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Power and Potential: MALDI Mass Spec at 30

The December 2017 SLAS Discovery Special Issue on Advances in MALDI Mass Spectrometry within Drug Discovery captures examples of diverse and exciting advances that inform us about the expanding range of applications of matrix assisted laser desorption/ionization time of flight (MALDI TOF) technology. The issue marks the 30th anniversary of MALDI TOF at a time when “the technology has gained momentum and researchers are recognizing MALDI and applying the technology in innovative ways,” says Co-Guest Editor Michael Scholle of SAMDI Tech (Chicago, IL, USA).

Co-Guest Editor Shannon Cornett, Ph.D., of Bruker Daltonics (Nashville, TN, USA) agrees that there is more widespread acceptance and recognition of the technology’s potential while noting that, “Not so long ago, there was a general sense in the community that MALDI is a powerful tool but primarily just for determining molecular weights.”

Applications for High-Throughput Screening (HTS)

Instrument and Software Improvements

Cornett and Scholle explain that the advances in speed, resolution and sensitivity have been vital to expanding applications of MALDI technology to biological questions from basic research through discovery of new therapeutics. “From a MALDI perspective, the key developments in the last few years have been in laser technology and acquisition speed,” says Cornett. He sees it as a quantum improvement that has made the technology applicable for HTS. Scholle agrees and points out that there has also been a multitude of instrument hardware innovations that have increased sensitivity and decreased downtime for maintenance.

Scholle goes on to point out that it is not all hardware and instrumentation driving this technology, but also the software advances that support the data acquisition and processing. He is pleased to see the paper in this special issue that presents a comprehensive informatics tool, MALDIViz, for managing the large amount of data generated by HTS-scale MALDI analysis and providing a visual interpretation. Scholle sees these types of solutions to bioinformatics bottlenecks as broadly applicable to programs in drug discovery, cell biology, assay development, buffer optimization and more.

In discussing the barriers to entry of MALDI TOF technology, Scholle puts the usual suspects on the list – cost and expertise. For high-end MALDI equipment, costs are predictably high and HTS screening systems also require additional equipment on the front end for sample handing. Fortunately, the mass spectrometry (MS) instruments of today have a much smaller footprint than in the past. When considering that every instrument has a learning curve, Scholle feels that the curve for gaining expertise in MALDI TOF is steeper and longer than standard laboratory instrumentation. In his experience, it takes two to three years for someone to acquire a breadth of experience with various analytes, buffers, matrices, instrument settings and so on. Cornett acknowledges the learning curve and also that he is seeing a shift to more biologists and biochemists wanting their hands on the technology rather that strictly analytical chemists and instrument-oriented people. He adds that, “at Bruker, we recognize that usability is key and that our customers want to sit down and collect data. Many of the operational challenges of five to 10 years ago have been dramatically improved.”

Despite the obvious barriers, most core facilities today have some MALDI instrumentation according to Cornett. Although many may only be used for quick checks of purity, which is beneficial to the overall facility as it does free up instruments with longer run times, such as liquid chromatography. In the last five years, Cornett notes he has seen an increase in dedicated, specific applications coming out directly for MALDI-based systems, especially in HTS screening.

Optimizing Assays

There are a number of components in any assay system that can be adjusted to obtain better data. The SLAS Discovery special issue includes two manuscripts highlighting buffer optimization and a front end rapid sample purification methodology.

The buffer investigation technical note by Chandler et al. looks at how common assay components may affect MALDI signal and therefore, assay sensitivity. MALDI offers the advantage of being a label-free analytical tool but it is known that assay components (buffers, detergents, etc.) can interfere with signal in ways that are not well understood. Scholle appreciates this paper for providing a starting point in understanding assay component compatibility with MALDI and looks forward to additional work in this direction, however, he also acknowledges that limiting buffer components may have negative effects on biochemical assays.

Cornett understands that buffers play a unique role in assays and thinks that “once you understand what you want a buffer to do and not do, you could design a system that serves analytical purposes without degrading the MALDI signal and that would be the ideal endpoint.”

Of course, most types of assays have some component that can interfere with results and cause false-positive or false-negative reporting. Many systems involve some sort of sample preparation and for MALDI-TOF, sample prep is critical, according to Scholle. Cleaner samples cause less interference and provide results with higher reproducibility. The paper by VanderPorten et al. describes the use of self-assembled monolayer desorption ionization (SAMDI) for a screening assay. This affinity purification approach in an HTS format not only purifies and enriches the samples, but also has the potential to decrease screening time and provide an alternative approach to hit finding efforts.

Scholle explains that the key message of the SAMDI paper is “that one can use MALDI to assay small molecule binding in a way that eliminates the need for a biochemical assay. SAMDI captures the protein-compound complex to defined surface chemistries in 384 and 1536 format, allowing for a simple wash to remove buffers, salts and detergents that often have a negative effect on MALDI signal. With SAMDI small molecule binding assays there is no need to set up an enzyme activity assay. Further, one can potentially determine small molecule binding to a protein of interest using 10-20 or more compounds per well. This would translate into a screening approach where a 1 million compound library can be screened in 50,000 wells within in a single day. In short, much less assay development is required when compared to a standard biochemical assay, sample preparation is fast and simple, compounds can be pooled, and large numbers of compounds can be screened at unprecedented speeds.”

Erica VanderPorten discusses this work in an on-demand SLAS Webinar, free for members.

