Advances in MALDI Mass Spectrometry within Drug Discovery

This special issue of SLAS Discovery is dedicated to MALDI TOF (matrix-assisted laser desorption/ionization time of flight), marking the 30th anniversary of this revolutionary soft ionization technique for analyzing nonvolatile biomolecules using mass spectrometry (MS). ¹ The technique was termed MALDI TOF by Hillenkamp and colleagues ² when describing their process of irradiating co-crystals of analyte and a matrix compound, usually a small organic acid, with a pulsed laser to desorb and ionize intact analyte molecules. In this approach, matrix–analyte mixtures are typically applied onto a metal MALDI TOF substrate plate in formats that are often amenable to standard laboratory liquid handling. The laser-absorbing matrix creates ions of the analyte in which its mass is determined by the time of flight to a detector. By combining the above, MALDI can be applied to the analysis of several types of biomolecules, including DNA, RNA, peptides, lipids, sugars, and proteins. From an experimental perspective, MALDI exhibits attractive characteristics that include tolerance to low concentrations of salts and detergents, straightforward sample preparation without the need for solid phase extraction, low fragmentation of the analyte, and the predominant formation of singly charge ions. This unique combination of properties has allowed MALDI to become an invaluable tool for analyzing many classes of nonvolatile compounds.

Matrix is a critical component of MALDI TOF MS that assists in the ionization of analytes. Compounds that make an effective matrix are more often discovered rather than chosen. Although the general properties of an effective matrix have been used to design an enhanced matrix compound, ³ possession of these ionization mechanisms does not necessarily guarantee a compound to be an effective matrix for all analytes. There are only three commonly used matrix compounds: α-cyano-4-hydroxycinnamic acid (α-CHCA or alpha-cyano); 3,5-dimethoxy-4-hydroxycinnamic acid (sinapinic acid); and 2,5-dihydroxybenzoic acid (DHB). Although dozens of other matrix compounds exist, a survey of these molecules would allow for determining the best outcome in an experiment (i.e., signal, lack of interference, ion polarity, etc.).

Common matrix compounds range in size from 200 to 400 Daltons, allowing for easy vaporization by a laser. Furthermore, the application of matrix molecules in MALDI can contribute to a strong chemical background of ions lower than 700–800 m/z from a variety of clustered matrix molecules. When analyzing biomolecules that are higher than 1000 m/z, the matrix background does not present significant interference with the intended analyte. When analyte molecules occupy the same m/z space as the interfering matrix clusters, however, it can be a challenge to differentiate analyte ions from background. Not only is the selection of matrix a key component of MALDI, but also the solvents and additives used and how matrix is applied to the substrate require careful optimization.

For many laboratories, MALDI is a tool of convenience when analyzing complex samples. MALDI analyses have a short turnaround time and often require very little sample preparation. For many complex samples, MALDI can provide the desired analytical information, thereby eliminating the need for other, more time-intensive analysis techniques. The versatility of MALDI, however, extends beyond simple determination of molecular weight. Combining MALDI MS and MS/MS methodologies with novel software tools enables a variety of different measurements, including identification and characterization of posttranslational modifications such as glycans and glycoproteins,^4–6 phosphopeptides,^7–8 as well as disulfide mapping in proteins as large as antibodies. ⁹ Using in-source fragmentation, top-down sequencing can also be carried out for small proteins, as well as determining >80% sequence coverage for antibodies. ¹⁰ Furthermore, detailed analysis of fractionated samples has enabled liquid chromatography (LC)-MALDI workflows for a variety of proteomic studies.^11–12

In recent years, several advances in instrumentation and methodologies have allowed MALDI to play a greater role in discovering and developing new therapeutics. MALDI instrumentation is becoming faster with laser speeds >10 kHz, sensitivities in the attomolar range, and resolution that allows detection of small posttranslational modifications on midsized proteins. These advances and future improvements will ensure that MALDI TOF plays a significant role from basic research through the discovery of new therapeutics. The focus of this MALDI TOF Mass Spectrometry special issue of SLAS Discovery is to highlight some of the recent studies using these novel MALDI techniques.

MALDI is now an established tool for clinical microbiology. Perhaps a lesser known extension of the clinical MALDI strategy is determining microorganism susceptibility and response to specific therapeutics by measuring changes in molecular markers. Authors Sharma and Bisht ¹³ use MALDI to characterize the secretory proteome of streptomycin-resistant Mycobacterium tuberculosis as a first approach to understanding how best to treat resistant strains.

High-throughput mass spectrometry (HTMS) combines standard liquid handling and the rapid speed of MALDI TOF (<1 s/spectrum) to screen libraries containing millions of compounds to determine their impact on target enzymes. MALDI can be a sensitive detection method for a variety of substrates and products in biochemical assays. Buffers, detergents, and carrier proteins are often required for optimal enzymatic activity in these assays, but they are more often detrimental to MALDI TOF signals and, ultimately, the hardware at certain concentrations. Chandler and coauthors ²⁰ survey a variety of buffers and additives and report their effect on detection of analytes. This data can be used to guide biochemical assay development that is funneled into an HTMS campaign. VanderPorten and coauthors ¹⁴ describe an HTS-compatible strategy using self-assembled monolayer desorption ionization (SAMDI) to screen non-covalent binding of small molecules to target proteins. Not only does this approach remove buffer components and enrich the analyte, but also SAMDI permits efficient screening of small molecule binders without the need for commonly used antibodies, fluorescence, or radioactivity labels.

An area of increasing interest is mass spectrometry imaging (MSI) of cell or tissue samples. With this approach, MALDI can be used to acquire two-dimensional (2D) arrays of hundreds of thousands of mass spectra over prepared sections of tissue. From these arrays of spectra, the intensity of any detected ion can be plotted onto the 2D grid to create label-free molecular maps. MALDI-MSI has been used to study numerous diseases ¹⁵ and therapeutics.^16–17 Grove and coauthors ¹⁸ use MALDI imaging to analyze sections of eye after treatment with brimonidine to gain insight into transit and distribution of the drug through the eye. Furthermore, Jones and coauthors ¹⁹ use MALDI-MSI to map the distribution of glycosphingolipids in a Gaucher disease model mouse brain, thereby providing significant insight into the pathology of this disease.

Scientists are combining disparate technologies to create synergies with MALDI TOF. For example, Jagadeesan and coauthors ²¹ have applied molecular imprint technology for the capture and detection of progastrin-releasing peptide. Jagadeesan and Ekström ²² have further developed a visualization tool for MALDI data that can be quickly applied to the analysis of large datasets. Other articles discuss the identification of inhibitors of salt-inducible kinases, ²³ in situ MALDI for screening the receptor tyrosine kinase cMET, ²⁴ and quantitative imaging of tryptophan and kynurenine metabolites in antitumor immune response. ²⁵

This issue represents a snapshot of innovation in the MALDI application space as it stands today. This exciting technology will continue to grow as instrumentation and scientific innovation further push the boundaries of MALDI TOF capabilities. Advances in MALDI speed, resolution, and sensitivity will continue to invigorate the imagination as we explore the future possibilities of developing this renewed interest in a powerful technology.