Metabolite imaging using matrix-enhanced surface-assisted laser desorption/ionization mass spectrometry (ME-SALDI-MS)

We describe here the use of a hybrid ionization approach, matrix-enhanced surface-assisted laser desorption/ionization mass spectrometry (ME-SALDI-MS) in bioimaging. ME-SALDI combines the strengths of traditional matrix-assisted laser desorption/ionization (MALDI) and SALDI and enables successful MS imaging of low-mass species with improved detection sensitivity. Using 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) as the MS standard, MS performances of MALDI, SALDI, and ME-SALDI are systematically compared. The analyte desorption and ionization mechanism in ME-SALDI is qualitatively speculated based on the observation of significantly reduced matrix background and improved survival yields of molecular ions. Improvements in detection sensitivity of low-mass species using ME-SALDI over MALDI in imaging are demonstrated with mouse heart and brain tissues.     

Matrix-assisted laser desorption/ionization imaging mass spectrometry for direct measurement of clozapine in rat brain tissue

Matrix-assisted laser desorption/ionization hyphenated with quadrupole time-of-flight (QTOF) mass spectrometry (MS) has been used to directly determine the distribution of pharmaceuticals in rat brain tissue slices which might unravel their disposition for new drug development. Clozapine, an antipsychotic drug, and norclozapine were used as model compounds to investigate fundamental parameters such as matrix and solvent effects and irradiance dependence on MALDI intensity but also to address the issues with direct tissue imaging MS technique such as (1) uniform coating by the matrix, (2) linearity of MALDI signals, and (3) redistribution of surface analytes. The tissue sections were coated with various matrices on MALDI plates by airspray deposition prior to MS detection. MALDI signals of analytes were detected by monitoring the dissociation of the individual protonated molecules to their predominant MS/MS product ions. The matrices were chosen for tissue applications based on their ability to form a homogeneous coating of dense crystals and to yield greater sensitivity. Images revealing the spatial localization in tissue sections using MALDI-QTOF following a direct infusion of (3)H-clozapine into rat brain were found to be in good correlation with those using a radioautographic approach. The density of clozapine and its major metabolites from whole brain homogenates was further confirmed using fast high-performance liquid chromatography/tandem mass spectrometry (HPLC-MS/MS) procedures.     

Repeat MALDI MS imaging of a single tissue section using multiple matrices and tissue washes

Matrix-assisted laser desorption ionization mass spectrometry imaging (MALDI MSI) techniques are continually being assessed with a view to improving the quality of information obtained from a given sample. A single tissue section will typically only be analyzed once by MALDI MSI and is then either used for histological staining or discarded. In this study, we explore the idea of repeat analysis of a single tissue section by MALDI MSI as a route toward improving sensitivity, structural characterization, and diversity of detected analyte classes. Repeat analysis of a single tissue section from a fresh frozen mouse brain is investigated with both α-cyano-4-hydroxycinnamic acid (CHCA) and para-nitroaniline (PNA). Repeat analysis is then applied to the acquisition of MALDI MSI and MALDI tandem mass spectrometry imaging employing collision induced dissociation (MS/MS imaging employing CID) from a formalin-fixed mouse brain section. Finally, both lipid and protein data are acquired from the same tissue section via repeat analysis utilizing CHCA, sinapinic acid (SA), and a tissue wash step. PNA was found to outperform CHCA as a matrix for repeat analysis; multiple lipids were identified using MS/MS imaging; both lipid and protein images were successfully acquired from a single tissue section.

On-tissue chemical derivatization of volatile metabolites for matrix-assisted laser desorption/ionization mass spectrometry imaging

Mass spectrometry imaging (MSI) of volatile metabolites is challenging, especially in matrix-assisted laser desorption/ionization (MALDI). Most MALDI ion sources operate in vacuum, which leads to the vaporization of volatile metabolites during analysis. In addition, tissue samples are often dried during sample preparation, leading to the loss of volatile metabolites even for other MSI techniques. On-tissue chemical derivatization can dramatically reduce the volatility of analytes. Herein, a derivatization method is proposed utilizing N,N,N-trimethyl-2-(piperazin-1-yl)ethan-1-aminium iodide to chemically modify short-chain fatty acids in chicken cecum, ileum, and jejunum tissue sections before sample preparation for MSI visualization.     

