Disclosed are various embodiments for transferring molecules from a surface for mass spectrometry and other sample analysis methods, and the like. A laser is focused onto a tip of an atomic force microscope to remove and capture a quantity of molecules from the surface, so they can be transferred to a mass spectrometer or another instrument for analysis.
International Mass Spectrometry Conference, Florence, Italy, August 28, 2018
Louisiana State University: Kermit K. Murray, Fabrizio Donnarumma, Kelin Wang, Carson W. Szot, Chao Dong,
Baylor University: Touradj Solouki, Michael E. Pettit
Matrix-assisted laser desorption/ionization (MALDI) mass spectrometry imaging (MSI) is a powerful method for determining the location of biomolecules in tissue; however, protein identification and quantification remains challenging. The goal of this project is to develop an imaging workflow that combines MALDI imaging with laser ablation microsampling for liquid chromatography tandem mass spectrometry.
In the combined workflow, MALDI imaging is used to identify regions of interest (ROI) from intact proteins. The ROI are sampled using infrared laser ablation and the captured material is analyzed by LC-MS/MS using data independent acquisition to identify and quantify the proteins. The data are cross-correlated to identify the localized proteins in the MALDI images.
Development of the combined approach is aimed at creating an automated system for ablation and capture and using it in a coupled workflow of MALDI imaging and LC MS/MS analysis. The infrared laser ablation and capture system uses a mid-infrared optical parametric oscillator laser with a custom reflective objective that has a large working distance and good numerical aperture. We have developed custom positioning software that allows MALDI MSI heat maps to be overlaid on camera images to co-register ROI ablation with the IR laser. Tissue sections are mounted on conductive microscope slides and either consecutive sections or MALDI analyzed sections can be used. Laser ablated proteins are digested with magnetic capture beads and the peptides released for analysis with a Waters nanoAcquity UPLC system coupled to a Synapt G2-HDMS.
MALDI MSI coupled with region-specific laser ablation sampling for LC MS/MS is a fast and versatile approach for spatially resolved tissue proteomics. We have demonstrated that proteins can be identified from spatially localized regions and are developing new methods for correlating the intact proteins observed in MALDI with the proteins identified by tandem mass spectrometry.
Novel Aspect: Coupled MALDI imaging with high precision infrared laser ablation capture for LC MS/MS for protein identification and quantification.
International Mass Spectrometry Conference, Florence, Italy, August 27, 2018
Kermit K. Murray, Remi O. Lawal, and Fabrizio Donnarumma
Louisiana State University, Baton Rouge, LA
We are using combined mid-infrared and deep-ultraviolet two-laser ablation coupled with electrospray ionization for ambient mass spectrometry of biomolecules in tissue. The goal is to increase nanoparticle production and improve sensitivity by using the UV laser to disrupt the tissue structure followed by IR ablation and ionization.
In this work, we are using a 193 nm ArF excimer laser to disrupt the tissue prior to irradiating with a 3000 nm IR optical parametric oscillator. Both lasers are focused onto the same target spot and separated in time by an adjustable delay. A nanospray needle is directed at the inlet on-axis of a modified quadrupole time-of-flight mass spectrometer.
The two-laser ablation system has been constructed and initial studies carried out for optimization of the system with peptide and protein standards. The lasers are mounted on an aluminum breadboard adjacent to the ion source and are focused onto the target with a single calcium fluoride lens for each beam. The dual-laser configuration can be operated either with the UV firing first to disrupt the covalent bonding in the tissue or with the IR firing first to heat the tissue. Initial studies with 193 nm laser ablation electrospray ionization demonstrate that the deep-UV is a much softer ionization method than might be anticipated and can produce ions from peptides and proteins without a matrix and with little fragmentation. Based on this interesting new result, initial experiments are aimed at improving the efficiency of the deep-UV ablation using IR laser pre-heating.
We have demonstrated that deep-UV and IR ablation coupled with electrospray ionization is a promising soft ionization method for large molecules with applications to tissue imaging. Continuing experiments are aimed at optimizing the UV and IR laser pulse energies and time delay to improve the sensitivity as well as improving the UV laser focus to improve spatial resolution.
Novel Aspect: Combined mid-infrared and deep-ultraviolet laser ablation on the same spot for laser ablation electrospray ionization imaging.
Infrared laser ablation sampling coupled with data independent high resolution UPLC-IM-MS/MS for tissue analysis
M. E. Pettit, F. Donnarumma, K. K. Murray, & T. Solouki, Analytica Chimica Acta in Press, DOI: 10.1016/j.aca.2018.06.066
Infrared laser ablation microsampling was used with data-dependent acquisition (DDA) and ion mobility-enhanced data-independent acquisition (HDMSE) for mass spectrometry based bottom-up proteomics analysis of rat brain tissue. Results from HDMSE and DDA analyses of the 12 laser ablation sampled tissue sections showed that HDMSE consistently identified approximately seven times more peptides and four times more proteins than DDA. To evaluate the impact of ultra-performance liquid chromatography (UPLC) peak congestion on HDMSE and DDA analysis, whole tissue digests from rat brain were analyzed at six different UPLC separation times. Analogous to results from laser ablated samples, HDMSE analyses of whole tissue digests yielded about four times more proteins identified than DDA for all six UPLC separation times.
Wang, K., Donnarumma, F., Baldone, M. D., & Murray, K. K. Infrared laser ablation and capture of enzymes with conserved activity. Anal Chim Acta, 1027, 41–46 (2018).
Infrared (IR) laser ablation at 3 μm wavelength was used to extract enzymes from tissue and quantitatively determine their activity. Experiments were conducted with trypsin, which was ablated, captured and then used to digest bovine serum albumin (BSA). BSA digests were evaluated using matrix-assisted laser desorption ionization (MALDI) mass spectrometry (MS) and sequence coverage of 59% was achieved. Quantification was performed using trypsin and catalase standards and rat brain tissue by fluorescence spectroscopy. Both enzymes were reproducibly transferred with an efficiency of 75 ± 8% at laser fluences between 10 and 30 kJ/m2. Trypsin retained 37 ± 2% of its activity and catalase retained 50 ± 7%. The activity of catalase from tissue was tested using three consecutive 50 μm thick rat brain sections. Two 4 mm2 regions were ablated and captured from the cortex and cerebellum regions. The absolute catalase concentration in the two regions was consistent with previously published data, demonstrating transfer of intact enzymes from tissue.