In living organisms, the compositions of biomolecules such as proteins, nucleic acids, sugars, lipids, and metabolites change continuously and dynamically in response to a variety of environmental factors. The levels of these molecules in living tissues are precisely regulated to maintain homeostasis. Therefore, in many cases, the distribution of these molecules in tissues or cells can provide valuable information for basic biological research, diagnosis of certain diseases, and identification of therapeutic targets. Conventionally, the biodistribution of such molecules has been examined by cell/tissue ‘homogenization’ methods, which provide only biochemical information, not information on the spatial distribution of target molecules in tissues. On the contrary, spectroscopic visualizations such as fluorescence tissue imaging can reveal the spatial distribution of molecules; however, they do not allow for molecular analysis. For these reasons, imaging mass spectrometry (IMS) has been developed and extensively studied; IMS allows for both visualization of tissues to give positional information on targets and molecular analysis of targets by affording the molecular weights (1-3). In addition, the advantages of mass spectrometry (MS) are manifold: i) MS provides chemical and structural information on targets, ii) MS can easily discriminate between true analytes and background and, therefore, eliminate false positive signals, and iii) MS can be used to monitor multiple analytes simultaneously. Matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) MS is particularly effective and is generally used for IMS due to its suitability for tissue analysis and the ‘soft’ ionization of large biomolecules such as proteins and oligonucleotides (4, 5). In general, a solution of organic matrix, including 2,5-dihydroxybenzoic acid, sinapinic acid, a-cyano-4-hydroxycinnamic acid, and 2,4,6-trihydroxyacetophenone, depending on the types of analytes present, is deposited on a thin section of a tissue, which is then scanned by MALDI-TOF MS to give a raster image of the distribution of biomolecules as revealed by relative signal intensities. However, the requirement for an organic matrix limits the applicability of MALDI IMS. The signals in MALDI IMS, in many cases, are strongly affected by the choice of a proper matrix and solvents, co-crystallization of a matrix and analytes, and, particularly, homogeneity of matrix deposition, leading to poor shot-to-shot and sample-to-sample reliability. Therefore, peak intensities, i.e. raster images, would not represent the real spatial distribution of target molecules in tissues. In addition, the requirement for an organic matrix hampers imaging of small molecules such as drugs and their metabolites owing to interference of the matrix in the low-mass region. Thus, various organic matrix-free LDI IMS systems have been developed to avoid the problems described above, mostly utilizing nanostructured surfaces and inorganic nanoparticles (NPs) as an alternative to the organic matrix.
This minireview starts with a brief introduction to organic matrix-free LDI MS for small molecule analysis and focuses on the progress that has been made in organic matrix-free LDI IMS methods, which are categorized into i) nanostructured surface-assisted LDI IMS and ii) inorganic nanoparticle-assisted LDI IMS. In addition, IMS methods using other ‘soft’ ionization methods such as desorption electrospray ionization (DESI) and laser ablation electrospray ionization (LAESI) will be discussed, followed by a brief discussion on the use of secondary ion mass spectrometry (SIMS), which have the advantages of little or no preparation, ease of implementation, and simplified analysis in ambient environments.
The main purpose of this minireview is to give the reader an unbiased description of the approaches for molecular level analyses of biological sample surfaces using MS, particularly for small molecules. This minireview, therefore, will not only offer a starting point for students and researchers entering this field but also be valued by active researchers requiring small molecule analyses of tissues in various areas including disease pathology, diagnostics, drug delivery systems, metabolomics, lipidomics, and pharmacokinetics.
Organic matrix-free LDI MS mostly utilizes nanostructured surfaces and inorganic NPs and is known as surface-assisted LDI (SALDI) MS (6-8). SALDI materials transfer sufficient energy from the irradiated laser to analytes for desorption/ionization without damaging the analytes and causing fragmentation, and therefore have been used as matrices for analysis of small molecules. In addition, nanostructures of the SALDI materials can provide efficient loading capacities due to large surface area, and analytes can be concentrated on the NPs by ionic strength, hydrophobic interactions, covalent binding, or bio-specific interactions through surface modifications, resulting in high sensitivity (9). As a pioneering work, Siuzdak and co-workers reported desorption-ionization on a porous silicon (DIOS) surface which was produced from flat nanocrystalline silicon through a simple etching procedure (10). Small molecule analytes including peptides (
The following two sections describe organic matrix-free LDI IMS with grafting, the distinct feature of SALDI MS in small molecule analysis, as discussed above: i) nanostructured surfaces on which thin tissues are deposited, and ii) inorganic NPs which are sprayed onto thin tissues (Fig. 1).