Distribution study of atorvastatin and its metabolites in rat tissues using combined information from UHPLC/MS and MALDI-Orbitrap-MS imaging

The combination of ultrahigh-resolution mass spectrometry imaging (UHRMSI) and ultrahigh-performance liquid chromatography coupled with tandem mass spectrometry (UHPLC/MS/MS) was used for the identification and the spatial localization of atorvastatin (AT) and its metabolites in rat tissues. Ultrahigh-resolution and high mass accuracy measurements on a matrix-assisted laser desorption/ionization (MALDI)-Orbitrap mass spectrometer allowed better detection of desired analytes in the background of matrix and endogenous compounds. Tandem mass spectra were also used to confirm the identification of detected metabolites in complex matrices. The optimization of sample preparation before imaging experiments included the tissue cryogenic sectioning (thickness 20 μm), the transfer to stainless steel or glass slide, and the selection of suitable matrix and its homogenous deposition on the tissue slice. Thirteen matrices typically used for small molecule analysis, e.g., 2,5-dihydroxybenzoic acid (DHB), 1,5-diaminonaphthalene (DAN), 9-aminoacridine (AA), etc., were investigated for the studied drug and its metabolite detection efficiency in both polarity modes. Particular matrices were scored based on the strength of extracted ion current (EIC), relative ratio of AT molecular adducts, and fragment ions. The matrix deposition on the tissue for the most suitable matrices was done by sublimation to obtain the small crystal size and to avoid local variations in the ionization efficiency. UHPLC/MS profiling of drug metabolites in adjacent tissue slices with the previously optimized extraction was performed in parallel to mass spectrometry imaging (MSI) measurements to obtain more detailed information on metabolites in addition to the spatial information from MSI. The quantitation of atorvastatin in rat liver, serum, and feces was also performed.

MALDI Mass Spectrometry Imaging of Neuronal Cell Cultures

Mass spectrometry imaging (MSI) provides the ability to detect and identify a broad range of analytes and their spatial distributions from a variety of sample types, including tissue sections. Here we describe an approach for probing neuropeptides from sparse cell cultures using matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) MSI—at single cell spatial resolution—in both MS and tandem MS modes. Cultures of Aplysia californica neurons are grown on an array of glass beads embedded in a stretchable layer of Parafilm M. As the membrane is stretched, the beads/neurons are separated physically and the separated beads/neurons analyzed via MALDI TOF MS. Compared with direct MS imaging of samples, the stretching procedure enhances analyte extraction and incorporation into the MALDI matrix, with negligible analyte spread between separated beads. MALDI tandem MSI using the stretched imaging approach yields localization maps of both parent and fragment ions from Aplysia pedal peptide, thereby confirming peptide identification. This methodology represents a flexible platform for MSI investigation of a variety of cell cultures, including functioning neuronal networks.     

A Caged In-Source Laser-Cleavable MALDI Matrix with High Vacuum Stability for Extended MALDI-MS Imaging

Insufficient vacuum stability of matrix chemicals is a major limitation in matrix-assisted laser desorption/ionization (MALDI) mass spectrometry imaging (MSI) of large tissue sample cohorts. Here, we designed and synthesized the photo-cleavable caged molecule 4,5-dimethoxy-2-nitrobenzyl-2,5-dihydroxyacetophenone (DMNB-2,5-DHAP) and employed it for lipid MALDI-MSI of mouse brain tissue sections. DMNB-2,5-DHAP is vacuum-stable in a high vacuum MALDI ion source for at least 72 h. Investigation of the uncaging process suggested that the built-in laser (355 nm) in the MALDI ion source promoted the in situ generation of 2,5-DHAP. A caging group is used for the first time in designing a MALDI matrix that is vacuum-stable, uncaged upon laser irradiation during the measurement process, and that boosts lipid ion intensity with MALDI-2 laser-induced postionization.     

