ARTICLE pubs.acs.org/jpr
MALDI-MS-Imaging of Whole Human Lens Capsule Maurizio Ronci,*,† Shiwani Sharma,‡ Tim Chataway,§ Kathryn P. Burdon,‡ Sarah Martin,‡ Jamie E. Craig,‡ and Nicolas H. Voelcker*,† †
School of Chemical and Physical Sciences, Flinders University, Bedford Park SA 5042, Australia Department of Ophthalmology, Flinders University, Bedford Park SA 5042, Australia § Flinders Proteomics Facility, School of Medicine, Flinders University, Bedford Park SA 5042, Australia ‡
bS Supporting Information ABSTRACT: The ocular lens capsule is a smooth, transparent basement membrane that encapsulates the lens and is composed of a rigid network of interacting structural proteins and glycosaminoglycans. During cataract surgery, the anterior lens capsule is routinely removed in the form of a circular disk. We considered that the excised capsule could be easily prepared for matrix-assisted laser desorption/ionization time-of-flight mass spectrometry imaging (MALDI-MSI) analysis. MALDI-MSI is a powerful tool to elucidate the spatial distribution of small molecules, peptides, and proteins within tissues. Here, we apply this molecular imaging technique to analyze the freshly excised human lens capsule en face. We demonstrate that novel information about the distribution of proteins by MALDI-MSI can be obtained from this highly compact connective tissue, having no evident histo-morphological characteristics. Trypsin digestion carried out on-tissue is shown to improve MALDI-MSI analysis of human lens capsules and affords high repeatability. Most importantly, MALDI-MSI analysis reveals a concentric distribution pattern of proteins such as apolipoprotein E (ApoE) and collagen IV alpha-1 on the anterior surface of surgically removed lens capsule, which may indicate direct or indirect effects of environmental and mechanical stresses on the human ocular lens. KEYWORDS: MALDI-MSI, human lens capsule, apolipoprotein E, collagen IV alpha-1, proteomics, trypsin digestion
’ INTRODUCTION Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry imaging (MALDI-MSI) is a powerful technique for analyzing the spatial distribution of molecules on biological surfaces and has emerged as a routine complement in proteomics workflows. Drugs and metabolites,1,2 peptides and proteins,3 6 lipids7,8 and other small molecules9 11 are the most common classes of target compounds suitable for MALDI-MSI analysis. Information about chemical structure and localization are simultaneously collected allowing nontargeted but systematic screening of thousands of molecules in a single experiment and at the same time the direct comparison with optical microscopy or immuno-histochemistry. Since its introduction in the late 1990s,5,12 remarkable methodological and technological improvements have been made.1,13 15 Such progress is elevating MALDI-MSI to a new level of performance, underpinning novel and exciting clinical applications in cancer research, neurodegeneration and fundamental applications in proteomics and drug discovery. With regards to clinical applications, MALDI-MSI has already proven to be an effective tool for surface analysis of different types of animal tissues and is making inroads toward the elucidation of molecular complexities of tissues. Fresh frozen samples, cryo-preserved as well as formalin-fixed paraffin-embedded (FFPE) tissues are suitable as a starting material for MALDI-MSI analysis.16 20 However, the suitability of unprocessed whole tissue has not been r 2011 American Chemical Society
ascertained. The human lens capsule is an excellent example of a tissue that is amenable to whole mount analysis. It is a smooth, transparent, homogeneous basement membrane that encapsulates the ocular lens, characterized by indistinct histo-morphological structure. The central anterior lens capsule is excised during the capsulorhexis step in routine cataract surgery in the form of a thin circular disk approximately 5.5 mm in diameter. Its thickness varies from 11 to 15 μm and increases with age.21 Due to its limited thickness, it is possible to analyze the tissue histologically or histochemically without sectioning. However, the tissue is composed of a rigid and highly cross-linked network of interacting collagen IV and laminin, further bound together by glycosaminoglycans (GAG) such