There is an increasing appreciation that every cell, even of the same type, is different. This complexity, when additionally combined with the variety of different cell types in tissue, is driving the need for spatially resolved omics at the single-cell scale. Rapid advances are being made in genomics and transcriptomics, but progress in metabolomics lags. This is partly because amplification and tagging strategies are not suited to dynamically created metabolite molecules. Mass spectrometry imaging has excellent potential for metabolic imaging. This review summarizes the recent advances in two of these techniques: matrix-assisted laser desorption ionization (MALDI) and secondary ion mass spectrometry (SIMS) and their convergence in subcellular spatial resolution and molecular information. The barriers that have held back progress such as lack of sensitivity and the breakthroughs that have been made including laser-postionization are highlighted as well as the future challenges and opportunities for metabolic imaging at the single-cell scale.

Femtosecond laser desorption postionization mass spectrometry using 7.9 eV single-photon ionization (7.9 eV fs-LDPI-MS) detected three of four drug compounds previously found to have very low ionization efficiencies by secondary ion mass spectrometry. Electronic structure calculations of the ionization energies and other properties of these four drug compounds predicted that all display ionization energies below the 7.9 eV photon energy, as required for single-photon ionization. The 7.9 eV fs-LDPI-MS of carbamazepine, imipramine, and verapamil all showed significant precursor (M+) ion signal, but no representative signal was observed for ciprofloxacin. Furthermore, 7.9 eV fs-LDPI-MS displayed higher M+ signals and mostly similar fragment ions compared with 70 eV electron impact mass spectrometry. Ionization and fragmentation patterns are discussed in terms of calculated wave functions for the highest occupied molecular orbitals. The implications for improving lateral resolution and sensitivity of MS imaging of drug compounds are also considered.

We report the soft laser extraction and production of highly charged peptide and protein ions for mass spectrometry directly from bulk liquid water at atmospheric pressure and room temperature, using picosecond infrared laser ablation. Stable ion signal from singly charged small molecules, as well as highly charged biomolecular ions, from aqueous solutions at low laser pulse fluence (∼0.3 J cm) is demonstrated. Sampling via single picosecond laser pulses is shown to extract less than 27 pL of volume from the sample, producing highly charged peptide and protein ions for mass spectrometry detection. The ablation and ion generation is demonstrated to be soft in nature, producing natively folded proteins ions under sample conditions described for native mass spectrometry. The method provides laser-based sampling flexibility, precision and control with highly charged ion production directly from water at low and near neutral pH. This approach does not require an additional ionization device or high voltage applied directly to the sample.

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The benzyltriphenylphosphonium (BTP) thermometer ion is utilized to characterize the fragmentation mechanisms of matrix-assisted laser desorption/ionization (MALDI) for femtosecond ultraviolet laser pulses. We demonstrate that the survival yield of BTP approaches unity under these conditions, which suggests that a minimal amount of fragmentation is occurring. It is also shown that the survival yield of BTP is insensitive to the laser fluence. However, the magnitude of fragmentation for the matrix increased notably for the same fluence range. These results indicate that the amount of energy transferred from the matrix ions to the BTP thermometer ions is minimal because the femtosecond desorption applied here occur within the stress-confinement regime. This observation is in agreement with recent molecular dynamics simulations which predict that it should be possible to separate both desorption and ionization processes in the regime of stress-confined desorption. Our results indicate that angiotensin is the largest biomolecule which could be routinely measured with these pulses. A mass upper-limit supports the hypothesis that ionization is hindered by the increased thermal gradients imposed in the lattice and associated velocity distribution within the ablation process from the much higher lattice heating rate with femtosecond pulses. This effect results in the temporal overlap between the neutral molecules and the matrix ions being too small to result in sufficient proton exchange for ionization. Graphical TOC Entry2

VUV action spectroscopy has recently gained interest for the study of peptides and proteins. However, numerous aspects of the fundamental processes involved in the photodissociation are yet to be understood. It can, for example, be expected that sulfur-containing amino-acid residues have a significant impact on the dissociation processes following photoionization because of their potential involvement in the transport of electron holes in proteins. In order to investigate the influence of the sulfur-containing methionine residue on the VUV photodissociation of a small peptide a VUV action spectroscopy study of gas-phase protonated methionine-enkephalin and leucine-enkephalin in the photon energy range of 6–35 eV was performed. The results show that upon non-ionizing photoexcitation, the fragmentation patterns of the two peptides are nearly identical, whereas significant differences were observed upon photoionization. The differences between the fragment yields and the identified specific dissociation channels for methionine-enkephalin could be explained by the high electron hole affinity of sulfur, which efficiently directs the radical to the methionine side chain. Additionally, for both peptides the presence of the intact photoionized precursor ions was confirmed by their isotopic patterns and the stability of these species could be evaluated. Graphic abstract

The deconvolution of low-resolution time-of-flight data has numerous advantages including the ability to extract additional information from the experimental data. We augment the well-known Lucy-Richardson deconvolution algorithm by various Bayesian prior distributions and show that a prior of second-differences of the signal outperforms the standard Lucy-Richardson algorithm by accelerating the rate of convergence by a factor of two and preserving the peak amplitude ratios of a larger fraction of the total peaks. A stopping criterion and boosting mechanism is implemented to ensure that these methods converge to the same final entropy and that local minima are avoided.Improvement by a factor of two in mass resolution of the signal allowed more accurate quantification of the spectra. The general method is demonstrated in this paper by the deconvolution of fragmentation pathway peaks of the benzyltriphenylphosphonium thermometer ion following femtosecond ultraviolet laser desorption.

The fragmentation mechanisms of matrix-assisted laser desorption/ionization (MALDI) for femtosecond ultraviolet laser pulses in a transmission geometry are characterized on the basis of the well-known benzyltriphenylphosphonium (BTP) thermometer ion. We demonstrate that the survival yield of BTP approaches unity under these conditions, which suggests that a minimal amount of fragmentation is occurring. It is shown that, while the survival yield of BTP is insensitive to the fluence within the studied fluence range, the magnitude of fragmentation for the matrix increased notably with increasing fluence. While nonlinear absorption and ionization are expected to lead to large matrix fragmentation rates, the high BTP survival yields indicate a reduced amount of energy being transferred from the matrix to these BTP thermometer ions. The femtosecond ablation employed here results in increased heating rates and occurs within the fully stress-confinement regime, which minimizes the matrix-analyte interaction during the ablation event. This interpretation is supported by our finding that angiotensin was the largest biomolecule which could be routinely be measured with femtosecond pulses. The spatio-temporal overlap between a neutral biomolecule and matrix ions resulting from this process is too short to result in sufficient proton exchange for ionization.<br>