Sample Preparation for IMS
Sample preparation in IMS is critically important to ensure reproducible tissue images with the highest achievable spatial resolution; however, current methods are often highly variable and time consuming. We are developing protocols for both matrix application and matrix-free sample preparation that provide high analyte specificity and high image fidelity across multiple preparations.
We developed protocols for pre-coating MALDI targets with matrix (Figure 1) [1,2], thereby improving image quality and providing standardized and high-throughput capabilities.
1. We achieved homogeneous matrix coating and small crystal sizes of approximately 1 µm with automatic spraying using a TM Sprayer modified with an electrospray ionization nozzle.
2. Automatic spraying provides fast throughput and allows for batch preparation at low cost.
We also pre-coated targets with analyte and class specific reactive reagents to derivatize specific classes of compounds, such as eicosanoids , sterols, and low molecular weight amines , including the anti-tuberculosis drug INH , amino acids, and neurotransmitters , to enhance their sensitivity and selectivity for imaging. For example, Figure 2 shows a rat brain cerebellum image of GABA derivatized with 4-hydroxy-3-methoxy-cinnamaldehyde (CA) and acquired by MALDI FTICR IMS. The above methods and derivatization protocols were validated using accurate mass measurements, MS/MS, and LC-MS/MS to distinguish target analytes from potentially interfering isobaric compounds.
Figure 2. Rat brain cerebellum images of CA derivatized GABA. Modified from: J Mass Spectrom 49 (8):665-73.
These techniques were further developed to analyze compounds that have been traditionally difficult to detect with MALDI IMS. One approach involves coupling a single chain fragment variable (scfv) antibody to a cleavable mass tag in order to locate analytes that bind to the antibody. For example, biotinylated scfv along with tagged-avidin was used to detect CYP1A1 and CYP1B1 in breast cancer tissue sections . The other approach utilizes a probe that is bound only to an active substrate, allowing localization of specific activities. For example, we have imaged the distribution of serine hydrolase activity in mouse embryo tissue via a fluorophosphonate probe linked to a dendrimer with photocleavable mass tags . The large single chain antibody library makes these methods amenable to potentially hundreds of specific proteins.
We developed a new matrix (E)-4-(2,5-dihydroxyphenyl)but-3-en-2-one (2,5-cDHA) that is vacuum stable and provides high sensitivity for detection of proteins, and we developed an automatic spraying method based on a seeding approach to acquire small matrix crystals (1-2 µm) on the surface. 2,5-cDHA is a cinnamate analogue of 2,5-DHA. The synthesis of 2,5-cDHA is straightforward using the Wittig reaction and gram scale quantities can be obtained easily. It was shown that 2,5-cDHA is stable in the instrument vacuum after 24 hours, no significant change of 2,5-cDHA coating was measured. The coating contains very small crystals ranging from 1 to 2 µm in diameter (Figure 3A). With the highly sensitive detection of proteins using 2,5-cDHA, stable coating in the vacuum, and small crystals of coating, 5 µm spatial resolution imaging of proteins can be accomplished with commercial instrument routinely as shown in the Figure 3B .
Figure 3 A) SEM pictures of 2,5-cDHA on rat brain show that crystals are in ~1-2 µm diameter; B) 5 µm spatial resolution ion images acquired in 7 hours with no signal fading (reconstructed from J Mass Spectrom. 2018; 53:1005-1012).
Hydrogel Enhanced Protein Identification for Imaging
Hydrogel microwells achieve simultaneous proteolytic digestion and extraction on-tissue within sampled regions (Figure 4). The microwells are currently fabricated at dimensions of 1 mm to 4 mm in diameter [10,11], and methods are in place to produce smaller diameter microwells. The microwell approach incorporates microwave-assisted digestion  and can extract peptides from membrane proteins  and FFPE tissue, achieving better performance than on-tissue enzyme spotting methods. The strategic advantage of the hydrogel microwell workflow is that it blends the traditional proteomic identification capabilities of LC-MS/MS with histology-directed IMS experiments.
Figure 4. Hydrogel workflow. Modified from: Anal Chem 85 (5):2717-2723.
Membrane Protein Imaging
Membrane proteins represent a challenging class of analytes for IMS. Our efforts are focused on improving detection of less abundant membrane proteins. To date we have established protocols for IMS analysis of membrane proteins from brain and ocular tissues that provide a completely unique protein profile from traditional IMS profiles (representing soluble proteins) and reveal modified membrane proteins [13,14]. To increase the number of membrane protein identifications, methods were established to digest proteins directly on tissue sections via spray coating technologies .
