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Supplementary Figure 1 Representative fluorescein imaging of the cornea and experimental setup for alkali exposure. (A) A representative bovine (approximate diameter 34 mm-left) and porcine (diameter~ 20 mm-right) eye is shown. (B) A representative enucleated bovine eye was stained using Fluorescein. (C) The same eye as in (B) after washing with PBS reflecting corneal epithelial integrity. (D) A representative enucleated porcine eye stained using Fluorescein. (E) The same eyeas in (D) after washing with PBS suggesting the eye to possess corneal epithelial integrity. The experimental set up used for exposure to alkali. (F) Lateral view of plexi-glass cover and the platform for alkali exposure to the enucleated eyes. (G) Frontal view of plexiglass box for exposure to the enucleated eye by approximately 5 ml of alkali solution.

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Supplementary Figure 2. (A) Estimation of soluble bovine corneal proteins by different biochemical methods. A test volume of 5 and 10 µl (represented by solid and hollow squares) of corneal protein extract [in 125 mM Tris-HCl (pH 7.0), 100 mM NaCl, 0.1% Triton X-100, 0.1% Genapol C-100, and 0.1% sodium dodecyl sulphate buffer] was estimated by various methods as indicated. Results indicate agreement in amount of protein measured in these two test volumes and the conformity to 1:2 ratio in protein amount estimated. Biochemical method varies for protein estimation due to presence of chemicals in extraction or assay buffers. Biuretreagent reacts with short-chain polypeptides, Lowry method involves reduction of the Folin-Ciocalteu reagent due to aromatic residues (mainly tryptophan, also tyrosine). Absorbance measurements for Biuret, Lowry and bicinchoninic acid (or BCA) are made at wavelength 540 nm, 750 nm and 562 nm respectively. (B) Estimation of soluble bovine corneal proteins by silver staining on a 4-15% PHAST gel and subsequent densitometric scan. The gel was over-stained in order to capture short-chain polypeptides and heterogeneous sparsely distributed polypeptide species that results in slightly higher estimation of proteins using Biuret method. Based on biochemical determinations 5µg equivalent proteins were loaded on each lane as indicated. Densitometric quantitationsindicated equal protein loading in each lane.

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Supplementary Figure 3. Representative porcine corneal normalized protein yield and profile following exposure to sodium hydroxide. (A) Representative protein yield (µg/mg) following exposure of cornea to distilled water (control),0.25M, 6M and 11M concentrations of NaOH indicated by diamonds, triangles, squares and circles respectively at indicated time intervals. The recovered protein amounts estimated by Bradford’s method were normalized to initial amount of wet tissue used. Results are the mean ± SD of three independent estimates. The results were found significantly different from 0.0 by the one-sample t-test: *P < 0.05. (B) Representative SDS-PAGE profile of total extracted protein following exposure to 11M NaOH. An equal amount of protein (10 µg) for each exposure time as indicated was separated on a 10% SDS-PAGE and subsequently stained with Coomassie blue. Arrow heads indicates protein bands that decrease in intensity after NaOH exposure, the bar and thick arrow indicates smeared protein aggregates, thin arrow represent a new protein band seen after NaOH exposure.


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Supplementary Figure 4. Representative porcine corneal normalized protein yield and profile following exposure to ammonium hydroxide. (A) Representative protein yield (µg/mg) following exposure of cornea to 0.25M, 6M and 11M concentrations of NH4OH indicated by triangles, squares and circles respectively at indicated time intervals. The recovered protein amounts estimated by Bradford’s method were normalized to initial amount of wet tissue used. Results are the mean ± SD of three independent estimates. The results were found significantly different from 0.0 by the one-sample t-test: *P < 0.05. (B) Representative SDS-PAGE profile of total extracted protein following exposure to 11M NH4OH. An equal amount of protein (10 µg) for each exposure time as indicated was separated on a 10% SDS-PAGE and subsequently stained with Coomassie blue. Arrow heads indicates protein bands that decrease in intensity after NH4OH exposure, the thin and thick arrow indicates new protein bands observed after NH4OH exposure.


