R.O. Lawal, F. Donnarumma, K.K. Murray, Deep-ultraviolet laser ablation electrospray ionization mass spectrometry, J. Mass Spectrom.54 (2019) 281–287. doi:10.1002/jms.4338.
A 193‐nm wavelength deep ultraviolet laser was used for ambient laser ablation
electrospray ionization mass spectrometry of biological samples. A pulsed ArF excimer
laser was used to ablate solid samples, and the resulting plume of the desorbed material
merged with charged electrospray droplets to form ions that were detected with a
quadrupole time‐of‐flight mass spectrometer. Solutions containing peptide and protein
standards up to 66‐kDa molecular weight were deposited on a metal target,
dried, and analyzed. No fragmentation was observed from peptides and proteins as
well as from the more easily fragmented vitamin B12 molecule. The mass spectra
contained peaks from multiply charged ions that were identical to conventional
electrospray. Deep UV laser ablation of tissue allowed detection of lipids from
untreated tissue. The mechanism of ionization is postulated to involve absorption of
laser energy by a fraction of the analyte molecules that act as a sacrificial matrix or
by residual water in the sample.
Analytica Chimica Acta Kelin Wang, Fabrizio Donnarumma, Scott W. Herke, Chao Dong, Patrick F. Herke, Kermit K. Murray
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.
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 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.
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.