as nidogen and perlecan,22 which confer elasticity and viscosity to the tissue. Thus the tissue requires harsh chemical treatment in order to break the crosslinks and release the proteins for mass spectrometric analysis. Two recently published conventional proteomics studies aimed to identify the protein constituents of pseudoexfoliative material accumulated on lens capsules affected by pseudoexfoliation (PEX) syndrome. The tissue was treated with 100% formic acid overnight followed by cyanogen bromide in 70% formic acid overnight before performing mass spectrometric analysis.23,24 A variety of proteins were identified in the capsule and the pseudoexfoliative material using this technique, Received: February 17, 2011 Published: June 13, 2011 3522
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Journal of Proteome Research however, a less destructive technique which would unravel the protein constituents of lens capsule, including the minor species, without requiring harsh treatment protocols and retaining spatial information would be highly desirable. The primary motivation behind the present study was to prove the principle that unsectioned human tissues, such as the whole mount human lens capsule, can be analyzed by MALDI-MSI to reveal protein localization. We obtain valuable information about the distribution of proteins after simple trypsin digestion and show the high repeatability of the digestion protocol on four different human lens capsule specimens by evaluating the distribution of the same set of peptides. Furthermore, we discover a novel differential concentric distribution of some of the structural and functional proteins across the lens capsule. In particular, we identify two main distribution regions corresponding to the pupillary area and to the iris area. We have validated these results using immunohistochemistry. With the proof of principle in hands, we are able to study the PEX-affected lens capsules and to characterize the protein composition and localization of the PEX material.
’ EXPERIMENTAL SECTION Materials
Unless stated otherwise, all solvents and chemicals were obtained from commercial sources at the highest purity available and used without further purification. MALDI-MS and LIFT-MS/ MS spectra were acquired on an Autoflex III TOF/TOF mass spectrometer (Bruker-Daltonics GmbH, Bremen, Germany) equipped with a SmartBeam 200 Hz laser, using R-cyano-4-hydroxycinnamic acid (CHCA) as matrix. Proteomics grade modified trypsin was purchased from Sigma (St. Louis, MO). R-Cyano-4hydroxycinnamic acid (CHCA), peptide standard mix and protein standard mix for MALDI-MS calibration were obtained from Bruker-Daltonics. ZipTip C18 micropipet tips were from Millipore (Billerica, MA). A Dionex Ultimate 3000 HPLC (Dionex Corp, Sunnyvale, CA) chromatographic system coupled to a Thermo Orbitrap XL linear ion trap mass spectrometer fitted with a nanospray source (Thermo Electron Corp, San Jose, CA) were used to perform LC MS/MS analysis. All buffers and solutions were freshly prepared in Milli-Q grade water. Specimen Collection
The anterior human lens capsules were collected from cataract patients at the time of cataract surgery following approval of the Southern Adelaide Health Service/Flinders University Human Research Ethics Committee, South Australia, Australia. Lens capsules were removed by capsulorhexis prior to phacoemulsification of the lens and stored in sterile balanced salt solution at 4 °C for later analysis. A total of 11 clinical samples, divided as follows, were used: 4 for the MALDI-MSI analysis, 3 (pooled) for validation by liquid chromatography tandem mass spectrometry and 4 for validation by immunohistochemistry. Lens Capsule Preparation
Human lens capsules were directly mounted on indium tin oxide (ITO) coated glass slides basement membrane surface up, and washed extensively with Milli-Q water, then 70% ethanol and finally 100% ethanol, one minute each. Trypsin solution (200 μL of a solution of 0.1 mg/mL trypsin in 10 mM (NH4)HCO3) was laid in several cycles through the ImagePrep (Bruker-Daltonics) piezoelectric automatic sprayer at room temperature, allowing the samples to partially dry between each cycle. Digestion was carried out for one hour. The samples were finally coated with a