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Supplementary Figure 5. Representative porcine corneal normalized protein yield and profile following exposure to calcium hydroxide. (A) Representative protein yield (µg/mg) following exposure of cornea to 0.25M, 6M and 11M concentrations of Ca(OH)2 indicated by triangles, squares and circles respectively at indicated time intervals. The recovered protein amounts estimated by Bradford’s method were normalized to initial amount of wet tissue used. Results are the mean ± SD of three independent estimates. The results were found significantly different from 0.0 by the one-sample t-test: *P < 0.05. (B) Representative SDS-PAGE profile of total extracted protein following exposure to 11M Ca(OH)2. An equal amount of protein (10 µg) for each exposure time as indicated was separated on a 10% SDS-PAGE and subsequently stained with Coomassie blue. Arrow head indicate protein band that decreased in intensity after Ca(OH)2 exposure, the thin and thick arrow indicates new protein bands observed after Ca(OH)2 exposure.


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Supplementary Figure 6. Representative porcine corneal normalized protein yield and profile following sodium hydroxide exposure and subsequent treatment. (A) Representative normalized protein yield (µg/mg) after corneal exposure to 11M NaOH for indicated time intervals followed by treatment with lime juice as detailed in the experimental procedures. The results were found significantly different from 0.0 by the one-sample t-test: *P < 0.05. (B) SDS-PAGE profile of total extracted protein following 11M NaOH and lime juice treatment as described in experimental procedure. (C) Representative normalized protein yield (µg/mg) after corneal exposure to 11M NaOH for indicated time intervals followed by treatment with vinegar as detailed in the experimental procedures. (D) Representative SDS-PAGE profile of total extracted protein following 11M NaOH and subsequent vinegar treatment. (A, C) The recovered protein amounts estimated by Bradford’s method were normalized to initial amount of wet tissue used. Results are the mean ± SD of three independent estimates. (B, D) An equal amount of protein (10 µg) for indicated exposure time was separated on a 10% SDS-PAGE and stained with Coomassie blue.


Supplementary Figure 7. Representative immunohistochemical analysis for Plexin D1(A, A’) and Dynein (B, B’) on human cornea. The images were taken at 20X magnification on a Leica TSP5 confocal microscope after probing with respective antibodies. The bottom panels (A’, B’) are merged pictures of DAPI and antibody stained images.

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Accession Number†

Protein MW (KDa)*

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P01834 Ig kappa chain C region 11.6 2.8 0 4480.4 604.62 0 832.29 959.65 0 0Q15653 NF-kappaB inhibitor beta variant 38, 36 2.5 976.52 998.02 1598.5 1025.92 0 913.53 933.65 0P13645 Cytokeratin-10 58.8 2.4 2393.85 1020.7 0 0 0 566.52 0 843.91AAT49050 Immunoglobulin heavy constant gamma 1-like protein 52 2.3 1027.68 28797 3657.97 0 2209.06 5557.98 6575.27 177.75P30838 Aldehyde dehydrogenase 50 2.2 0 0 1458.06 22616.8 1339.35 3640.71 2200.5 3806.35Q9NYC9 Dynein heavy chain 9 512, 503 1.0 1921.84 14172 13889 2446.48 1921.84 14171.9 13889.5 2446.68XP_002348035 Hypothetical protein 100.3 0.8 15563.7 29088 30148.2 26978.4 22041.6 39247.1 31981.1 28847.4P81947 Alpha tubulin 50.1 0.8 2286.88 0 812.39 2717.39 1450.08 1155.56 2793.2 1741.22Q14694 Ubiquitin carboxyl-terminal hydrolase 10 87 0.7 1020.33 5430.2 1376.56 1904.57 722.69 6097.41 6037.78 1471.52P02768 Human Serum Albumin 69, 47 0.7 2840.09 5658.5 1706.84 10427.2 2277.63 5728.95 8075.6 11435.8Q9Y4D7 Plexin-D1 212, 196 0.7 1020.33 5430.2 1376.56 1904.57 722.69 6097.41 6037.78 1471.52Q8IZT6 Abnormal spindle-like microcephaly-associated protein 409, 218 0.6 0 12617 3320.38 2372.82 1921.84 14171.9 13889.5 2446.48O60938 Keratocan 40.5 0.5 0 15459 4694.93 2384.95 2172.34 14720.9 26894.3 3177.96P51884 Lumican 38.4 0.3 0 11981 3870.84 0 769.8 38883.2 20551.6 0P07585 Decorin variant A 39.7,

