Matrix pre-coated targets for high throughput MALDI imaging of proteins

Application note

Received: 25 November 2013 Revised: 7 February 2014 Accepted: 22 February 2014 Published online in Wiley Online Library

(wileyonlinelibrary.com) DOI 10.1002/jms.3354

Matrix pre-coated targets for high throughputMALDI imaging of proteinsJunhai Yanga and Richard M. Capriolia,b,c*

We have developed matrix pre-coated targets for imaging proteins in thin tissue sections by matrix-assisted laser desorption/ionization mass spectrometry. Gold covered microscope slides were coated with sinapinic acid (SA) in batches in advance and
were shown to be stable for over 6months when kept in the dark. The sample preparation protocol using these SA pre-coatedtargets involves treatment with diisopropylethylamine (DIEA)-H2O vapor, transforming the matrix layer to a viscous ionicliquid. This SA-DIEA ionic liquid layer extracts proteins and other analytes from tissue sections that are thaw mounted to thistarget. DIEA is removed by the immersion of the target into diluted acetic acid, allowing SA to co-crystallize with extractedanalytes directly on the target. Ion images (3–70 kDa) of sections of mouse brain and rat kidney at spatial resolution downto 10μm were obtained. Use of pre-coated slides greatly reduces sample preparation time for matrix-assisted laserdesorption/ionization imaging while providing high throughput, low cost and high spatial resolution images. Copyright ©2014 John Wiley & Sons, Ltd.
Additional supporting information may be found in the online version of this article at the publisher’s web-site.

Keywords: MALDI IMS; imaging; pre-coated; target; proteins

* Correspondence to: Richard M. Caprioli, Mass Spectrometry Research Center,Vanderbilt University, 465 21st Avenue South, 9160 MRB 3, Nashville, TN37232-8575, USA. E-mail: [email protected]

a Department of Biochemistry, Vanderbilt University, Nashville, Tennessee, USA

b Departments of Chemistry, Pharmacology and Medicine, Vanderbilt University,Nashville, Tennessee, USA

c Mass Spectrometry Research Center, Vanderbilt University, Nashville, Tennessee,USA

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Introduction

Matrix-assisted laser desorption/ionization imaging mass spec-trometry (MALDI IMS) is a highly effective discovery tool becauseit provides spatial information on a variety of compoundssimultaneously without the need for a priori knowledge of thesecompounds. Such molecular mapping can be accomplished at5–10μm spatial resolution from thin tissue sections of nearlyany size with unmatched molecular specificity. Although carefulsample preparation is important in achieving high qualityimages, it can be time consuming and generally requires specialequipment for matrix deposition, e.g. nebulizer,[1,2] modifiedinkjet printer,[3] robotic droplet depositor,[4] chemical printer,[5]

spray depositor[6] and vibrational vaporizer.[7] In addition,although high molecular weight species (30 to over 200 kDa)have been measured directly from tissue sections underspecial conditions,[8–11] imaging high molecular weight species(>50 kDa) remain challenging. In general, 30min or more isrequired to prepare a tissue section for imaging, making highthroughput, robust and cost effective matrix deposition methodhighly desirable.

Matrix pre-coated MALDI targets have been reported forcontinuous sample deposition,[12,13] lipid imaging using 2,5-dihydroxybenzoic acid,[14] derivatization reagents[15] and appli-cation of 1,5-diaminonaphthelene.[16] These matrix pre-coatedtargets are suitable for imaging lipids, small peptides andpharmaceuticals but have not proven effective for imagingproteins.

We report here a sample preparation method employingtargets homogenously pre-coated with sinapinic acid (SA) forimaging proteins (~3–70 kDa) with high spatial resolution (downto 10μm) and high throughput because there is no matrixdeposition step at the time of analysis, only a fast hydrationand rinsing step. Briefly, SA on a pre-coated slide is converted

J. Mass Spectrom. 2014, 49, 417–422

to an ionic liquid through exposure to an organic diisopro-pylethylamine (DIEA) vapor, and thin tissue sections are placedon top. A single step of immersing the target into diluted aceticacid (1%) for 60 s completes the sample preparation (Fig. 1).Matrix pre-coated slides were prepared by casting matrixsolution on clean gold coated microscope slides. The samplepreparation protocol was evaluated for the thickness of matrixcoating, the thickness of tissue section, reproducibility ofimaging of serial sections and shelf life of the prepared slides.Ion images are presented for mouse brain, rat kidney and ratbrain sections.