Novel approach to enhance sensitivity in surface‐assisted laser desorption/ionization mass spectrometry imaging using deposited organic‐inorganic hybrid matrices

Sample pretreatment is key to obtaining good data in matrix‐assisted laser desorption/ionization mass spectrometry imaging (MALDI‐MSI). Although sublimation is one of the best methods for obtaining homogenously fine organic matrix crystals, its sensitivity can be low due to the lack of a solvent extraction effect. We investigated the effect of incorporating a thin film of metal formed by zirconium (Zr) sputtering into the sublimation process for MALDI matrix deposition for improving the detection sensitivity in mouse liver tissue sections treated with olanzapine. The matrix‐enhanced surface‐assisted laser desorption/ionization (ME‐SALDI) method, where a matrix was formed by sputtering Zr to form a thin nanoparticle layer before depositing MALDI organic matrix comprising α‐cyano‐4‐hydroxycinnamic acid (CHCA) by sublimation, resulted in a significant improvement in sensitivity, with the ion intensity of olanzapine being about 1800 times that observed using the MALDI method, comprising CHCA sublimation alone. When Zr sputtering was performed after CHCA deposition, however, no such enhancement in sensitivity was observed. The enhanced sensitivity due to Zr sputtering was also observed when the CHCA solution was applied by spraying, being about twice as high as that observed by CHCA spraying alone. In addition, the detection sensitivity of these various pretreatment methods was similar for endogenous glutathione. Given that sample preparation using the ME‐SALDI‐MSI method, which combines Zr sputtering with the sublimation method for depositing an organic matrix, does not involve a solvent, delocalization problems such as migration of analytes observed after matrix spraying and washing with aqueous solutions as sample pretreatment are not expected. Therefore, ME‐Zr‐SALDI‐MSI is a novel sample pretreatment method that can improve the sensitivity of analytes while maintaining high spatial resolution in MALDI‐MSI.     

In situ isobaric lipid mapping by MALDI–ion mobility separation–mass spectrometry imaging

The highly diverse chemical structures of lipids make their analysis directly from biological tissue sections extremely challenging. Here, we report the in situ mapping and identification of lipids in a freshwater crustacean Gammarus fossarum using matrix‐assisted laser desorption/ionization (MALDI) mass spectrometry imaging (MSI) in combination with an additional separation dimension using ion mobility spectrometry (IMS). The high‐resolution trapped ion mobility spectrometry (TIMS) allowed efficient separation of isobaric/isomeric lipids showing distinct spatial distributions. The structures of the lipids were further characterized by MS/MS analysis. It is demonstrated that MALDI MSI with mobility separation is a powerful tool for distinguishing and localizing isobaric/isomeric lipids.     

The improved performance of MALDI‐TOF MS on the analysis of herbal saponins by using DHB‐GO composite matrix

Matrix‐assisted laser desorption/ionization time‐of‐flight mass spectrometry (MALDI‐TOF MS) is an excellent analytical technique for rapid analysis of a variety of molecules with straightforward sample pretreatment. The performance of MALDI‐TOF MS is largely dependent on matrix type, and the development of novel MALDI matrices has aroused wide interest. Herein, we devoted to seek more robust MALDI matrix for herbal saponins than previous reported, and ginsenoside Rb1, Re, and notoginsenoside R1 were used as model saponins. At the beginning of the present study, 2,5‐dihydroxybenzoic acid (DHB) was found to provide the highest intensity for saponins in four conventional MALDI matrices, yet the heterogeneous cocrystallization of DHB with analytes made signal acquisition somewhat “hit and miss.” Then, graphene oxide (GO) was proposed as an auxiliary matrix to improve the uniformity of DHB crystallization due to its monolayer structure and good dispersion, which could result in much better shot‐to‐shot and spot‐to‐spot reproducibility of saponin analysis. The satisfactory precision further demonstrated that minute quantities of GO (0.1 μg/spot) could greatly reduce the risk of instrument contamination caused by GO detachment from the MALDI target plate under vacuum. More importantly, the sensitivity and linearity of the standard curve for saponins were improved markedly by DHB‐GO composite matrix. Finally, the application of detecting the Rb1 in complex biological sample was exploited in rat plasma and proved it applicable for pharmacokinetic study quickly. This work not only opens a new field for applications of DHB‐GO in herbal saponin analysis but also offers new ideas for the development of composite matrices to improve MALDI MS performance.     