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solution of 7 mg/mL CHCA in TFA 0.2%/ACN 50:50 using the ImagePrep piezoelectric automatic sprayer. MALDI-MSI Analysis
Mass spectrometric imaging analysis was performed in linear positive (LP) mode in the range 1 10 kDa with a spatial resolution of 100 μm. Fleximaging 2.1 (build 25) (BrukerDaltonics) was used to control Flexcontrol 3.3 (build 85) during the acquisition. Fleximaging was used to extract the ion intensity map images, after processing the data sets by baseline subtraction, normalization and data reduction. ClinProTools 2.2 (build 83) was used as spectra class analysis and visualization tool (details of instrumental parameters are provided as Supporting Information). Protein Identification by On-tissue MALDI-LIFT-MS/MS
To increase mass accuracy on the peptides signals, a reflectron positive (RP) mode spectrum was recorded in the pupillary region of the lens and in the external ring (see the paragraph “MALDI-MSI analysis of human lens capsule” and Figure 1b for the definition of regions). Signals selected for fragmentation were chosen after the evaluation of the RP spectra. In particular, the minimum requirements to obtain good fragmentation spectra and positive results for the database search were: intensity higher than 4000 arbitrary units and the absence of any other signal inside a mass window of about 1% of the selected mass (details for instrumental parameters of MALDI-LIFT-MS/MS acquisitions are provided as Supporting Information). Immunohistochemistry
Samples were mounted on SuperFrost Plus slides (Menzel Glaser), washed three times with Milli-Q water and allowed to stick to the slide for 30 min. They were then fixed in prechilled acetone/methanol (1:1) at 20 °C for 5 min, treated with 0.1 M KCl HCl (pH 1.5) for 10 min to increase antigen accessibility, rinsed briefly with phosphate buffer saline (1 PBS; 150 mM NaCl, 5 mM NaH2PO4, pH 7.4) and incubated for 1 h with the blocking solution (1% BSA in 1 PBS). After washing twice with 1 PBS for 5 min each the samples were treated with 0.05% hydrogen peroxide in 1 PBS for 10 min to block endogenous peroxidases, washed two times in 1 PBS and subsequently incubated for 1 h at room temperature with anti-ApoE (1:500) (Calbiochem, Merck Pty, Vic, Australia) or anti-ZO-1 (1:250) (Zymed Laboratories, Invitrogen, Vic, Australia) primary antibody. Samples were rinsed two times with 1 PBS and incubated in humidified tray for 1 h at RT with Dual Link Reagent (Dako). Samples were first washed two times in 1 PBS, then in running tap water, then 2 times in absolute ethanol for 5 min and finally two times in xylene before being mounted in Depex (Gurr, BDH). Images were taken on an Olympus BX50 Bright Field Imaging Microscope equipped with Q Imaging Digital Camera using a 2 objective. MS/MS Analysis of Dissected Lens Capsules
According to the MALDI-MSI distribution results and to the anatomic evidence, the pupillary region of the lens capsule was dissected using a surgical sterile punch (2 mm diameter) as schematically shown in Figure 3. The excised central region and the peripheral ring were separately transferred into 500 μL Eppendorf tubes and washed twice with Milli-Q water. 40 μL of 100 mM Tris buffer (pH 7.5) containing 6 M urea were added to each tube. Cysteines were reduced and alkylated with 50 μL 100 mM dithiothreitol and 50 μL 55 mM iodoacetamide, respectively. After derivatization, the samples were incubated overnight at 37 °C with 20 μL of a 50 mM ammonium bicarbonate buffer containing 3523
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Figure 1. Optical image of the lens capsule acquired in bright field and relative ion intensity maps of several peptides, chosen for visualization purposes, showing differential distribution (panel a, scale bar 1 mm). Three main distribution regions are noticeable: core, external ring and edge, schematically represented in panel b. Panel c shows the gel/stack view of all the spectra corresponding to the core (pupillary) region and to the peripheral ring (iris) region, grouped in two classes. The spectrum view shows the overlaid average spectra for each class (red line = pupillary core; green line = external ring) with two zoom inserts pointing to the signals at m/z 1394 and 1925, which present opposite distribution.