Experimental and materials

Ethanol, methanol, acetonitrile and acetic acid were purchasedfrom Fisher Scientific (Suwanee, GA), xylene from Acros (MorrisPlains, NJ), isopropanol, DIEA, potassium hydroxide andhydrogen peroxide (30%) from Sigma-Aldrich (Milwaukee, WI).SA was purchased from Oakwood Products, Inc (SC) andrecrystallized twice with 70% acetonitrile. Water-white glassmicroscope slides with a titanium adhesion layer and 50 nm thick

Copyright © 2014 John Wiley & Sons, Ltd.

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Figure 1. Sample preparation with a sinapinic acid pre-coated slide (yellow, SA; green, SA-DIEA-H2O).

J. Yang and R. M. Caprioli

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Au coating were purchased from Deposition Research Lab. Inc.(St. Charles, MO). Frozen rat brain, mouse brain, chicken liverand rat kidney were obtained from Pel Freez (Rogers, AR) andstored at �80°C.

Preparation and use of matrix pre-coated slides on goldcoated slides

Each gold coated microscope slide (7.5 × 2.5 cm) is cut by a dia-mond glass cutter to the size desired by the user. We typicallycut each slide to give five targets of 2.5 × 1.5 cm. The targets werecleaned by immersing them into a solution of hydrogen peroxide(30%) and saturated potassium hydroxide in isopropanol at 2 : 1ratio for 3min, rinsed with MilliQ water (from a MilliQ Advantage

Figure 2. (A) 3μm thick chicken liver sections placed on slides with differenbrain sections imprinted on SA pre-coated slides with a coating of 2.7mg/cm

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A10 Ultrapure Water Purification System, Millipore, Billerica, MA)and absolute alcohol and air dried.

Sinapinic acid coating of the targets was performed by im-mersing them into a 3ml of SA solution (100mg SA in 10mlH2O, 5ml ethanol, 1ml acetic acid and 650 μl chloroform) in aPetri dish (35 × 10mm). The solution was left for evaporationfor about 2 h in the hood. The targets were then rinsed withMilliQ water and dried under ambient conditions. The weightof the gold coated slides was measured before and after theSA coating, the difference of the weight was divided by the areaof the slides (2.5 cm× 1.5 cm= 3.75 cm2) to give the weight/areaof each slide. Generally, this procedure provides a coating of SAof approximately 2.6–2.7mg/cm2. The targets were carefullyplaced on a piece of emery paper (8000 grit, Scientific

t amounts of sinapinic acid (SA) coating and (B) different thicknesses of rat2. Average spectra were taken from 100 locations on the tissue.

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Matrix pre-coated targets for MALDI imaging MS

Instrument Services, Ringoes, NJ) with the matrix side down andgently polished to remove larger crystals. The pre-coatedtargets were preserved under dark conditions at room temper-ature, and when stored in this way, they gave equivalent perfor-mance when stored for over 6months.

Each SA pre-coated target is placed in a 35 × 10mm Petri dish,and this dish was placed in a 60ml glass jar (Fisher, 02-911-763)containing 200 μl of DIEA and 900 μl of H2O under roomtemperature. The jar was then sealed for 7min. The targetwas taken out and placed in the cryostat chamber to coolto temperature of �20°C. Thin sections of different tissueswere placed on the top of the matrix coating, and the slidewas immediately immersed into 20ml of 0°C cold 1% AcOHfor 60 s. The solution was agitated for 10 s so that the tissueon the surface was removed. The slide was rinsed again in asecond bath of 1% AcOH and dried under ambient conditionsfor subsequent imaging.

Serial tissue sections were subjected to the standard hematox-ylin and eosin (H&E) staining.[17] Optical images of the H&Estained section were acquired with a Mirax slide scanner fromZeiss at 0.23μm resolution.

Matrix-assisted laser desorption/ionization mass spectrometryanalyses were performed on a Bruker Autoflex Speed massspectrometer in positive ion linear mode using FlexControl 3.3software. Approximately 100–200 laser shots/spot were acquired

Figure 3. Average spectra from a rat kidney from the images in Fig. 4. (A) 3

J. Mass Spectrom. 2014, 49, 417–422 Copyright © 2014 John W

with a 1 kHz repetition rate Smartbeam II Nd:YAG laser (355 nm).Image production and analysis was performed using FlexImaging3.0 and FlexAnalysis 3.3.

Results and discussion

Activation of the stored SA pre-coated slide was performed bytreatment with an organic amine (DIEA) and water vapor,converting SA on the surface to an ionic liquid (SA-DIEA-H2O).After tissue placement on the activated slide, the liquid matrixextracts proteins from tissue section. The SA-DIEA-H2O layer isviscous, minimizing delocalization of compounds after tissueplacement. Results presented in the following examples showno significant delocalization at the spatial resolutions tested.When the slide is immersed into diluted AcOH, DIEA is removedfrom the liquid film and the SA precipitates and co-crystallizeswith the extracted proteins.

