RNA Sampling from Tissue Sections using Infrared Laser Ablation

K. Wang, F. Donnarumma, S.W. Herke, C. Dong, P.F. Herke, K.K. Murray, RNA sampling from tissue sections using infrared laser ablation, Anal. Chim. Acta. 1063 (2019) 91–98. doi:10.1016/j.aca.2019.02.054.

RNA Sampling from Tissue Sections using Infrared Laser Ablation
RNA Sampling from Tissue Sections using Infrared Laser Ablation

RNA was obtained from discrete locations of frozen rat brain tissue sections through infrared (IR) laser ablation using a 3-μm wavelength in transmission geometry. The ablated plume was captured in a microcentrifuge tube containing RNAse-free buffer and processed using a commercial RNA purification kit. RNA transfer efficiency and integrity were evaluated based on automated electrophoresis in microfluidic chips. Reproducible IR-laser ablation of intact RNA was demonstrated with purified RNA at laser fluences of 3-5 kJ/m2 (72±12% transfer efficiency) and with tissue sections at a laser fluence of 13 kJ/m2 (79±14% transfer efficiency); laser energies were attenuated ∼20% by the soda-lime glass slides used to support the samples. RNA integrity from tissue ablation was >90% of its original RIN value (∼7) and the purified RNA was sufficiently intact for conversion to cDNA and subsequent qPCR assay.

Patent: Tip Enhanced Laser Assisted Sample Transfer for Biomolecule Mass Spectrometry

Link

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.

Tip-enhanced laser ablation and capture of DNA

F. Cao, F. Donnarumma, K.K. Murray, Tip-enhanced laser ablation and capture of DNA, Appl. Surf. Sci. 476 (2019) 658–662. doi:10.1016/j.apsusc.2019.01.104.

Abstract: Tip-enhanced laser ablation was used to extract DNA plasmid for polymerase chain reaction (PCR) amplification. A 532 nm nanosecond laser was directed onto a gold coated atomic force microscopy (AFM) tip 10 nm above a sample surface to ablate a 7.1 kbp green fluorescent protein (GFP) plasmid DNA sample on a glass coverslip. The ablated material was captured on a metal ribbon 300 µm above the sample surface. The ablation craters had diameters from 1 to 2 µm and an average volume of 0.14 µm3. PCR and nested PCR were employed for the amplification of the ablated DNA. The quantity of sample from each ablation crater for PCR amplification was 20 ag.

Tip-enhanced laser ablation and capture of DNA
The proposed technology allows topographical imaging with atomic force microscopy (AFM) and extraction of DNA via tip-enhanced laser ablation using the same tip. Plasmid DNA is imaged with a gold coated tip and extracted using a pulsed laser with a sampling size of 1 µm. The captured DNA can be amplified by polymerase chain reaction (PCR) and nested PCR.

Broadband ion mobility deconvolution for rapid analysis of complex mixtures

M.E. Pettit, M.R. Brantley, F. Donnarumma, K.K. Murray, T. Solouki, Broadband ion mobility deconvolution for rapid analysis of complex mixtures, Analyst. 143 (2018) 2574–2586. doi:10.1039/c8an00193f.

High resolving power ion mobility (IM) allows for accurate characterization of complex mixtures in high-throughput IM mass spectrometry (IM-MS) experiments. We previously demonstrated that pure component IM-MS data can be extracted from IM unresolved post-IM/collision-induced dissociation (CID) MS data using automated ion mobility deconvolution (AIMD) software [Matthew Brantley, Behrooz Zekavat, Brett Harper, Rachel Mason, and Touradj Solouki, J. Am. Soc. Mass Spectrom., 2014, 25, 1810-1819]. In our previous reports, we utilized a quadrupole ion filter for m/z-isolation of IM unresolved monoisotopic species prior to post-IM/CID MS. Here, we utilize a broadband IM-MS deconvolution strategy to remove the m/z-isolation requirement for successful deconvolution of IM unresolved peaks. Broadband data collection has throughput and multiplexing advantages; hence, elimination of the ion isolation step reduces experimental run times and thus expands the applicability of AIMD to high-throughput bottom-up proteomics. We demonstrate broadband IM-MS deconvolution of two separate and unrelated pairs of IM unresolved isomers (viz., a pair of isomeric hexapeptides and a pair of isomeric trisaccharides) in a simulated complex mixture. Moreover, we show that broadband IM-MS deconvolution improves high-throughput bottom-up characterization of a proteolytic digest of rat brain tissue. To our knowledge, this manuscript is the first to report successful deconvolution of pure component IM and MS data from an IM-assisted data-independent analysis (DIA) or HDMSE dataset.

IMSC 2018: Infrared Laser Ablation Microsampling Coupled with MALDI Imaging

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.

IR laser ablation microsampling

Introduction
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.

Methods
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.

Results
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.

Conclusions
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 28, 2018

Louisiana State University: Kermit K. Murray, Fabrizio Donnarumma, Kelin Wang, Carson W. Szot, Chao Dong,
Baylor University: Touradj Solouki, Michael E. Pettit

Introduction
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.

Methods
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.

Results
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.

Conclusions
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.

IMSC 2018: Combined Infrared and Ultraviolet Ablation Electrospray Ionization Mass Spectrometry

International Mass Spectrometry Conference, Florence, Italy, August 27, 2018

IMSC 2018 : Combined Infrared and Ultraviolet Ablation Electrospray Ionization Mass Spectrometry
IMSC 2018 : Combined Infrared and Ultraviolet Ablation Electrospray Ionization Mass Spectrometry

Kermit K. Murray, Remi O. Lawal, and Fabrizio Donnarumma
Louisiana State University, Baton Rouge, LA

Introduction

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.

Methods

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.

Results

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.

Conclusions

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, Infrared laser ablation sampling coupled with data independent high resolution UPLC-IM-MS/MS for tissue analysis, Anal. Chim. Acta. 1034 (2018) 102–109. doi:10.1016/j.aca.2018.06.066.

Infrared laser ablation sampling coupled with data independent high resolution UPLC-IM-MS/MS for tissue analysis 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.

ASMS 2018: MALDI Imaging and Laser Ablation Sampling for Analysis of Fungicide Distribution in Apples

ThOC 8:50

Igor Pereira, Bijay Banstola, Kelin Wang, Boniek Gontijo Vaz, Fabrizio Donnarumma, and Kermit K. Murray

Louisiana State University & Federal University of Goiás

Imazalil is a postharvest fungicide widely used for mold control in apples and has been classified as a potential cancerogenic in the NIH Hazardous Substances Data Bank. Imazalil is applied to apples by dipping, drenching, or spraying using concentrations between 1000 and 2000 ppm. The fungicide can penetrate the exocarp (outer skin) and diffuse into the interior mesocarp region of the fruit. MS analysis of imazalil treated apples is commonly conducted using the entirety of the fruit, which results in loss of localization information and does not provide any information about the rate of penetration. MALDI imaging can provide localization of imazalil and laser ablation can extract the compound with minimal sample prep for subsequent ESI MS.

MALDI image of fungicide in apple
MALDI image showing penetration of fungicide in an apple after 1, 4, and 7 days.
Igor Pereira

Infrared laser ablation and capture of enzymes with conserved activity

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).

Graphical abstract: Infrared laser ablation and capture of enzymes with conserved activity
Graphical abstract: Infrared laser ablation and capture of enzymes with conserved activity

Abstract

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.