Imaging MS (IMS) is a technology that provides information on the localization of molecules in a sample. It has become a powerful way to profile and characterize the spatial distribution of biocompounds (including peptides, proteins, lipids and metabolites) through the direct analysis of thin tissue sections [Citation1,Citation2]. This semiquantitative technique is distinctive due to its capability to preserve the spatial localization of diagnostic molecules and biomarkers, information that is lost when tissue extracts or homogenates are used. Recent application of this technology to the analysis of thin tissue sections clearly shows retention of spatial and anatomic relationships permitting the complex interaction between diseased cells and their environment to be studied at the molecular level. From the systematic analysis of a single tissue section, molecular-specific maps directly correlated with tissue architecture may be simultaneously obtained for hundreds of different endogenous compounds. The potential of this type of analysis in which the spatial distribution of specific molecular species can be mapped throughout a tissue section is particularly exciting for the study of disease [Citation3,Citation4]. While this capability is routinely available for known individual proteins via immunohistochemistry, IMS offers the potential for the simultaneous analysis of many molecular species (proteins, lipids, metabolites) present in a single specimen regardless of the availability of specific antibodies. IMS technology offers an entirely new and highly precise means of analyzing tissues. In addition, this technology permits the imaging of the tissue distribution of administered pharmaceutical compounds [Citation5], as well as following the cellular response to drugs and biologics thus opening new possibilities for the measurement of concomitant molecular changes in specific tissues after systemic drug administration [Citation6].
Classical (MALDI) IMS approaches only allow to broadly analyze classes of biomolecules. Based on the matrix employed and its mode of deposition on tissue sections, different classes of biomolecules (proteins, peptides or lipids) can be analyzed. For example, current methods of IMS of lipids do not allow specific targeting of a chosen class of lipid, which may be of high value for diagnosis, prognosis or as indicators of response to therapy. Strategies to improve specificity and sensitivity limitations in IMS and open the technique for the analysis of novel classes of endogenous molecules need to be developed. To incorporate some degree of molecular specificity in IMS, several approaches can be proposed.
Alternative ionizing agents: based on their structure, some biocompounds have high affinities for various (metal) cations. This unique property can be exploited to selectively ionize and detect such biocompounds by MS even when present in complex mixtures. An example is cholesterol for which a preferential ionization with silver ions (formation of [M+Ag]+ molecular ions) has been observed. We have recently taken advantage of this unique property and demonstrated that the addition of metallic silver on the surface of tissue sections allows to specifically detect cholesterol, fatty acids as well as other olefin containing molecules present within tissue sections by IMS [Citation7]. In this case, a thin layer (~20 nm) of metallic silver is deposited on the tissue sections. The sections are then analyzed by IMS under laser desorption ionization (LDI) conditions.
We have also recently optimized a novel method specific for the specific detection and IMS of triglycerides contained within tissue sections present in high abundance within the lipid droplets characteristic of fatty liver disease [Dufresne et al., Submitted]. In this approach, a sodium salt is first deposited using an automated liquid spray system followed by the deposition of an approximately 20 nm layer of gold using a metal sputter system. Targeted lipid compounds are analyzed by LDI MS and IMS. In this system, gold is used as a desorption agent and triglycerides are ionized by capturing a sodium ion (formation of [M+Na]+ molecular ions). With this approach, the sensitivity of triglyceride detection from tissue sections improved by a factor of over 200 fold with respect to analysis by MALDI MS.