Assessing tissue thickness and the amount of matrix coating

Tissue sections of the same thickness (liver sections 3μm thick,Fig. 2(A)) were placed on the matrix pre-coated slide with differ-ent amounts of matrix coating (weight of matrix/cm2). Itwas observed that the peak intensity in the average mass

–30 kDa and (B) 30–70 kDa. These were acquired from two serial sections.

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spectrum is maximized when the thickness of the coating is1.1mg/cm2 or more.In our protocol, the tissue section was removed at the last step,

and therefore, the thickness of tissue section has little impact onthe mass spectral profile (Fig. 2(B)). This thickness independenceis different from the pre-coated target for imaging lipids,[16]

where the tissue section is preserved on the surface and thethinner the section, the better quality of the mass spectrum.Indeed, in the current case where the same protocol was usedexcept that the tissue sections were kept on the SA slide, weobserved a reduced sensitivity (Fig. S1).A rat kidney section of 12μm thickness was imaged at 120μm

spatial resolution producing high quality ion images in the m/zrange between 3000 and 70 000 (average spectrum shown inFig. 3). All the ion images matched well with the optical imagesof H&E stained serial sections (Fig. 4). Similar results wereobtained for mouse brain images when imaged in the 3–70 kDarange at both low spatial resolution (100μm, shown in Figs. S2and S3) and high spatial resolution (10μm, shown in Fig. 5).Matrix pre-coated slides can be made beforehand in batches,

enabling quality control assessment of each slide either opti-cally or gravimetrically. Using targets with carefully controlledcoating thickness, matrix crystal size distribution and surface

Figure 4. Representative m/z ion images from 3 to 30 and 30 to 70 kDa frombelow the images.


homogeneity, high reproducibility can be achieved, as demon-strated in Fig. 6, where four serial rat brain sections wereplaced onto SA pre-coated slides that had been stored for 1,2, 30 and 180 days. Nearly identical images were obtaineddemonstrating that SA pre-coated slides can provide reproduc-ible performance and have great stability over at least severalmonths.

We have also investigated the effectiveness of different targetmaterials for pre-coated slides, including: glass, stainless steel,copper, aluminum, silicon and gold, following application of SAsolution. A gold surface gave the best performance using a ho-mogenous SA coating. Although homogenous SA coatings canbe obtained on almost any surface using the TM sprayer, we havedetermined that gold surfaced slides with an SA coating had suf-ficient stability to sustain the transformation to ionic form andsurvive the immersion into the 1% AcOH. On non-gold surfaces,at the final step of immersing into the AcOH, the SA coatingsometimes peeled off the surface resulting in a failed samplepreparation.

For SA pre-coated slides, under the condition where SA wasnot transformed to SA-DIEA-H2O, or just transformed to SA-DIEAwithout water in the layer, the mass spectra produced were ofsignificantly poorer quality.

two serial sections of a rat kidney (scale bar: 2mm). m/z values are noted


Figure 5. Ion images at 10μm spatial resolution from a mouse brain section. m/z values are noted below the images. Scale bars: 200μm.

Figure 6. Reproducibility and stability of sinapinic acid (SA) pre-coated targets. Four serial sections of a rat brain were imaged at 150μm spatialresolution using SA pre-coated slides stored for 1, 2, 30 and 180 days. m/z values are noted above each column.

Matrix pre-coated targets for MALDI imaging MS

J. Mass Spectrom. 2014, 49, 417–422 Copyright © 2014 John Wiley & Sons, Ltd. wileyonlinelibrary.com/journal/jms

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Conclusions

The SA pre-coated slides can be prepared in advance andpreserved under dark conditions for a long period, a significantconvenience for the end user. These targets can be used toimage proteins at high spatial resolution (down to 10μm) andimage high molecular weight species up to about 70 kDa.Because there is no need for expensive matrix coating roboticsfor matrix deposition, the use of pre-coated slides is cost effectiveand does not require special expertise on the part of the user.

Acknowledgement

The authors acknowledge funding from the National Institutesof Health (grant # NIH/NIGMS 5R01 GM058008-14 and 5P41GM103391-03).

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Supporting information

Additional supporting information may be found in the onlineversion of this article at the publisher’s web-site.


Matrix pre-coated targets for high throughput MALDI imaging of proteins

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