Substrate-Mediated Laser Ablation under Ambient Conditions for Spatially-Resolved Tissue Proteomics

Abstract

Numerous applications of ambient Mass Spectrometry (MS) have been demonstrated over the past decade. They promoted the emergence of various micro-sampling techniques such as Laser Ablation/Droplet Capture (LADC). LADC consists in the ablation of analytes from a surface and their subsequent capture in a solvent droplet which can then be analyzed by MS. LADC is thus generally performed in the UV or IR range, using a wavelength at which analytes or the matrix absorb. In this work, we explore the potential of visible range LADC (532 nm) as a micro-sampling technology for large-scale proteomics analyses. We demonstrate that biomolecule analyses using 532 nm LADC are possible, despite the low absorbance of biomolecules at this wavelength. This is due to the preponderance of an indirect substrate-mediated ablation mechanism at low laser energy which contrasts with the conventional direct ablation driven by sample absorption. Using our custom LADC system and taking advantage of this substrate-mediated ablation mechanism, we were able to perform large-scale proteomic analyses of micro-sampled tissue sections and demonstrated the possible identification of proteins with relevant biological functions. Consequently, the 532 nm LADC technique offers a new tool for biological and clinical applications.

Pulse Duration Effects on Solution-Phase Protein Desorption in Laser Electrospray Mass Spectrometry

“…However, the intensities accessible via the 55 fs and 56 ps pulses result in a high photon density capable of driving nonresonant, multiphoton absorption to overcome the band gap of quartz or couple into the molecular system of the myoglobin . Previous studies have shown that no mass spectral signal for bradykinin or phosphocholine was achieved from silica glass using a ns pulse with laser pulse energies from 0 to 2.4 mJ without the addition of a resonantly absorbing matrix . Regardless of wavelength, the efficiency of ns laser ablation relies upon the presence of a resonance within the sample or substrate .…”

“…A similar saturation in the mass spectral signal was observed in the 532 nm, 4 ns laser ablation energy optimization of aqueous bradykinin peptide, phosphatidyl choline, and lysozyme C protein from stainless-steel substrates. 42 At the highest power tested, the 55 fs pulse shows an increase in signal intensity with a large standard deviation (305.0 ± 232.6), while a decrease in signal was observed for the 56 ps pulse (58.0 ± 2.1). Previous investigations showed that comparable mass spectral signal intensities for absorbing (∼10 3 cm −1 ) and nonabsorbing (∼0.2 cm −1 ) analytes were similar when using equivalent laser fluences from stainless-steel substrates.…”

“…Previous investigations showed that comparable mass spectral signal intensities for absorbing (∼10 3 cm −1 ) and nonabsorbing (∼0.2 cm −1 ) analytes were similar when using equivalent laser fluences from stainless-steel substrates. 42,43 It was concluded that the ablation mechanism for nonabsorbing samples was substrate-mediated and was not due to the direct coupling of the laser radiation with the sample. In this study, the removal of the myoglobin is related to the optical properties of the irradiated substrate and the corresponding pulse duration.…”

“…46 Previous studies have shown that no mass spectral signal for bradykinin or phosphocholine was achieved from silica glass using a ns pulse with laser pulse energies from 0 to 2.4 mJ without the addition of a resonantly absorbing matrix. 42 Regardless of wavelength, the efficiency of ns laser ablation relies upon the presence of a resonance within the sample or substrate. 24 A weak absorption in the visible region by liquid water contributes to absorption at ∼790 nm.…”

In situ mass spectrometry analysis of intact proteins and protein complexes from biological substrates

“…Analysis of material by laser ablation/droplet capture was demonstrated in a spatially resolved proteomics strategy [71]. When combined with a nanoPOTS workflow, hundreds of proteins were identified from 50 μm diameter laser-dissected tissue samples; however, the workflow inherently relies on enzymatic digestion and liquid chromatography separation to achieve these numbers.…”