sequencing grade trypsin (Sigma) at 0.5 mg/mL. Five μL of 10% TFA were added to quench the digestion process. The digested samples were first analyzed by MALDI-TOF-MS and subsequently by tandem mass spectrometry (details for instrumental parameters of LC MS/MS analysis are provided as Supporting Information). Before MALDI-MS analysis, peptides were concentrated and desalted by reverse phase extraction using ZipTip C18 according to the manufacturer guidelines, and eluted with 2 μL 50% ACN/ TFA 0.2% containing CHCA at 5 mg/mL directly on the MTP Ground Steel 384 target (Bruker Daltonics).
’ RESULTS AND DISCUSSION MALDI-MSI of Human Lens Capsule
Generally, the detection sensitivity of standard MALDI-MS analysis on purified samples is in the attomole range for peptides and in the femtomole range for proteins. For on-tissue MALDI-MSI experiments, the detection sensitivity drops drastically because of ion suppression effects due to the large chemical complexity
of the microenvironments invariably encountered in tissues. This phenomenon emphasizes the crucial role of the sample preparation method in enhancing detection sensitivity on-tissue,25,26 particularly when dealing with compact tissues such as the lens capsule. Human lens capsules were first analyzed following a conventional MALDI-MSI approach focusing on proteins in the low mass range 1 30 kDa. Less than five mass signals were present in the resulting spectra. All the signals were below 10 kDa and none of them matched the mass of proteins known to be present in the lens capsule (data not shown). These preliminary experiments indicated that standard protocols are not suited for this type of tissue. In situ trypsin digestion was subsequently employed to release peptides from the cross-linked protein network of the lens capsule. On-tissue trypsin digestion has been applied to proteomic analysis by MALDIMSI to “unlock” cross-linked proteins in formalin-fixed paraffinembedded (FFPE) stored samples and to enable the direct identification of peptides from the sections through MS/MS.27,28 Moreover, also for spray coating trypsin deposition methods, besides robotic nanospotting, it has been shown that the distribution of 3524
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Table 1. List of Identified Proteins by On-tissue MALDI-LIFT-MS/MS UniProtKB/ Swiss-Prot accession number
sequence coverage MSMS (%)
mass error precursor/rms fragments (ppm)
peptides list R.LGPLVEQGR.V R.AQAWGERLR.A R.LGPLVEQGRVR.A K.ILYHGYSLLYVQGNER.A R.GLDGYQGPDGPR.G
pI
Mw (kDa)
Mascot score MS/MS
Apolipoprotein E
P02649 APOE_HUMAN
5.65
36.132
70
6
Collagen alpha-1 (IV) Collagen alpha-2 (IV) Clusterin
P02462 CO4A1_HUMAN P08572 CO4A2_HUMAN P10909 CLUS_HUMAN
8.55
160.514
60
0
17/125 14/308 16/203 26/963
8.89
167.449
33
0
11/180
5.89
52.461
119
8
Laminin subunit beta-2 Laminin subunit alpha-5 Metalloproteinase Inhibitor 3 Vitronectin
P55268 LAMB2_HUMAN O15230 LAMA5_HUMAN P35625 TIMP3_HUMAN P04004 VTNC_HUMAN
6.07
195.854
97
1
6.63
399.540
28
0
5/200 16/138 33/121 18/305 29/131 7/271
R.RPHFFFPK.V R.ASSIIDELFQDR.F K.LFDSDPITVTVPVEVSR.K R.QLDALLEALKLKR.A R.GAVADTRDTEQTLYQVQER.M R.LGLVWAALQGAR.T
9.00
24.129
93
8
5.55
54.271
102
7
10/782 13/145 21/161 24/211 10/92 48/61
K.YQYLLTGR.V R.WDQLTLSQR.K R.RVDTVDPPYPR.S R.FEDGVLDPDYPR.N R.DVWGIEGPIDAAFTR.I R.DWHGVPGQVDAAMAGR.I
protein name
peptides generated by the digestion procedure matches the original localization of the undigested proteins and diffusion across the tissue is limited.29 Interestingly, our results indicate that proteins in the lens capsule are differentially distributed across different areas. In particular, three main regions were recognized: a central region (core), a wide circular section around the center (external ring) and one thinner ring close to the border of the lens capsule (edge). Figure 1a shows the distribution of some of the most representative signals for each area. In the first row, some of the signals specifically located in a peripheral ring region are shown (m/z 1185, 1925, 2213, 3745); in the second row, the ones found in the pupillary