1. Grove KJ, Frappier SL, Caprioli RM (2011) Matrix pre-coated MALDI MS targets for small molecule imaging in tissues. J Am Soc Mass Spectrom 22 (1):192-195. PMCID: 4151471. Return to text.
2. Yang J, Caprioli RM (2013) Matrix precoated targets for direct lipid analysis and imaging of tissue. Anal Chem 85 (5):2907-2912. PMCID: 3632340. Return to text.
3. Manna JD, Reyzer ML, Latham JC, Weaver CD, Marnett LJ, Caprioli RM (2011) High-throughput quantification of bioactive lipids by MALDI mass spectrometry: application to prostaglandins. Anal Chem 83 (17):6683-6688. PMCID: 3165080. Return to text.
4. Chacon A, Zagol-Ikapitte I, Amarnath V, Reyzer ML, Oates JA, Caprioli RM, Boutaud O (2011) On-tissue chemical derivatization of 3-methoxysalicylamine for MALDI-imaging mass spectrometry. J Mass Spectrom 46 (8):840-846. PMCID: 3174490. Return to text.
5. Manier ML, Reyzer ML, Goh A, Dartois V, Via LE, Barry CE, Caprioli RM (2011) Reagent precoated targets for rapid in-tissue derivatization of the anti-tuberculosis drug isoniazid followed by MALDI imaging mass spectrometry. J Am Soc Mass Spectrom 22 (8):1409-1419. PMCID: 3424619. Return to text.
6. Manier ML, Spraggins JM, Reyzer ML, Norris JL, Caprioli RM (2014) A derivatization and validation strategy for determining the spatial localization of endogenous amine metabolites in tissues using MALDI imaging mass spectrometry. J Mass Spectrom 49 (8):665-673. PMCID: 4126081. Return to text.
7. Thiery G, Mernaugh RL, Yan H, Spraggins JM, Yang J, Parl FF, Caprioli RM (2012) Targeted multiplex imaging mass spectrometry with single chain fragment variable (scfv) recombinant antibodies. J Am Soc Mass Spectrom 23 (10):1689-1696. PMCID: 3525520. Return to text.
8. Yang J, Chaurand P, Norris JL, Porter NA, Caprioli RM (2012) Activity-based probes linked with laser-cleavable mass tags for signal amplification in imaging mass spectrometry: analysis of serine hydrolase enzymes in mammalian tissue. Anal Chem 84 (8):3689-3695. PMCID: 3328658. Return to text.
9. Yang J , Norris JL , Caprioli R, Novel vacuum stable ketone-based matrices for high spatial resolution MALDI imaging mass spectrometry Journal of mass spectrometry : JMS. 2018 Aug 21; 53 (10). 1005-1012
10. Harris GA, Nicklay JJ, Caprioli RM (2013) Localized in situ hydrogel-mediated protein digestion and extraction technique for on-tissue analysis. Anal Chem 85 (5):2717-2723. PMCID: 3595595. Return to text.
11. Taverna D, Pollins AC, Sindona G, Caprioli RM, Nanney LB (2014) Imaging mass spectrometry for assessing cutaneous wound healing: Analysis of pressure ulcers. J Proteome Res. PMCID: Not Listed. Return to text.
12. Taverna D, Norris JL, Caprioli RM (2015) Histology-directed microwave assisted enzymatic protein digestion for MALDI MS analysis of mammalian tissue. Anal Chem 87 (1):670-676. PMCID: 4287167. Return to text.
13. Nicklay JJ, Harris GA, Schey KL, Caprioli RM (2013) MALDI imaging and in situ identification of integral membrane proteins from rat brain tissue sections. Anal Chem 85 (15):7191-7196. PMCID: 3782084. Return to text.
14. Schey KL, Grey AC, Nicklay JJ Mass Spectrometry of membrane proteins: a focus on aquaporins. Biochemistry 53 (22):3807-3817. Return to text.
15. Wenke JL, Schey KL Microwave-Assisted Enzymatic Digestion On-Tissue for Membrane Protein Analysis with MALDI Imaging Mass Spectrometry. In: 61st American Society for Mass Spectrometry Conference on Mass Spectrometry and Allied Topics, Minneapolis, MN, 2013.