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.
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.
The role of laser wavelength in atomic force microscopy (AFM) tip-enhanced laser ablation was studied using an apertureless tip and a nanosecond pulsed laser. An optical parametric oscillator (OPO) laser wavelength tunable from 410 to 2400 nm was used to irradiate a gold-coated silicon AFM probe held 15 nm above the surface of an anthracene film. The absorption of laser energy by the tip at 532 nm is sufficient to melt the gold coating and increase the diameter of the tip from about 100 nm to approximately 1 µm. The ablation crater volume was measured and found to have a maximum at 500 nm and an approximately linear drop to 800 nm. Craters could not be produced between 800 and 1200 nm and the crater was slightly smaller at 450 nm compared to 500 nm. A crater rim was observed with a volume comparable to that of the crater but lower density. The mechanism of ablation is postulated to be the result of energy absorption by the tip through plasmon resonance of the gold coating followed by melting of the anthracene by ballistic, contact, or radiative heating of the anthracene film.
We have developed a new ionization approach for matrix-assisted ionization with high temporal resolution using an electrically actuated pulsed valve. Matrix and analyte samples are deposited on a thin metal foil and placed at the inlet of an ambient ionization mass spectrometer. When the pulsed valve is actuated, a short puff of high pressure gas impinges on the foil and ejects particulate from the sample on the opposite side. Highly charged ions are formed from the particles at the mass spectrometer inlet. Using this source, multiply charged protein ions are produced within a selectable 4 second time window.
A systematic method for evaluation of MALDI profiling and imaging was developed and applied to the use of three surfactants, sodium dodecyl sulfate (SDS), Triton X-100, and Tween 20, on rat brain tissue. For profiling studies, mass spectra were acquired from regular arrays of spots with manually deposited surfactant and matrix. The studies recorded the total number of peaks in the mass spectra from 2 to 20 kDa and compared the number of peaks and peak intensities with and without surfactant. It was found that SDS decreases the total number of peaks at all concentrations but does lead to an increase in the number of peaks below 5 kDa. Triton X-100 at 0.05% concentration yielded the highest number of peaks and highest number of new peaks, with the best results above 5 kDa. Correlation of the increase in signal with the estimated hydrophobicity suggests that Triton X-100 improves mass spectrometry quality through an increase in the intensity of hydrophobic protein peaks. Tween 20 provided good performance at 0.05% concentration across all mass ranges. For imaging studies, multiple images were obtained and the integrated intensity ratio for images obtained with and without surfactant was compared for 10 selected peaks. It was found that SDS tends to degrade imaging performance whereas Triton X-100 and Tween 20 improved performance compared to no surfactant, especially above 7 kDa.
Mass spectrometry imaging (MSI) was introduced more than five decades ago with secondary ion mass spectrometry (SIMS) and a decade later with laser desorption/ionization (LDI) mass spectrometry (MS). Large biomolecule imaging by matrix-assisted laser desorption/ionization (MALDI) was developed in the 1990s and ambient laser MS a decade ago. Although SIMS has been capable of imaging with a moderate mass range at sub-micrometer lateral resolution from its inception, laser MS requires additional effort to achieve a lateral resolution of 10 lm or below which is required to image at the size scale of single mammalian cells. This review covers untargeted large biomolecule MSI using lasers for desorption/ionization or laser desorption and post-ionization. These methods include laser microprobe (LDI) MSI, MALDI MSI, laser ambient and atmospheric pressure MSI, and near-field laser ablation MS. Novel approaches to improving lateral resolution are discussed, including oversampling, beam shaping, transmission geometry, reflective and through-hole objectives, microscope mode, and near-field optics.
We have developed a mid-infrared laser ablation sampling technique for nano-flow liquid chromatography coupled with tandem mass spectrometry proteomic profiling of discrete regions from biological samples. Laser ablation performed in transmission geometry was used to transfer material from 50-μm thick tissue sections mounted on a glass microscope slide to a capturing solvent. Captured samples were processed using filter-aided sample preparation and enzymatically digested to produce tryptic peptides for data-dependent analysis with an ion trap mass spectrometer. Comparison with ultraviolet laser capture microdissec- tion from neighboring regions on the same tissue section revealed that infrared laser ablation transfer has higher reproducibility between samples from different consecutive sections. Both techniques allowed for proteomics investigation of different orga- nelles without the addition of surfactants.