Article pubs.acs.org/ac
Hydroxyflavones as a New Family of Matrices for MALDI Tissue Imaging Xiaodong Wang,† Jun Han,† Albert Chou,† Juncong Yang,† Jingxi Pan,† and Christoph H. Borchers*,†,§ †
University of Victoria - Genome British Columbia Proteomics Centre, Vancouver Island Technology Park, #3101-4464 Markham St., Victoria, BC V8Z 7X8, Canada § Department of Biochemistry and Microbiology, University of Victoria, Petch Building Room 207, 3800 Finnerty Rd., Victoria, BC V8P 5C2, Canada S Supporting Information *
ABSTRACT: The discovery of new matrices that are suitable for in situ analysis of low molecular-weight compounds by matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) is an important technological aspect of tissue imaging. In this work, ten natural flavonoid compounds, including flavone and nine of its mono- or polyhydroxylsubstituted analogues (3-hydroxyflavone, 5-hydroxyflavone, 3,7-dihydroxyflavone, chrysin, 7,3′,4′-trihydroxyflavone, fisetin, luteolin, quercetin, and morin) were evaluated as potential MALDI matrices for the profiling and imaging of endogenous lipids in mouse liver, using a Fourier transform ion cyclotron resonance (FTICR) mass spectrometer with a 355-nm Nd:YAG UV laser, in the positive ion mode. When an electronic sprayer was used for matrix coating and with a high-pH (0.1−0.5% ammonia hydroxide) matrix solvent, eight of the ten compounds, all of which had at least one OH group at the C3 or C5 position of the flavone structure, enabled the successful detection of 77 to 161 phospholipids and other lipids. The best results were observed with two penta-OH flavones (i.e., quercertin and morin). Taking quercetin as an example, this matrix showed characteristics superior to those of commonly used MALDI matrices, such as DHB (2,5-dihydroxybenzoic acid), CHCA (α-cyano-4-hydroxycinnamic acid), and 2mercaptobenzothiazole (2-MBT). These characteristics were: μm-sized matrix crystals, uniform matrix coating, low volatility in the high vacuum (∼10−7 mbar) source, good chemical stability, low yield of matrix-related ions, low matrix consumption, low power threshold for laser desorption/ionization, and improved safety of handling. The use of quercetin led to improved lipid imaging, with 212 lipids being successfully imaged from rat brain in a single experiment and with asymmetric distributions of some lipids in left and right brain hippocampus being observed for the first time.
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DHB (2,5-dihydroxybenzoic acid) is another widely used matrix for lipid imaging in both positive and negative ion modes.9 However, the nonhomogeneous crystallization and large crystal size of DHB can cause molecular delocalization and poor spot-to-spot reproducibility.10 With spray matrix deposition, 2-mercaptobenzothiazole (2-MBT)11 and, more recently, dithranol,12 have been found to be two useful matrices for MALDI imaging. pNA is a matrix that has recently been shown for MALDI tissue imaging of endogenous lipids in the positive ion mode on an intermediate pressure MS system,13 but it is too volatile for high-vacuum MALDI sources which are typical for Fourier transform ion cyclotron resonance (FTICR) MS instruments. In addition, ionic matrices have also been reported for tissue imaging of lipids by MALDI-MS.14,15 For example, the use of
issue imaging by matrix-assisted laser desorption/ ionization mass spectrometry (MALDI-MS) is now a well-established analytical technique that has become a powerful tool for simultaneously determining the spatial localization and distribution patterns of endogenous compounds (e.g., lipids) directly from the surface of a thinly cut tissue section.1−4 Despite the enormous progress that has been made in tissue imaging, there is still an unmet need for MALDI matrices suitable for the imaging of low molecular-weight compounds. The screening and discovery of new MALDI matrices has been an important aspect of mass spectrometric imaging.1 Compounds such as 9-aminoacridine (9-AA),5 paranitroaniline (pNA),6 and 2,5-dihydroxyacetophenone (DHA)7 are currently the popular MALDI matrices for MALDI imaging of low-molecular-weight compounds in the negative ion MS detection mode. In the positive ion mode, α-cyano-4hydroxycinnamic acid (CHCA) has been widely used for detection of lipids, but the high abundance of matrix-related background signals limits its wide application in tissue imaging.8 © 2013 American Chemical Society
Received: May 31, 2013 Accepted: July 15, 2013 Published: July 15, 2013 7566
dx.doi.org/10.1021/ac401595a | Anal. Chem. 2013, 85, 7566−7573
Analytical Chemistry
Article
(TFA), formic acid, and ammonia hydroxide (NH4OH), were also purchased from Sigma-Aldrich. Tissue Cutting and Matrix Coating. The frozen tissues were sectioned at −20 °C into slices of 12 μm thickness in a Microm HM500 cryostat (Waldorf, Germany). Serial tissue slices were immediately thaw-mounted on ITO-coated microscopic glass slides obtained from Bruker Daltonics (Bremen, Germany). Before matrix deposition, the tissue sections were dried under vacuum at a pressure of 10 mbar for 15 min. Matrix coating was carried out with a Bruker Daltonics ImagePrep electronic matrix sprayer (Bremen, Germany) as previously described,12 with slight modifications. Briefly, up to thirty cycles of matrix coating (3 s spray, 60 s incubation, and 90 s drying time) were applied with the ImagePrep sprayer for matrix application in order to obtain a uniform matrix layer. The ten flavonoid compounds to be used as the MALDI matrices were prepared as saturated solutions for 3-hydroxyflavone and 5hydroxyflavone (