Applications in Medicine

Clinical Microbiology

A PubMed search quickly shows that MALDI-TOF is well established as a tool in clinical microbiology for identifying pathogens. In the paper by Sharma and Bisht, the application of this technology is extended into microbiology by analyzing the secretary proteome of resistant Mycobacterium tuberculosis and identifying a number of overexpressed proteins that could be responsible for resistance. The hope is that these proteins will be explored further as potential drug targets or biomarkers of resistance.

Imaging Mass Spectrometry (IMS)

The special issue has two reports that highlight applications of IMS in pharmacology and pathology. Grove et al. analyze sections of the eye after a treatment to gain insight into a drug’s transit and distribution pattern. Jones et al. use IMS to locate lesions in the brain, providing more detailed information about the pathologic process in an inherited disease.

Cornett has been involved with IMS for some time and explains that the bulk of literature over the past 10 years has been using the technology to look at various diseases, but primarily cancers. “At this point researchers are able to evaluate the molecular signature of just a few cells. The hope is that we can utilize those signatures to detect molecular changes before they can be visualized with microscopy techniques. This capability could improve the accuracy of diagnosis and treatment. Of course, that is a really long-term goal but it is also a driving force. From a pharmaceutical development perspective, IMS provides far more detailed information directly from the tissue.”

It is not exactly intuitive to think about a mass spectrometer creating tissue images as microscopy does but Cornett broke it down this way, “Imagine that you have tissue on a slide at 50x magnification and you take a digital picture and that as you click on each pixel you could get a unique molecular signature of lipids, proteins or drugs present in that precise location. That approximates the level of detail that a mass spectrum could produce. MS can detect hundreds to thousands of compounds per pixel and from that can generate data.”

Cornett admits that there are still challenges with sensitivity but from a sample preparation standpoint, MALDI is somewhat forgiving and tissue is not exactly a pure sample. He says, “the faster lasers have helped with sensitivity because you are able to acquire more spectra and perform signal averaging to improve sensitivity.”

In addition to needed increases in sensitivity, an area that Cornett feels has the potential to take IMS to the next level is standardization. At the moment he sees research groups using many different protocols, each treating tissue sections a little differently. As a part of developing standardized workflows, Cornett points out the need to handle and section tissue in a way that is consistent and minimizes molecular contamination because MS takes tissue analysis beyond the cellular level, to the molecular level.

Scholle sees the MALDI imaging arena as having great potential. He has seen this area grow as the resolution of MALDI has improved, but also credits software advances for making the combination a very powerful technology. He feels that “the implications, to biology and pathology, are going to be profound and that we are only at the beginning.”

Future Directions

The SLAS Discovery special issue highlights a wide range of advances and applications of MALDI-TOF technology. The field is rapidly progressing and evolving as it gains acceptance across divergent fields. Both Scholle and Cornett feel that while there is an increase in acceptance for this technology, there remains a substantial contingent of people who could benefit from a better understanding of the technology so that they could imagine how to apply it to the questions they are trying to answer.

Scholle believes that the vision of both the user and instrument manufacturers have converged to help advance the technology. He says, “It’s about the manufacturers listening to the market. The MALDI end users provide feedback to the manufacturers and scientific community through applications and publications, informing manufacturers on how instruments are being used and providing insight on improvements towards areas of future market interest. Scholle believes that this is the model that has brought us to the current state of MALDI and will also guide its advancement.”

Learn More in the December 2017 SLAS Discovery Special Issue

The SLAS Discovery Special Issue on Advances in MALDI Mass Spectrometry within Drug Discovery features nine original research reports and one technical note from Belgium, France, Germany, India, Sweden, the UK and the US and is now available at SLAS Discovery Online for SLAS Premier members, SLAS Discovery subscribers and pay-per-view readers. Free public access becomes available one year after final publication. The following papers are included in the special issue:

Microenvironment Tumor Metabolic Interactions Highlighted by qMSI: Application to the Tryptophan-Kynurenine Pathway in Immuno-Oncology
Ait-Belkacem et al.

Identifying Inhibitors of Inflammation: A Novel High-Throughput MALDI-TOF Screening Assay for Salt Inducible Kinases (SIKs)
Heap et al.

Integration of an In Situ MALDI-Based High-Throughput Screening Process: A Case Study with Receptor Tyrosine Kinase c-MET
Beeman et al.

Identification of Small-Molecule Noncovalent Binders Utilizing SAMDI Technology
VanderPorten et al.

Tissue Localization of Glycosphingolipid Accumulation in a Gaucher Disease Mouse Brain by LC-ESI-MS/MS and High-Resolution MALDI Imaging Mass Spectrometry
Jones et al.

Secretory Proteome Analysis of Streptomycin-Resistant Mycobacterium Tuberculosis Clinical Isolates
Sharma and Bisht

Application of Imaging Mass Spectrometry to Assess Ocular Drug Transit
Grove et al.

MALDI-Viz - A Comprehensive Informatics Tool for MALDI-MS Data Visualization and Analysis
Jagadeesan and Ekström

Filter Plate-Based Screening of MIP SPE Materials for Capture of the Biomarker Pro-Gastrin Releasing Peptide
Jagadeesan et al.

A Systematic Investigation of the Best Buffers for Use in Screening by MALDI–Mass Spectrometry
Chandler et al.

 

SLAS2018, Feb 3-7 in San Diego, CA, also addresses this technology. In the Advances in Bioanalytics and Biomarkers Track there is a session on Label Free Bioanalytical Techniques in Drug Discovery that includes presentations on mass spectrometry and other techniques.

November 27, 2017