Here we describe a novel surface sampling technique termed pressurized liquid extraction surface analysis (PLESA), which in combination with a dedicated high resolution shotgun lipidomics routine enables both quantification and in-depth structural characterization of molecular lipid species extracted directly from tissue sections.
Surface analysis techniques have gained significant attention over the last couple of years with LESA being an interesting alternative to techniques such as SIMS or MALDI imaging. This note demonstrates practical applications for LESA in the material sciences.
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.
Why did you incorporate the TriVersa® NanoMate® into your laboratory
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.
To whom would you recommend the TriVersa® NanoMate® for their research?
I would recommend the TriVersa® NanoMate® to anyone with a mass spectrometer who uses nanoelectrospray.
Do you have any publications or presentations using the TriVersa® NanoMate®?
Publication Highlight
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.
Other Publications:
Hale et al. Native mass spectrometry imaging and in situ top-down identification of intact proteins directly from tissue. J Am Soc Mass Spec. DOI: 10.1021/jasms.0c00226
Havlikova et al. Direct identification of bacterial and human proteins from infected wounds in living 3D skin models. Sci Rep. DOI: 10.1038/s41598-020-68233-6
Haque et al. Self-incompatibility triggers irreversible oxidative modification of proteins in incompatible pollen. Plant Physiology. DOI: 10.1104/pp.20.00066
Sisley et al. LESA cyclic ion mobility mass spectrometry of intact proteins from thin tissue sections. Anal. Chem. DOI: 10.1021/acs.analchem.9b05169
Hale et al. Native LESA TWIMS-MSI: Spatial, conformational, and mass analysis of proteins and protein complexes.J Am Soc Mass Spec. DOI: 10.1021/jasms.9b00122
Kocurek et al. Electroporation and mass spectrometry: A new paradigm for in situ analysis of intact proteins from living yeast colonies. Analytical Chemistry/ DOI: 10.1021/acs.analchem.9b04365
Griffiths et al. Comprehensive LESA mass spectrometry imaging of intact proteins by integration of cylindrical FAIMS. Analytical Chemistry. DOI: 10.1021/acs.analchem.9b05124
Havlikova et al. Quantitative imaging of proteins in tissue by stable isotope labeled mimetic liquid extraction surface analysis mass spectrometry. Analytical Chemistry. DOI: 10.1021/acs.analchem.9b04148
Griffiths et al. LESA MS imaging of heat-preserved and frozen tissue: Benefit of multistep static FAIMS. Analytical Chemistry. DOI: 10.1021/acs.analchem.8b02739
Rosting et al. High field asymmetric waveform ion mobility spectrometry in nontargeted bottom-up proteomics of dried blood spots. J Proteom Res. DOI: 10.1021/acs.jproteome.7b00746
Sarsby et al. Liquid extraction surface analysis mass spectrometry coupled with field asymmetric waveform ion mobility spectrometry for analysis of intact proteins from biological substrates. Analytical Chemistry. DOI: 10.1021/acs.analchem.5b01151
Griffiths et al. Liquid extraction surface analysis field asymmetric waveform ion mobility spectrometry mass spectrometry for the analysis of dried blood spots. Analyst. DOI: 10.1039/C5AN00933B
Sarsby et al. Top-down and bottom-up identification of proteins by liquid extraction surface analysis mass spectrometry of healthy and diseased human liver tissue. J Am Soc Mass Spec. DOI:10.1007/s13361-014-0967-z
Randall et al. Direct analysis of intact proteins from Escherichia coli colonies by liquid extraction surface analysis mass spectrometry. Analytical Chemistry. DOI: 10.1021/ac503349d
Edwards et al. Compound heterozygotes and beta‐thalassemia: Top‐down mass spectrometry for detection of hemoglobinopathies. PROTEOMICS. DOI: 10.1002/pmic.201300316
Martin et al. Dried blood spot proteomics: Surface extraction of endogenous proteins coupled with automated sample preparation and mass spectrometry. J Am Soc Mass Spec. DOI:10.1007/s13361-013-0658-1
Edwards et al. Hemoglobin variant analysis via direct surface sampling of dried blood spots coupled with high-resolution mass spectrometry. Analytical Chemistry. DOI: 10.1021/ac1030804
A: One of the topics of our lab is the interaction of insect pheromones and their analogues with pheromone-binding proteins (PBPs) which are part of an extremely sensitive multi-component pheromone detection system.
We utilize binding assays to determine the function of the PBPs through affinity measurements of the protein-ligand receptors, calculate binding constants, the spatial arrangement of the complex and do modeling. In addition, point-mutated PBPs are used for a better understanding of the contribution of individual amino acids to the binding event.
Q: How does the TriVersa NanoMate® (TVNM) align with your research goals?
A: The TVNM enabled us to develop a high-throughput method to study protein-ligand-interactions for large series of different pheromones and their analogues.
As the binding energies involved are very low and we need to preserve the native structure of the molecules, the soft-ionization conditions of the TVNM are perfect for us. Further, these studies are difficult with classic electrospray, due to the stickiness of the samples. They create problems from short cleaning cycles and produce contaminations.
In contrast, the established method with the TVNM is reliable and stable, and it eliminates the sticky sample issues. In addition, multiple experiments with very low quantities of protein (1 nmol) at different cone-voltage conditions are possible.
We recently added the LESA™ (Liquid Extraction Surface Analysis) capability to the TVNM. This enables us to detect putative signal molecules on leaf surfaces and to track down their production and storage sites by comparing the data with extracts from samples derived from the inner compartments of the leaves.
Q: To whom would you recommend the TriVersa NanoMate for their research?
A: I would recommend the TriVersa NanoMate to everybody because it is a universal source. With direct infusion, coupling for fraction collection and surface analysis, it may replace all ionization sources.