core region (m/z 1394, 1426, 1733, 1831); then signals appearing to correspond to the peripheral edge of the lens capsule (m/z 2753, 2697) or evenly distributed on the surface (m/z 1601, 3594); finally, some peptides had distributions that do not follow an obvious pattern (m/z 1475, 1201, 1522, 2527). Figure 1b shows the schematic of a lens capsule with the differential areas highlighted by dotted lines. Using the spectra from the two main identified regions (pupillary core and peripheral ring), we performed a comparative analysis using ClinProtools. Data were processed by applying baseline subtraction, data reduction and recalibration. Figure 1c shows the gel/stack view of all the spectra and the average spectrum for each class, with two inserts pointing to exemplary mass signals with opposite distribution at m/z 1394 and 1925. As reported in the literature22 the core structural components of the lens capsule self-assemble into a three-dimensional cross-linked network that provides structural and protective support to the developing and mature lens. Based on previous work on a different type of crosslinked tissue (FFPE),18 we assume that the peptides originated from the same regions as their parent proteins and that the digestion process does not significantly affect their distribution. If that assumption holds true, valuable information about localization of proteins can be extracted. To identify proteins from the most abundant peptide signals, MS/MS fragmentation experiments were carried out next. Protein Identification
Identification by On-tissue MALDI-LIFT-MS/MS. In contrast to MALDI-MSI analysis on intact proteins, the identification of
proteins directly from the tissue surface is made possible after digestion with trypsin, provided that peptides signals are adequately intense and sufficiently isolated. MALDI-MS analysis of the lens capsule resulted in the appearance of feature-rich spectra from which we were able to match peptide peaks to several known human proteins. More than 200 signals were present in the average spectra. Peptide fragmentation analysis was carried out directly from the digested lens capsule surface. More than 40 peptides were fragmented and 18 peptide sequences returned a positive match after database interrogation: metalloproteinase inhibitor 3, alpha-1 and alpha-2 chains of collagen IV, apolipoprotein E, vitronectin, clusterin, laminin beta-2 and laminin alpha-5 were unambiguously identified (Table 1). The considerable number of MS/MS spectra returning no significant match in the database search was probably due to the high molecular complexity of the tissue surface after trypsin digestion, leading to signal overlap from isobar peptides or other biomolecules. Unexpected post-translational modifications or unspecific truncations as well as the random cross-linking of peptides through free thiol oxidation could also represent the reasons for unsuccessful identifications. The identification of these proteins as constituents of the lens capsule is in agreement with the data available in literature, obtained through conventional proteomic approaches.22 24 Evaluation of Repeatability
IUPAC defines repeatability as the closeness of agreement between independent results obtained with the same method on identical test material, under the same conditions (same analyst, reagents, equipment, and instruments performed within a short period of time) and it usually refers to the standard deviation of simultaneous duplicates or replicates.30 Repeatability, as applied to MALDI-MSI analysis, for obvious reasons, cannot be treated in the same way as for other analytical method validation studies. Moreover, statistical analysis is difficult to apply because no quantitative data are associated with the imaging results. A few studies have been performed to assess the analytical performances of MALDI MSI at the intact protein level31 and, to the best of our knowledge, the only study describing peptides diffusion along the tissue due to enzymatic digestion refers to a trypsin coating procedure different from the one reported here.27 Therefore, repeatability in the context of this 3525