Affinity surfaces: a strategy to increase specificity in IMS is to transfer proteins from a tissue section to an affinity surface in a regiospecific manner. Only proteins with an affinity for the surface chemistry will be retained, therefore the chemical nature of the surface is critical. In the first article on MALDI IMS, Caprioli et al. reported the regiospecific transfer of peptides from fresh rat pituitary on a C18 coated surface [Citation8]. In subsequent work, Chaurand et al. transferred proteins from fresh tissues to a carbon embedded polyethylene membrane in a regiospecific manner [Citation9,Citation10]. After transfer, the polyethylene membrane was rinsed and only proteins with affinity for the medium were retained and observed. In a similar approach, Bouamrani et al. thaw-mounted tissue section on different SELDI surfaces and observed significant variations in protein signal expression as a function of surface chemistry [Citation11]. We have recently developed a method allowing the region-specific transfer of proteins from tissue sections onto a nitrocellulose membrane [Citation12]. When analyzing the nitrocellulose membrane by MALDI IMS after the transfer process, we have found that only a subset of proteins detected by a direct MALDI IMS analysis of the tissue sections were observed. Further, some protein signals detected by conventional IMS analysis of the section were more readily detected after transfer. Interestingly, a subset of proteins was uniquely observed after transfer potentially indicating their preferential affinity for the nitrocellulose membrane.
On-tissue derivatization: several targeted on-tissue chemistries designed for IMS of specific endogenous or exogenous molecules have being developed. Most of the studies have focused on the derivatization of large molecules using photocleavable tags [Citation13,Citation14]. In this experiment, a primer probe is linked to a photocleavable reporter ion (with UV absorption close to the laser wavelength) that is further analyzed by LDI IMS to report the localization of the probe. These strategies have been mainly applied to analyses of high MW proteins, oligosaccharides, and mRNA [Citation13,Citation14]. The selectivity of the approach is based on the probe affinity since the MS detection is performed on a reporter ion rather than on the target molecule. The main interest of this approach is its multiplex capacity to detect and image several targets [Citation15]. The use of derivatizing reagents with a fixed charge or with high proton affinity is an efficient way to improve the ionization efficiency of the targeted analytes [Citation16]. More recently, a method that uses secondary ion mass spectrometry [Citation17] or LDI MS [Citation18] to image antibodies tagged with isotopically pure elemental metal reporters has been developed. With this approach, up to 100 targets over a five-log dynamic range can be simultaneously imaged [Citation17,Citation18].
On-tissue derivatization of low MW endogenous and administered molecules has also been developed. For instance, 1,1’-thiocarbonyldiimidazole treatment of tissue from mice dosed with 3-methoxysalicylamine (3-MoSA) allowed imaging of the drug’s spatial distribution as well as its pharmacokinetic profile in different organs [Citation19]. In this case, the resulting oxothiazolidine derivative is detected with much greater sensitivity by MALDI MS than 3-MoSA itself. In another example, trans-cinnamaldehyde was used to selectively derivatize isoniazid, a first-line medication for treatment of tuberculosis, within a lung tissue section [Citation20]. With this simple procedure, the MS signal was enhanced to a point where an IMS time-course analysis of drug distribution could be performed. In a third example, semi-quantitative spatial information for multiple free amino acids in tissue slices was obtained after on-tissue derivatization with p-N,N,N-trimethylammonioanilyl N’-hydroxysuccinimidyl carbamate iodide to increase their ionization efficiency and detection sensitivity [Citation21]. More recently, on-tissue derivatization of endogenous corticosteroids has been performed using a Girard T reagent [Citation22]. In addition to the gain in sensitivity, the derivatization tags may help to shift the MW of the target molecules beyond the spectral region of matrix interferences [Citation20]. Although to date only a few studies have been published, target-specific derivatization strategies are foreseen to significantly grow.
Since the beginning of this millennium, IMS technology has seen a tremendous evolution with the fast developments of novel methods and instrumentation [Citation2]. IMS is now being applied to a wide range of applications ranging from plant science to the detection of biomarkers from clinical biopsies in multiple human diseases. The implementation of some degree of specificity within the IMS process is necessary in many instances to accurately determine the distribution and abundance of high value compounds. This especially true in the clinical setting for diagnosis, prognosis and the assessment of response to therapy.
Financial & competing interests disclosure
The author has no relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript. This includes employment, consultancies, honoraria, stock ownership or options, expert testimony, grants or patents received or pending, or royalties.
No writing assistance was utilized in the production of this manuscript.
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