27.3, 23, 19, 8.2

0.2 0 2410.2 20861.8 0 3036.7 41192 67063.6 3629.92

Q15582 Transforming growth factor, 68kDa variant 74.7 0.2 0 4529.2 0 4711.06 3894.13 7402.49 28686.7 15666.6

Control 11M NaOH

†NCBI accession number is expressed in Italics and SwissProt accession number is provided. Ratio refers to total area is control divided by total area in alkali treated group. *Molecular weight for all isoform has been listed.

Supplementary Table 1. Proteins identified in control and alkali treated cornea


Supplementary mass spectrometry method Mass Spectrometry. To identify the proteins and perform quantification, protein bands separated on 10% SDS-PAGE were excised, the gel slices were destained with 50% acetonitrile /water and were suspended in 0.5M triethylammoniumbicarbonate (TEAB; number 17902 Sigma Chemical Co., St. Louis, MO) pH8.5 and reduced with 10 mM Tris-(2-carboxyethyl) phosphine (TCEP; Sigma Chemical Co., St. Louis). The proteins were subsequently alkylated in the dark using 55mM solution of iodoacetamide (Catalog No-RPN6302V; GE Healthcare Inc., Buckinghamshire, England) and in-gel digested with sequencing grade modified trypsin (catalog number V5113, Promega Corporation, Madison, WI 0.1ug/15ul in 15mM N-ethylmorpholin) overnight at 37ºC. The peptides were extracted twice with 50µl 0.1%Triflouroracetic acid/60% acetonitrile and finally with 30 µl of acetonitrile and dried in a SpeedVac. The extracted peptides were incubated with 8 plex iTRAQ Reagents, separate peptides isolated from different bands were subjected to incubation with reagents 113, 114, 115 and 116 (4381557; 4381557; 4381557; 4381560; ABI, Foster City, CA) from peptides derived from protein band of control cornea and with reagents 117, 118, 119 and 121 (4381561; 4381562; 4381563; 4381564) for peptides derived from alkali exposed corneal protein bands in 0.5M TEAB containing 60% v/v isopropanol. The incubation mixtures were dried in a SpeedVac, mixed together and loaded onto a slurry of 500µL of cation exchange buffer in 12 mM Ammonium Formate in 25% acetonitrile at pH 2.5-3.0, and subjected to chromatographic separation. 2D-LC separations: SCX separations of peptides were performed on a passivated Waters 600E HPLC system, using a 4.6 X 250 mm PolySULFOETHYL Aspartamide column (PolyLC, Columbia, MD) at a flow rate of 1 ml/min. Buffer A contained 10 mM ammonium formate, pH 2.7, in 20% acetonitrile/80% water. Buffer B contained 666 mM ammonium formate, pH 2.7, in 20% acetonitrile/80% water. The gradient was Buffer A at 100% (0-22 minutes following sample injection), 0% to 40% Buffer B (16-48 min), 40% to 100% Buffer B (48-49 min), then isocratic 100% Buffer B (49-56 min), then at 56 min switched back to 100% A to re-equilibrate for the next injection. The first 26 ml of eluant (containing all flow-through fractions) was combined into one fraction, then 14 additional 2-ml fractions were collected. All 15 of these SCX fractions were dried down completely to reduce volume and to remove the volatile ammonium formate salts, then resuspended in 9 µl of 2% (v/v) acetonitrile, 0.1% (v/v) trifluoroacetic acid and filtered prior to reverse phase C18 nanoflow-LC separation. For second dimension separation by reverse phase nanoflow LC, each SCX fraction was autoinjected onto a Chromolith CapRod column (150 X 0.1 mm, Merck) using a 5 µl injector loop on a Tempo LC MALDI Spotting system (ABI-MDS/Sciex). Buffer C was 2% acetonitrile, 0.1% trifluoroacetic acid, and Buffer D was 98% acetonitrile, 0.1% trifluoroacetic acid.The elution gradient was 95% C/ 5% D (2ul per minute flowrate from 0-3 min, then 2.5ul per minute from 3-8.1 min), 5% D to 38% D (8.1-40 min), 38% D to 80% D (41-44 min), 80% D to 5% D (44-49 min) (initial conditions). Flow rate was 2.5 µl/min during the gradient, and an equal flow of MALDI matrix solution was added post-column (7 mg/ml recrystallized CHCA (a-cyano-hydroxycinnamic acid), 2 mg/ml ammonium phosphate, 0.1% trifluoroacetic acid, 80% acetonitrile). The combined eluant was automatically