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Journal of Proteome Research manuscript is intended as an assessment on the variability of peptides distribution on independently analyzed samples, since trypsin
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digestion represents the critical step that could result in the loss of peptide localization. Four human lens capsules were independently analyzed to evaluate repeatability of the experiment. The distribution of the signals corresponding to the identified peptides was assessed for each replicate. Figure 2 shows the virtual images of the distributions for the same peptides on each replicate. The same peptides, assigned after peptide fragmentation analysis to the same proteins, showed very similar distribution on the different analyzed samples. Five peptides attributed to ApoE (m/z 1214, 1223, 1313, 1442 and 1685) were found exclusively in the pupillary region of the lens capsule for all replicates (Figure 2a) while for the alpha-1 chain of collagen IV (m/z 1116, 1385, 1513 and 1925) were always found to be more abundant in the peripheral ring (Figure 2b). These results are highly coherent not only from the point of view of the retention of localization after the digestion, but also because the same set of peptides were generated on independent samples. The above data suggest that peptide localization can be reproducibly ascertained by MALDI-MSI following trypsin digestion of the specimen. Validation Experiments
Figure 2. Repeatability study: (a) ion intensity maps of peptide mass signals corresponding to Apo E and the (b) collagen IV alpha-1 on four independently analyzed samples (1 4). The mass values signed with a “*” were identified by MALDI-LIFT-MS/MS directly on-tissue. Considering the intersubject variability and the differences in shape of the excised lens capsules, the results obtained for the distribution patterns strongly suggest that the tryptic digestion is repeatable and does not produce significant diffusion of the peptides (scale bars 1 mm).
Immunohistochemistry. To validate our results and test our initial assumption that the digestion process does not significantly affect protein distribution, immunohistochemical labeling was performed for apolipoprotein E on whole mount lens capsules. Immunolabeling, without antigen retrieval, resulted in no staining in any region of the lens capsule (data not shown). Considering the nature of this tissue, compact and highly cross-linked, such result is not surprising and in fact is coherent with our first MALDI-MSI tests in which we aimed to image intact proteins. Hence, to increase antigen accessibility, KCl-HCl treatment was used for antigen retrieval32 before primary antibody incubation. The immunostaining pattern confirms the presence of ApoE only in the pupillary region (Figure 4a). Similar staining was not seen on lens capsules immunolabeled for ZO-1, an intracellular protein absent in the lens capsule (Figure 4b), or on negative control capsules (Figure 4c), which prove specificity of the anti-ApoE immunostaining. The immunohistochemistry data is in agreement with MALDI-MSI results. It also confirms that, for the raster size used in the MALDI-MSI analysis (100 μm), unavoidable
Figure 3. Schematic workflow of human lens capsule dissection. 3526
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Figure 4. Immunohistochemical labeling of human lens capsule with (a) the anti-ApoE primary antibody and (b and c) negative controls. The staining in the central area in panel a confirms the higher abundance of ApoE in the pupillary region as shown by the MALDI-MSI results. Specificity of this staining is proven by absence of similar staining on lens capsule (b) immune-labeled with ZO-1 or (c) incubated only with the secondary antibody. The border of the lens capsule in panel b and c has been highlighted for visualization purposes. Representative images from independent experiments are shown. Images were taken with a 2 objective. Scale bar 1 mm.