Co-NC as adsorbent and matrix providing the ability of MALDI MS to analyze volatile compounds

Traditional matrix does not allow matrix-assisted laser desorption/ionization mass spectrometry(MALDI MS) to analyze volatile compounds,because volatile analytes may vaporize during the sample preparation process or in the high vacuum circumstance of ion source.Herein,we reported a Co and N doped porous carbon material(Co-NC) which were synthesized by pyrolysis of a Schiff base coordination compound.Co-NC could simultaneously act as adsorbent of volatile compounds and as matrix of MALDI MS,to provide the capability of MALDI MS to analyze volatile compounds.As adsorbent,Co-NC could stro ngly adsorb and enrich the volatile compounds in perfume and herbs,and hold them even in the high vacuum circumstance.On the other hand,Co-NC could absorb the energy of the laser,and then transfer the energy to the analyte for desorption and ionization of analyte in both negative and positive ionization modes.Additionally,the background interferences were avoided in the low-mass region(<500 Da) when using Co-NC as matrix,overcoming the challenges of MALDI MS analysis of small molecule compounds.In summary,Co-NC as matrix tremendously extended the application of MALDI MS.     

Ionic (liquid) matrices for matrix-assisted laser desorption/ionization mass spectrometry—applications and perspectives

A large number of matrix substances have been used for various applications in matrix-assisted laser desorption/ionization mass spectrometry (MALDI MS). The majority of matrices applied in ultraviolet-MALDI MS are crystalline, low molecular weight compounds. A problem encountered with many of these matrices is the formation of hot spots, which lead to inhomogeneous samples, thus leading to increased measurement times and hampering the application of MALDI MS for quantitative purposes. Recently, ionic (liquid) matrices (ILM or IM) have been introduced as a potential alternative to the classical crystalline matrices. ILM are equimolar mixtures of conventional MALDI matrix compounds such as 2,5-dihydroxybenzoic acid (DHB), α-cyano-4-hydroxycinnamic acid (CCA) or sinapinic acid (SA) together with organic bases [e.g., pyridine (Py), tributylamine (TBA) or N,N-dimethylethylenediamine (DMED)]. The present article presents a first overview of this new class of matrices. Characteristic properties of ILM, their influence on mass spectrometric parameters such as sensitivity, resolution and adduct formation and their application in the fields of proteome analysis, the measurement of low molecular weight compounds, the use of MALDI MS for quantitative purposes and in MALDI imaging will be presented. Scopes and limitations for the application of ILM are discussed.     

Qualitative and quantitative analysis of low molecular weight compounds by ultraviolet matrix-assisted laser desorption/ionization mass spectrometry using ionic liquid matrices

A major problem hampering the use of matrix-assisted laser desorption/ionization (MALDI) mass spectrometry for quantitative measurements is the inhomogeneous distribution of analytes and matrices in solid sample preparations. The use of ionic liquids as matrices for the qualitative and quantitative analysis of low molecular weight compounds like amino acids, sugars and vitamins was investigated. The ionic liquid matrices are composed of equimolar combinations of classical MALDI matrices (sinapinic acid, alpha-cyano-4-hydroxycinnamic acid or 2,5-dihydroxybenzoic acid) with organic bases. These matrix systems allow a homogenous sample preparation with a thin ionic liquid layer having negligible vapour pressure. This leads to a facilitated qualitative and quantitative measurement of the analytes compared with classical solid matrices.     

Quantum dots assisted laser desorption/ionization mass spectrometric detection of carbohydrates: qualitative and quantitative analysis

A quantum dots (QDs) assisted laser desorption/ionization mass spectrometric (QDA‐LDI‐MS) strategy was proposed for qualitative and quantitative analysis of a series of carbohydrates. The adsorption of carbohydrates on the modified surface of different QDs as the matrices depended mainly on the formation of hydrogen bonding, which led to higher MS intensity than those with conventional organic matrix. The effects of QDs concentration and sample preparation method were explored for improving the selective ionization process and the detection sensitivity. The proposed approach offered a new dimension to the application of QDs as matrices for MALDI‐MS research of carbohydrates. It could be used for quantitative measurement of glucose concentration in human serum with good performance. The QDs served as a matrix showed the advantages of low background, higher sensitivity, convenient sample preparation and excellent stability under vacuum. The QDs assisted LDI‐MS approach has promising application to the analysis of carbohydrates in complex biological samples. Copyright © 2016 John Wiley & Sons, Ltd.     