spotted onto a stainless steel MALDI target plate every 6 seconds (0.6 µl per spot), for a total of 370 spots per original SCX fraction. Mass Spectrometry analysis: After drying the sample spot mentioned above, thirteen calibrant spots (ABI 4700 Mix) are added to each plate manually. MALDI target plates (15 per experiment) were analyzed in a data-dependent manner on an ABI 4800 MALDI TOF-TOF. As each plate is entered into the instrument, a plate calibration/ MS Default calibration update is performed, and then the MS/MS default calibration is updated. MS spectra were taken from 5500 MALDI Spots, averaging 500 laser shots per spot at Laser Power 3100. A total of 3249 MS/MS spectra was taken with up to 2500 laser shots per spectrum at Laser Power 3600, with CID gas Air at 1.2 to 1.3 x 10-6 Torr. After the MS and MS/MS spectra from all 15 plates in a sample set have been acquired, protein identification and quantitation was performed using the Paragon algorithm as implemented in Protein Pilot 3.0 software (from ABI/MDS-Sciex) and Matrix Sciences Mascot algorithm version 2.1. ProteinPilot Software was utilized for searches with following search parameters: Cys Alkylation – Iodoacetamide; ID Focus – Biological Modifications; Search Effort – Thorough. The Jan 1st 2010 Human NCBI database Sequences containing 512785 Protein Sequences, plus 156 common lab contaminants. For estimation of “False Discovery Rate” (FDR), simultaneous search was performed on a concatenated Decoy database which is the exact reverse of each protein sequence in the database plus 156 common human and lab contaminants. Total Protein Sequences searched in Database plus contaminants plus concatenated Reverse Decoy Database: 1025652. The protein identifications at 95% confidence level were retained. The preset “Thorough” (iTRAQ or Identification) search settings were used where identifications must have a ProteinPilot Unused Score > 1.3 (>95% Confidence interval). In addition, the only protein IDs accepted MUST have a local “FDR” estimation of no higher than 5%, as calculated from the slope of the accumulated Decoy database hits by the PSPEP (Proteomics System Performance Evaluation Pipeline) program1. This local FDR estimate is much more stringent than p<0.05 or 95% confidence scores in Mascot, Sequest, ProteinPilot, or the aggregate False Discovery Rate estimations (2 X number of Decoy database IDs/Total IDs at any chosen threshold score) commonly used in the literature, and combined with the ProGroup algorithm included in ProteinPilot gives a very conservative and fully MIAPE-compliant list of proteins identified (i.e., Mascot and other lists of "Proteins ID'd at p<0.05" will produce more numerous "significant" IDs from the same data, but those larger lists are highly likely to contain many more False Positive IDs). Supplementary Reference 1. Tang, W.H., Shilov, I.V. & Seymour, S.L. Nonlinear fitting method for

determining local false discovery rates from decoy database searches. J Proteome Res. 2008; 7: 3661-7.


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