peptides diffusion caused by on-tissue trypsin digestion remain limited and does not misrepresent the original protein distribution. Mass Spectrometric Analysis of Dissected Lens Capsules. Further validation of differential distribution of the identified proteins was carried out through conventional proteomics approaches. For this purpose, the core pupillary and peripheral ring regions were dissected from three human lens capsules as described in the Experimental Section. The excised pupillary regions and the complementary rings were pooled together, trypsin digested and analyzed both by MALDI-MS, after ZipTip extraction, and LC MS/MS. All the proteins identified by on-tissue MALDI-LIFT-MS/MS were confirmed by tandem mass spectrometry and, as expected, the total number of identified proteins through this approach was higher. Overall, LC MS/MS data returned 59 identified proteins (197 unique peptides) for the dissected core region of the lens capsule and 50 (216 unique peptides) for the peripheral ring, with a false discovery rate, calculated after the score adjustment by Mascot Percolator, of 1.02% and 0.46%, respectively (the complete list of proteins for the two regions are provided as Supporting Information, Supplementary Table 1 and 2). Interestingly, several proteins virtually show the same distribution pattern: alpha-Crystallin B chain, C-X-C motif chemokine 14, carbohydrate sulfotransferase 14, metalloproteinase inhibitor 3, serine protease HTRA1, lamin-A/C, semaphorin-3B and meteorin were identified with higher confidence (higher number of peptides) in the pupillary fraction while basement membrane-specific heparan sulfate proteoglycan core protein, RPE-spondin, lysyl oxidase homologue 1 and nidogen-1 with higher confidence in the periphery, suggesting the presence of structural or functional differences between the different areas of the human lens capsule. Biological Significance of the Localization of Apolipoprotein E and Collagen IV Alpha-1
ApoE is commonly considered to be a lipid transport molecule but it also has roles in immunomodulation and regulation of cell
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growth and differentiation.33 Additionally, its key roles in the brain, varying from neural function to neurite outgrowth and repair from injury, have been suggested.34 The protein is upregulated in response to oxidative stress and appears to act as an antioxidant.35 ApoE is expressed in ocular tissues and is a component of the Bruch’s membrane, the basement membrane of the retinal pigment epithelium in the posterior segment of the eye.36 It is also a component of the vascular extracellular matrix.37 It interacts with the extracellular matrix proteins and regulates their synthesis and degradation.38 Several hypotheses could be formulated to explain the preferential occurrence of ApoE in the core region of the lens capsule including constant exposure of this region to environmental stress such as ultraviolet irradiation from sunlight and regulation of the thickness of the part of the lens capsule in the visual axis. Whether these hypotheses hold true remains to be determined. Collagen IV alpha-1 is a member of the type IV collagen family which aggregates in nonfibrillar structures and is characterized by disulfide and lysyl-derived cross-links.39 It is broadly distributed in most basement membranes including the lens capsule and is involved in maintaining their integrity, stability and functionality.22,40,41 The thickness of the anterior lens capsule varies concentrically and changes with age.21 It has been reported that collagen IV alpha-1 is present on the anterior surface of the anterior lens capsule in higher amounts compared to the posterior surface.42 Our MALDI MSI results are not only in agreement with those findings but add novel insight regarding the distribution of collagen IV alpha-1 on the lens capsule whole mount. Abundance of this structural protein in the periphery of the lens capsule may correlate with greater thickness of this region compared to that of the pupillary region or with the contact with iris, which is known to express it in the basement membrane of blood vessels.43
’ CONCLUSIONS MALDI-MSI analyses to date have only been performed on tissue sections with highly defined histo-morphological features. The correlation between the histology of a tissue and the MALDI-MSI signal distribution is often taken for granted. Clearly, establishing such a correlation significantly improves the characterization of different tissue regions. However, as we have shown here differential protein distributions may occur even if tissues do not have evident histomorphological characteristics. Our study also establishes the feasibility of obtaining protein distribution information from a freshly harvested tissue without sectioning. Our simple and reliable digestion protocol for MALDI-MSI allows protein identification through on-tissue MS/MS peptide fragmentation. Furthermore, through the application of MALDI-MSI, we have found evidence of a differential spatial distribution of proteins on the anterior surface of the human lens capsule as a function of the anatomical position, which is a novel finding with implications for physiology of the human eye. In particular, this study reveals the spatial distribution of several proteins on the anterior surface of the lens capsule including ApoE and collagen IV alpha-1. These proteins were identified through direct MS/MS fragmentation on-tissue and by liquid chromatography tandem mass spectrometry. Their presence in this membrane is in agreement with previous reported data obtained through conventional proteomic approaches. ApoE is more abundant in the central area of the lens capsule corresponding to the pupillary region. This region is constantly exposed to environmental stresses. Other proteins such as the collagen IV alpha-1 are preferentially located in the peripheral ring surrounding the core, a 3527
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Journal of Proteome Research region in contact with the iris and hence relatively protected from UV irradiation, but under mechanical shear stress from the constant movement of the iris under different ambient illumination conditions. On the basis of these findings, we speculate that constant exposure of the anterior lens capsule to environmental stresses in the pupillary region and mechanical stress in the iris-overlaid region drives the differential concentric distribution of proteins on the human anterior lens capsule surface. This hypothesis will be the subject of further investigations in the future.
