The main reason for purchasing the TriVersa NanoMate® is its automated nanoESI capability. The added benefit is its ease of use. We act as a service lab to the chemistry department, and often we have inexperienced users with a variety of different samples to be analyzed. The TriVersa NanoMate® makes it easy for users to do what needs to be done, very quickly.
Q: To whom would you recommend the TriVersa NanoMate® for their research?
A: I would recommend the TriVersa NanoMate® to anyone who is looking for an easy to use nanoelectrospray source, and who would like to automate and to speed up their process.
Our goal is to provide the research community at IRB Barcelona and their co-workers with state-of-the-art tools and methodologies for the MS analysis of a broad range of biological species, from large proteins and DNA to small molecules. The final purpose is to get insight into these molecules’ identity, structure, interaction with other molecules and biological function in order to help in drug design, protein mechanism elucidation and in the search for biomarkers. We have implemented methods specialized in top-down proteomics and we are pioneers in this MS strategy in Spain.
As a core facility, we are responsible for working with different biologic molecules, and we are required to change methods constantly and efficiently.
Originally, we purchased the TriVersa® NanoMate® for its chip-based direct infusion mode for noncovalent interaction analysis, but we learned quickly that it could be applied to other areas of our research. Prior to using the TriVersa® NanoMate®, the steps involved in collecting fractions were painful and time-consuming. With the TriVersa® NanoMate®, we can run LC/fraction collection or infusion without changing the setup and wasting time with stabilization. We do not experience the problems typical with traditional nanoelectrospray sources.
One aspect of the TriVersa® NanoMate® that impressed us was the ability to analyze complicated top-down samples with the LC compatibility. It is not possible to analyze these samples on an LC time scale, and the fraction collection capability allowed us to analyze in a way that was not possible previously.
We use the TriVersa® NanoMate® for everything; noncovalent interactions, top-down, middle-down, bottom-up, basic infusion, LC coupled to fraction collection. The instrument is useful in all of its different set-ups, especially without having to change sources and waiting for a stable spray.
The reliability of the system is one of the greatest benefits especially for people who have to change frequently between applications. We have found the spray sensing feature to be very valuable because we know our precious samples will not be lost.
Characterization of Human Sperm Protamine Proteoforms Through a Combination of Top-Down and Bottom-Up Mass Spectrometry Approaches
Soler-Ventura et al. J Proteome Res, 2020, 19(1), 221-237. DOI: 10.1021/acs.jproteome.9b00499
Identified the sperm protamine proteoforms profile, including their post-translational modifications, in normozoospermic individuals using a top-down MS approach and a proteinase-K-digestion-based bottom-up MS approach.
Q: WHAT IS THE FOCUS OF YOUR LABS RESEARCH?
A: The laboratory is split 50/50 between proteomics and lipidomics research. While working on protein analysis, such as identifying protein interaction networks or characterizing the proteomes of organisms that are related very distantly to organisms with sequenced genomes, we also attempt to better quantify the lipidome of various organelles, cells and tissues.
Q: WHAT ARE YOUR PRIMARY GOALS?
A: In lipidomics, we forge the alliance with developmental biology. The primary goal of the group is to combine lipidomics with developmental biology. As organisms grow and develop from a single cell, newly differentiated tissues require their own unique membrane lipid composition. We hope to characterize these tailored changes to better understand how inherited defects in lipid metabolism cause disease. We are equally interested in lipidomes of membrane microdomains and the biological significance of its remarkable complexity.
Q: WHY DID YOU INCORPORATE THE TRIVERSA NANOMATE® INTO YOUR LABORATORY?
A: We had a need for automated nanoflow direct-infusion capabilities. Shotgun lipidomics relies on low and stable flow rates, and the TriVersa NanoMate® has this demonstrated ability. We have purchased three additional instruments because they allow us to rapidly switch between lipids and proteomic analysis.
Q: DO YOU HAVE ANY PUBLICATIONS OF PRESENTATIONS USING THE TRIVERSA NANOMATE®?
Publication Highlight
2021: Hormone-Sensitive Lipase Couples Intergenerational Sterol Metabolism to Reproductive Success
Christoph Heier Is a corresponding author , Oskar Knittelfelder, Harald F Hofbauer, Wolfgang Mende, Ingrid Pörnbacher, Laura Schiller, Gabriele Schoiswohl, Hao Xie, Sebastian Grönke, Andrej Shevchenko, Ronald P Kühnlein
Hormone-sensitive lipase (Hsl) was identified as an ancestral regulator of SE degradation, which improves intergenerational sterol transfer and reproductive success in flies.
Other Publications:
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