Mass spectrometry imaging of triglycerides in biological tissues by laser desorption ionization from silicon nanopost arrays

Mass spectrometry imaging (MSI) is used increasingly to simultaneously detect a broad range of biomolecules while mapping their spatial distributions within biological tissue sections. Matrix‐assisted laser desorption ionization (MALDI) is recognized as the method‐of‐choice for MSI applications due in part to its broad molecular coverage. In spite of the remarkable advantages offered by MALDI, imaging of neutral lipids, such as triglycerides (TGs), from tissue has remained a significant challenge due to ion suppression of TGs by phospholipids, e.g. phosphatidylcholines (PCs). To help overcome this limitation, silicon nanopost array (NAPA) substrates were introduced to selectively ionize TGs from biological tissue sections. This matrix‐free laser desorption ionization (LDI) platform was previously shown to provide enhanced ionization of certain lipid classes, such as hexosylceramides (HexCers) and phosphatidylethanolamines (PEs) from mouse brain tissue. In this work, we present NAPA as an MSI platform offering enhanced ionization efficiency for TGs from biological tissues relative to MALDI, allowing it to serve as a complement to MALDI‐MSI. Analysis of a standard lipid mixture containing PC(18:1/18:1) and TG(16:0/16:0/16:0) by LDI from NAPA provided an ~49 and ~227‐fold higher signal for TG(16:0/16:0/16:0) relative to MALDI, when analyzed without and with the addition of a sodium acetate, respectively. In contrast, MALDI provided an ~757 and ~295‐fold higher signal for PC(18:1/18:1) compared with NAPA, without and with additional Na⁺. Averaged signal intensities for TGs from MSI of mouse lung and human skin tissues exhibited an ~105 and ~49‐fold increase, respectively, with LDI from NAPA compared with MALDI. With respect to PCs, MALDI provided an ~2 and ~19‐fold increase in signal intensity for mouse lung and human skin tissues, respectively, when compared with NAPA. The complementary coverage obtained by the two platforms demonstrates the utility of using both techniques to maximize the information obtained from lipid MS or MSI experiments.     

Uncovering matrix effects on lipid analyses in MALDI imaging mass spectrometry experiments

The specific matrix used in matrix‐assisted laser desorption/ionization imaging mass spectrometry (MALDI IMS) can have an effect on the molecules ionized from a tissue sample. The sensitivity for distinct classes of biomolecules can vary when employing different MALDI matrices. Here, we compare the intensities of various lipid subclasses measured by Fourier transform ion cyclotron resonance (FT‐ICR) IMS of murine liver tissue when using 9‐aminoacridine (9AA), 5‐chloro‐2‐mercaptobenzothiazole (CMBT), 1,5‐diaminonaphthalene (DAN), 2,5‐Dihydroxyacetophenone (DHA), and 2,5‐dihydroxybenzoic acid (DHB). Principal component analysis and receiver operating characteristic curve analysis revealed significant matrix effects on the relative signal intensities observed for different lipid subclasses and adducts. Comparison of spectral profiles and quantitative assessment of the number and intensity of species from each lipid subclass showed that each matrix produces unique lipid signals. In positive ion mode, matrix application methods played a role in the MALDI analysis for different cationic species. Comparisons of different methods for the application of DHA showed a significant increase in the intensity of sodiated and potassiated analytes when using an aerosol sprayer. In negative ion mode, lipid profiles generated using DAN were significantly different than all other matrices tested. This difference was found to be driven by modification of phosphatidylcholines during ionization that enables them to be detected in negative ion mode. These modified phosphatidylcholines are isomeric with common phosphatidylethanolamines confounding MALDI IMS analysis when using DAN. These results show an experimental basis of MALDI analyses when analyzing lipids from tissue and allow for more informed selection of MALDI matrices when performing lipid IMS experiments.     

A Mechanism for Ionization of Nonvolatile Compounds in Mass Spectrometry: Considerations from MALDI and Inlet Ionization

Mechanistic arguments relative to matrix-assisted laser desorption/ionization (MALDI) mass spectrometry (MS) address observations that predominately singly charged ions are detected. However, recently a matrix assisted laser ablation method, laserspray ionization (LSI), was introduced that can use the same sample preparation and laser as MALDI, but produce highly charged ions from proteins. In MALDI, ions are generated from neutral molecules by the photon energy provided to a matrix, while in LSI ions are produced inside a heated inlet tube linking atmospheric pressure and the first vacuum region of the mass spectrometer. Some LSI matrices also produce highly charged ions with MALDI ion sources operated at intermediate pressure or high vacuum. The operational similarity of LSI to MALDI, and the large difference in charge states observed by these methods, provides information of fundamental importance to proposed ionization mechanisms for LSI and MALDI. Here, we present data suggesting that the prompt and delayed ionization reported for vacuum MALDI are both fast processes relative to producing highly charged ions by LSI. The energy supplied to produce these charged clusters/droplets as well as their size and time available for desolvation are determining factors in the charge states of the ions observed. Further, charged droplets/clusters may be a common link for ionization of nonvolatile compounds by a variety of MS ionization methods, including MALDI and LSI.     

