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.
F. Donnarumma, F. Cao, & K. K. Murray, J. Am. Soc. Mass Spectrom. (2015) DOI: 10.1007/s13361-015-1249-0
We have developed a laser ablation sampling technique for matrix-assisted laser desorption ionization (MALDI) mass spectrometry and tandem mass spectrometry (MS/MS) analyses of in-situ digested tissue proteins. Infrared laser ablation was used to remove biomolecules from tissue sections for collection by vacuum capture and analysis by MALDI. Ablation and transfer of compounds from tissue removes biomolecules from the tissue and allows further analysis of the collected material to facilitate their identification. Laser ablated material was captured in a vacuum aspirated pipette-tip packed with C18 stationary phase and the captured material was dissolved, eluted, and analyzed by MALDI. Rat brain and lung tissue sections 10 μm thick were processed by in-situ trypsin digestion after lipid and salt removal. The tryptic peptides were ablated with a focused mid-infrared laser, vacuum captured, and eluted with an acetonitrile/water mixture. Eluted components were deposited on a MALDI target and mixed with matrix for mass spectrometry analysis. Initial experiments were conducted with peptide and protein standards for evaluation of transfer efficiency: a transfer efficiency of 16% was obtained using seven different standards. Laser ablation vacuum capture was applied to freshly digested tissue sections and compared with sections processed with conventional MALDI imaging. A greater signal intensity and lower background was observed in comparison with the conventional MALDI analysis. Tandem time-of-flight MALDI mass spectrometry was used for compound identification in the tissue.
C. A. Seneviratne, S. Ghorai & K. K. Murray, Rapid Commun. Mass Spectrom. in press
RATIONALE: Ambient mass spectrometry can detect small molecules directly, but complex mixtures can be a challenge. We have developed a method that incorporates small molecule separation based on laser desorption with 80 capture on a solid-phase microextraction (SPME) fiber for injection into a gas chromatography/mass spectrometry 81 (GCMS) system.
METHODS: Samples on a metal target were desorbed by a 3 μm mid-infrared laser focused to a 250 μm spot and 1.2 mJ pulse energy. The desorbed material was aspirated into a metal tube suspended 1 mm above the laser spot and captured 84 on a SPME fiber. The collected material was injected into a GC/MS instrument for analysis.
RESULTS: We have developed a versatile approach for ambient laser desorption sampling onto SPME for GC/MS analysis. The performance of the laser desorption SPME capture GC/MS system was demonstrated for small molecule 87 standards, a mixture of nitroaromatic explosives, and collected cigarette smoke.
CONCLUSIONS: The utility of ambient laser desorption sampling onto SPME for GC/MS was demonstrated. The performance of the method was evaluated by preparing calibration standards of caffeine over a range from 200 to 90 1000 ng. Laser desorption ambient sampling of complex mixtures was accomplished using SPME GC/MS.
Atomic force microscope (AFM) tip-enhanced laser ablation was used to transfer molecules from thin films to a suspended silver wire for off-line mass spectrometry using laser desorption ionization (LDI) and matrix-assisted laser desorption ionization (MALDI). An AFM with a 30 nm radius gold-coated silicon tip was used to image the sample and to hold the tip 15 nm from the surface for material removal using a 355 nm Nd:YAG laser. The ablated material was captured on a silver wire that was held 300 μm vertically and 100 μm horizontally from the tip. For the small molecules anthracene and rhodamine 6G, the wire was cut and affixed to a metal target using double-sided conductive tape and analyzed by LDI using a commercial laser desorption time-of-flight mass spectrometer. Approximately 100 fg of material was ablated from each of the 1 μm ablation spots and transferred with approximately 3% efficiency. For larger polypeptide molecules angiotensin II and bovine insulin, the captured material was dissolved in saturated matrix solution and deposited on a target for MALDI analysis.
Al Ghafly, Siraj, Das, Regmi, Magut, Galpothdeniya, Murray, Warner, Rapid Commun. Mass Spectrom. 2014, 28, 2307; DOI: 10.1002/rcm.7027.
Detection of hydrophobic peptides remains a major obstacle for matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS). This stems from the fact that most matrices for MALDI are hydrophilic and therefore have low affinities for hydrophobic peptides. Herein, 1-aminopyrene (AP) and AP-derived group of uniform materials based on organic salts (GUMBOS) as novel matrices for MALDI-MS analyses of peptides were investigated for hydrophobic and hydrophilic peptides.
A number of solid-phase AP-based GUMBOS are synthesized with variable hydrophobicity simply by changing the counterions. Structures were confirmed by use of 1H NMR and electrospray ionization mass spectrometry (ESI-MS). 1-Octanol/water partition coefficients (Ko/w) were used to measure the hydrophobicity of the matrices. A dried-droplet method was used for sample preparation. All spectra were obtained using a MALDI-TOF mass spectrometer in positive ion reflectron mode.
A series of AP-based GUMBOS was synthesized including [AP][chloride] ([AP][Cl]), [AP][ascorbate] ([AP][Asc]) and [AP][bis(trifluoromethane)sulfonimide] ([AP][NTf2]). The relative hydrophobicities of these compounds and α-cyano-4-hydroxycinnamic acid (CHCA, a common MALDI matrix) indicated that AP-based GUMBOS can be tuned to be much more hydrophobic than CHCA. A clear trend is observed between the signal intensities of hydrophobic peptides and hydrophobicity of the matrix.
MALDI matrices of GUMBOS with tunable hydrophobicities are easily obtained simply by varying the counterion. We have found that hydrophobic matrix materials are very effective for MALDI determination of hydrophobic peptides and, similarly, the more hydrophilic peptides displayed greater intensity in the more hydrophilic matrix.