By utilizing the DIOS technique, which allows for small molecule analysis, Liu
As an advanced use of DIOS, nanostructure initiator MS (NIMS) was introduced by the Siuzdak group for porous silicon-based mass analysis (20). NIMS uses a nanostructured silicon surface composed of roughly 10 nm pores to trap initiator molecules, such as fluorinated siloxane, lauric acid, and polysiloxane. The NIMS surface is exposed to laser irradiation resulting in vaporization or fragmentation of the initiator molecules and subsequent desorption/ionization of the absorbed analyte on the NIMS surface. The lipids (
In addition to porous silicon-based surfaces, carbon-based surfaces have also been used for matrix-free IMS. Many types of carbon-based materials, including functionalized carbon nanotubes and graphene oxides, have been suggested as alternative matrices for matrix-free IMS due to their absorption properties and efficient energy transfer to analytes. Kim
The performance of inorganic NPs as matrices for LDI MS depends strongly on their size, morphology, composition, and concentration. In this respect, AuNPs and AgNPs are the most often studied and widely used materials in LDI-MS because their size is readily tunable and various shapes and compositions can be prepared depending on the researcher’s purpose. Goto-Inoue
AgNPs are also actively used for IMS. Hayasaka
Since the first introduction of the graphite surface-assisted detection of proteolytic digests (
NPs of metal oxides have been utilized for SALDI materials due to their unique structures and compositions. The Setou group demonstrated IMS of lipids and peptides at cellular resolution (15 mm) using extremely small iron oxide NPs (3.7 nm in diameter) flanked by amorphous silicates with hydroxyl and amino groups on their surfaces (45). These hydrophilic functional groups facilitate the ionization of adsorbed analytes through not only efficient energy transfer but also preferential sodium/potassium adduct formation. This material was further used to determine the distribution of sulfatide (
Recently, metal oxide laser ionization (MOLI) MS was reported as an organic matrix-free system for lipid analysis using powders of various metal oxides such as ZnO, MgO, FexOy, CoxOy, and CuO as an alternative to organic matrices (48). In this method, lipid molecules were ionized by protonation or sodiation which can be attributed to Lewis acid-base interactions between analytes and metal oxide. As such, the MOLI MS method can offer a new approach for the analysis of lipids. For example, CaO as a matrix replacement provided reproducible lipid cleavage, enabling lipid profiling for bacterial identification (49, 50). MOLI MS was also applied to IMS by Basu
The Cooks group first introduced the DESI method for analysis of diverse analytes including small non-polar compounds, peptides, and proteins (52). In this method, solvent microdroplets were electrosprayed onto the sample surface, and the impact of electrosprayed charged particles on the surface resulted in desorption and ionization of analytes. The resulting desorbed gas-phase ions were then transferred to a distant mass spectrometer which gave mass spectra similar to normal ESI MS. Using DESI MS, small molecule RDX (
Another approach to electrospray ionization-based IMS, the combination of infrared (IR) laser ablation with electrospray ionization (LAESI), was introduced by the Vertes group (59). In LAESI-MS, biological and medical analytes and organisms with sufficient water content are analyzed using a mid-IR laser at 2940 nm, corresponding to the frequency of the O-H bond’s vibration in water, resulting in strong absorption of the wavelength by the water. Because the sample absorbs mid-IR energy, a gas phase plume is created from the sample surface. These laser-ablated particulates from the sample surface then interact with electrospray droplets, which provide a source of ions, allowing ionization of the laser-ablated particulates and subsequent analysis by a detector. Using this method, excretion of the antihistamine fexofenadine (
SIMS is a desorption and ionization technique used to analyze the composition of surfaces by sputtering the surfaces with an energetic primary ion beam and analyzing secondary ions emitted from the surfaces (1, 64, 65). These secondary ions can directly produce high-resolution chemical images, so this platform is well-suited for the analysis of surface composition of biological materials. Although LDI- and ESI-based methods are widely used for visualization of molecular distribution on biological surfaces due to their efficiency and simplicity, these methods produce limited-resolution raster images. In this respect, SIMS ionization is advantageous over these methods, as it allows not only high mass resolution but also high spatial resolution of low molecular weight analytes. In terms of spatial resolution, a commonly used LDI method is capable of resolution as small as 5-100 mm because it uses laser light which has a focused spot size as small as 1 mm. However, SIMS offers enhanced resolution because it uses a primary ion beam that can be focused as sharply as 10 nm, allowing IMS of single cells and even different organelles within cells. For example, Todd
This paper was supported by Konkuk University in 2017.
The authors have no conflicting interests.