Aniline/α‐cyano‐4‐hydroxycinnamic acid is a highly versatile ionic liquid for matrix‐assisted laser desorption/ionization mass spectrometry

The performance of a matrix‐assisted laser desorption/ionization (MALDI) ionic liquid matrix (ILM) consisting of α‐cyano‐4‐hydroxycinnamic acid (CHCA) and aniline (ANI) was evaluated to assess whether it could offer possible advantages over conventional matrices. Ultraviolet (UV), Fourier transform infrared (FTIR), nuclear magnetic resonance (NMR) and laser desorption/ionization mass spectrometry (LDI‐MS) experiments were carried out with the aim of confirming the structure of the ANI‐CHCA ILM. Different model analytes such as amino acids, peptides, proteins, lipids, phospholipids, synthetic polymers, and sugars were tested. Mass spectra with similar or improved signal‐to‐noise (S/N) ratio (compared to CHCA) were invariably obtained demonstrating the potential of this ILM as a general purpose matrix. Furthermore, protein identification by peptide mass fingerprinting (PMF) and database search was facilitated compared to CHCA since higher scores and increased sequence coverage were observed. Finally, a complex lipid mixture (i.e. a raw extract of a milk sample) analysed by MALDI‐MS showed improved S/N ratio, a reduced chemical noise and a limited formation of matrix‐clusters. Copyright © 2009 John Wiley & Sons, Ltd.     

MALDI imaging in human skin tissue sections: focus on various matrices and enzymes

Matrix-assisted laser/desorption ionization (MALDI) mass-spectrometric imaging (MSI), also known as MALDI imaging, is a powerful technique for mapping biological molecules such as endogenous proteins and peptides in human skin tissue sections. A few groups have endeavored to apply MALDI-MSI to the field of skin research; however, a comprehensive article dealing with skin tissue sections and the application of various matrices and enzymes is not available. Our aim is to present a multiplex method, based on MALDI-MSI, to obtain the maximum information from skin tissue sections. Various matrices were applied to skin tissue sections: (1) 9-aminoacridine for imaging metabolites in negative ion mode; (2) sinapinic acid to obtain protein distributions; (3) α-cyano-4-hydroxycinnamic acid subsequent to on-tissue enzymatic digestion by trypsin, elastase, and pepsin, respectively, to localize the resulting peptides. Notably, substantial amounts of data were generated from the distributions retrieved for all matrices applied. Several primary metabolites, e.g. ATP, were localized and subsequently identified by on-tissue postsource decay measurements. Furthermore, maps of proteins and peptides derived from on-tissue digests were generated. Identification of peptides was achieved by elution with different solvents, mixing with α-cyano-4-hydroxycinnamic acid, and subsequent tandem mass spectrometry (MS/MS) measurements, thereby avoiding on-tissue MS/MS measurements. Highly abundant peptides were identified, allowing their use as internal calibrants in future MALDI-MSI analyses of human skin tissue sections. Elastin as an endogenous skin protein was identified only by use of elastase, showing the high potential of alternative enzymes. The results show the versatility of MALDI-MSI in the field of skin research. This article containing a methodological perspective depicts the basics for a comprehensive comparison of various skin states.

High‐Spatial Resolution Mass Spectrometry Imaging: Toward Single Cell Metabolomics in Plant Tissues

Mass spectrometry imaging (MSI) is a powerful tool that has advanced our understanding of complex biological processes by enabling unprecedented details of metabolic biology to be uncovered. Through the use of high‐spatial resolution MSI, metabolite localizations can be obtained with high precision. Here we describe our recent progress to enhance the spatial resolution of matrix‐assisted laser desorption/ionization (MALDI) MSI from ∼50 μm with the commercial configuration to ∼5 μm. Additionally, we describe our efforts to develop a ‘multiplex MSI’ data acquisition method to allow more chemical information to be obtained on a single tissue in a single instrument run, and the development of new matrices to improve the ionization efficiency for a variety of small molecule metabolites. In combination, these contributions, along with the efforts of others, will bring MSI experiments closer to achieving metabolomic scale.