Imaging mass spectrometry (IMS) techniques produce a visual, spatial representation of the molecular composition of a biological sample. Advances in instrumentation and computing power, as well as experimental expertise, allow maps of hundreds of proteins, carbohydrates, lipids, small molecules and virtually any other MS-compatible molecule to be created from a single sample, without use of specific, targeted reagents.
Researchers have devised dozens of IMS strategies, including variants that do and do not require sample preparation and that work at either atmospheric pressure or in a vacuum. One popular approach is matrix-assisted laser desorption ionization (MALDI)-IMS, developed in 1997 by Richard Caprioli, now the Stanford Moore Chair in Biochemistry and Director of the Mass Spectrometry Research Center at Vanderbilt University School of Medicine, and his team. Biocompare.com spoke with him about the adaptability of IMS to different sample types and research needs.
Here are some tips on how get a sample prepared and ready to image, liberally adapted from that conversation and Caprioli’s recent review on the subject [1].
Get involved early
MALDI-IMS relies on an ionizable mixture of matrix and sample that is taken up and analyzed by a mass spectrometry, one spot at a time, to create a series of 2D images representing the sample’s molecular distribution, one image per molecule.
Ideally, the tissue to be imaged is perfectly arranged on a slide or tissue array, awaiting the matrix to be spotted atop. But in reality, many variables can affect this step, beginning with the way the sample is originally acquired and preserved. If you can, communicate with the people harvesting and preparing the samples. It’s best to immediately flash-freeze the sample by laying it on top of a piece of foil floating on a cryogen (like liquid nitrogen) and then wrapping it in the foil and immersing it in the cryogen bath; this helps preserve both molecular and histological (morphological) integrity.
Fixed tissue also can be used, and because most tissue is preserved in this way, many protocols have been developed for the purpose. But be aware that crosslinking that occurs during the process will hinder downstream protein analysis via MS.
Media used to embed the sample also can have an effect. Polymeric solutions like the popular “Optimal Cutting Temperature” (OCT) (e.g., Fisherbrand™ HistoPrep™ Frozen Tissue Embedding Media) can cause contamination and ion suppression. So rather than embedding the frozen sample in OCT, use it just as a glue to secure the sample to the cryotome chuck. If embedding the sample is unavoidable, wash the OCT from the sample with ethanol before applying MALDI matrix.
Start with the basics
A host of matrices and solvent solutions have been developed for specialized applications and difficult MALDI-IMS analytes, but start with the basics. Typically a solution of organic acid (most commonly 2,5-dihydroxybenzoic acid (DHB), α-cyano-4-hydroxycinnamic acid (CHCA) or 3,5-dimethoxy-4-hydroxycinnamic acid (SA, sinapinic acid)) in an organic solvent (e.g., methanol, ethanol or acetonitrile), with 0.1% trifluoroacetic acid, is applied to the sample, where it extracts and co-crystalizes with the analytes. The matrix captures the laser energy and transfers it to the analytes, allowing the latter to desorb and ionize, and subsequently enter the MS analyzer.
The basic IMS protocol was developed for hydrophilic proteins and peptides and works for many small molecule drugs, metabolites and lipids, as well, but you may need to optimize conditions a bit. And if you’re looking at hydrophobic, transmembrane or otherwise insoluble proteins, a MALDI-compatible (for example, non-ionic) detergent may be in order.
Your best bet: Consult the literature to see what matrices, solvents, detergents and conditions have been used to successfully image similar samples, and go from there.
Choose all or some
When laying down the matrix, consider what you hope to see. Will you be imaging the entire specimen, in which case matrix needs to be applied uniformly across the sample? Or will you be looking just at a few discrete, previously selected areas of interest (often in collaboration with a biologist or pathologist)? Although the latter (termed “histology-directed molecular imaging”) doesn’t provide as complete a picture as the former—molecular data is superposed on a microscope or other image—it significantly reduces both data acquisition time and the torrent of the data that needs to be collected.
Use a noncontact printer
There are many ways to lay a matrix atop tissue samples, the most common of which are spraying and spotting. Manual spraying can be easy and inexpensive, but it is the least reproducible. An automated (robotic) sprayer allows protocols to be fine-tuned and repeated after they’re optimized. But with continuous spraying, the analytes may migrate laterally, making their localization less precise. To get around this issue, try using an automated spotter instead of a sprayer. Because cross-contamination is often a concern, look specifically for a noncontact printer. (It’s faster, too.)
With MALDI-IMS there are different options for, and differing opinions about, everything from sample prep and matrix addition to imaging and data analysis. Start with the tried and true, and then tweak and optimize for your samples and analytes of interest.
Reference
1. Norris, JL, Caprioli, RM, “Analysis of tissue specimens by matrix-assisted laser desorption/ionization imaging mass spectrometry in biological and clinical research,” Chem Rev, 113(4):2309-42, 2013. [PMID: 23394164]
Image: Courtesy of the Mass Spectrometry Research Center, Vanderbilt University