’ ASSOCIATED CONTENT
bS
Supporting Information Detailed instrumental parameters for MALDI-MSI and LC-ion trap MS/MS runs, MALDI-LIFT-MS/MS spectra (Supplementary Figure 1) and full tables of peptides identified for the core (Supplementary Table 1) and the periphery (Supplementary Table 2) of the lens capsule. This material is available free of charge via the Internet at http://pubs.acs.org.
’ AUTHOR INFORMATION Corresponding Author
*Dr. Maurizio Ronci, GPO Box Adelaide, SA 5001, Australia. Phone: +61 (08) 8201 2955. Fax: +61 (08) 8201 2905. E-mail: maurizio.ronci@flinders.edu.au. Prof. Nicolas Voelcker, GPO Box Adelaide, SA 5001, Australia. Phone: +61 (08) 8201 5338. Fax: +61 (08) 8201 2905. E-mail: nico.voelcker@flinders.edu.au.
’ ACKNOWLEDGMENT This work was supported by National Health and Medical Research Council (NHMRC) project #535044. J.E.C. is supported in part by NHMRC Practitioner Fellowship and KPB supported by NHMRC Career Development Award. ’ REFERENCES (1) Sugiura, Y.; Setou, M. Imaging mass spectrometry for visualization of drug and endogenous metabolite distribution: toward in situ pharmacometabolomes. J. Neuroimmune Pharmacol. 2010, 5 (1), 31–43. (2) Hsieh, Y.; Chen, J.; Korfmacher, W. A. Mapping pharmaceuticals in tissues using MALDI imaging mass spectrometry. J. Pharmacol. Toxicol. Methods 2007, 55 (2), 193–200. (3) Taban, I. M.; Altelaar, A. F.; van der Burgt, Y. E.; McDonnell, L. A.; Heeren, R. M.; Fuchser, J.; Baykut, G. Imaging of peptides in the rat brain using MALDI-FTICR mass spectrometry. J. Am. Soc. Mass Spectrom. 2007, 18 (1), 145–51. (4) Goodwin, R. J.; Pennington, S. R.; Pitt, A. R. Protein and peptides in pictures: imaging with MALDI mass spectrometry. Proteomics 2008, 8 (18), 3785–800. (5) Caprioli, R. M.; Farmer, T. B.; Gile, J. Molecular imaging of biological samples: localization of peptides and proteins using MALDITOF MS. Anal. Chem. 1997, 69 (23), 4751–60. (6) Cornett, D. S.; Reyzer, M. L.; Chaurand, P.; Caprioli, R. M. MALDI imaging mass spectrometry: molecular snapshots of biochemical systems. Nat. Methods 2007, 4 (10), 828–33. (7) Meriaux, C.; Franck, J.; Wisztorski, M.; Salzet, M.; Fournier, I. Liquid ionic matrixes for MALDI mass spectrometry imaging of lipids. J. Proteomics 2010, 73 (6), 1204–18. (8) Jackson, S. N.; Ugarov, M.; Egan, T.; Post, J. D.; Langlais, D.; Albert Schultz, J.; Woods, A. S. MALDI-ion mobility-TOFMS imaging of lipids in rat brain tissue. J. Mass Spectrom. 2007, 42 (8), 1093–8.
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