Supplementary methods
4D separation liquid chromatography tandem mass spectrometry
Immunoaffinity depletion of major plasma proteins from patients and controls
The top six abundant proteins (serum albumin, immunoglobulin G, immunoglobulin A,
transferrin, haptoglobin, and antitrypsin) were depleted using a MARS column (Agilent
Technology, Santa Clara, CA, USA).{Kim, 2009 #35} Plasma (40 μL) was diluted 1:5 with a
proprietary “buffer A” and loaded onto the MARS column. The unbound fraction was
concentrated by ultrafiltration using a Microcon filter (3000-Da cutoff; Millipore, MA, USA).
Gel-eluted liquid fraction entrapment electrophoresis (GELFrEE)
Protein concentrations were determined by the Bradford method. Subsequently, an equal
amount of concentrated unbound fraction from each HC sample and equal amounts of protein
from each AMD sample were mixed separately. Further details on the GELFrEE system can
be obtained from publications by Tran et al.{Lee, 2009 #36;Tran, 2008 #37} A total of 500
μg of protein was loaded onto a 1 cm gel column, cast to 12% T (with a 3-cm stacking gel
cast to 4% T) and electrophoresed at 240 V for 2 h. Following elution of the dye front
(fraction ‘zero’), additional collections were performed at 5, 15, 30, 50, and 120 min, yielding
a total of 5 fractions.
Isoelectrofocusing of tryptic peptides
Ten GELFrEE fractions (five from HC and five from AMD) were adjusted to the same
protein concentration, reduced with 10 mM DTT for 30 min at 37°C and then alkylated with
40 mM iodoacetamide for 1 h in the dark at 25°C. After the samples were diluted 10-fold
with 50 mM NH4HCO3, trypsin was added at a ratio of 1:40 (w:w), followed by incubation
overnight at 37°C. A 3100 OFFGEL Fractionator and OFFGEL Kit pH 3–10 (Agilent
Technologies) with a 12-well setup were used for isoelectrofocusing of the tyrptic peptides.
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Peptides were diluted in 1.8 mL of the focusing buffer containing only 5% (v/v) glycerol,
which deviated from the supplier’s protocol. IPG strips were rehydrated by adding 40 μL of
peptide IPG strip rehydration solution per well for 15 min. Then, 150 μL of sample was
loaded in each well. Peptide focusing was performed until 20 kVh was reached, with a
maximum voltage of 8,000 V and maximum current of 50 μA. After focusing, 12 peptide
fractions and both ends (total: 14 fractions) were withdrawn, and wells were rinsed with 150
μL of H2O/MeOH/TFA (49/50/1 v/v) for 15 min. Rinsing solutions were pooled with their
corresponding peptide fractions and concentrated in a SpeedVac prior to liquid
chromatography tandem mass spectrometry (LC-MS/MS) analysis.
Reversed phase LC-MS/MS analysis
LC-MS/MS was performed on an Agilent nanoflow-1200 series HPLC system connected to
a linear ion trap mass spectrometer (LTQ, Thermo Scientific, San Jose, CA, USA). Peptides
were reconstituted with 10 μL of 0.4% acetic acid, after which an aliquot (1 μL) was injected
into a reversed phase Magic C18aq column (12 cm  75 μm) equilibrated with 95% solvent A
(0.1% formic acid in H2O) + 5% solvent B (0.1% formic acid in acetonitrile). The peptides
were eluted in a linear gradient of 10% to 30% solvent B over 80 min. The MS survey was
scanned from 300 to 2000 m/z, followed by six data-dependent MS/MS scans with the
following options: isolation width, 1.5 m/z; normalized collision energy, 25%; dynamic
exclusion duration, 180 s. In total, 140 LC-MS/MS runs (2 pooled samples  5 GELFrEE
fractions  14 OFFGEL fractions) were performed. The acquired MS/MS spectra were
compared against the human International Protein Index database, including known
contaminants (IPI, version 3.44, European Bioinformatics Institute, www.ebi.ac.uk/IPI),
using SEQUEST (TurboSequest version 27, revision 12) with a mass tolerance of ± 0.5 Da
for MS/MS and ± 3.0 Da for MS. Searches were performed allowing the options of no
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enzyme, a fixed modification of carbamidomethylation at cysteine (+57 Da), and a variable
modification of methionine oxidation (+16 Da). Peptide assignment was performed with the
Trans-Proteomic
Pipeline
(TPP,
version
4.0,
http://www.proteomecenter.org).
The
SEQUEST search output was used as the input for Peptide-Prophet and Protein-Prophet.
Peptides with probabilities greater than 0.05 were included in the subsequent Protein-Prophet
analysis, and proteins having protein probabilities of more than 0.9 were gathered. This
probability corresponded to an FDR of 0.007 for the healthy control dataset and an FDR of
0.012 for the AMD dataset.
Quantification and statistical analysis
The relative abundance of identified proteins was estimated on the basis of spectral counts
(the number of MS/MS spectra matched to the protein concerned). Spectral count data were
extracted using the Protein Prophet program and exported to MS-EXCEL. Afterward, the
normalized spectral abundance factor (NSAF) for each protein was obtained considering the
total
numbers
of
spectra
for
AMD
patients
and
HCs
(29,836
and
30,083,
respectively).{Paoletti, 2006 #38;Yang, 2012 #39} We calculated an NSAF for each protein
as follows:
in which the total number of tandem MS spectra matching peptides from protein k (SpC)
was divided by the protein’s length (L) and then divided by the sum of SpC/L for all N
proteins. The abundance ratio for each protein was calculated as protein ratio = (average
NSAF of the protein in AMD) / (average NSAF of the protein in HC).
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In order to filter out differentially expressed proteins (DEPs) that were statistically
significant, G-tests adjusted by the William’s correction were performed. The spectral counts
for each protein were first normalized as
where (SpC)i∈AMD and (SpC)i∈HC denote spectral counts of the i-th protein from the AMD
and HC groups, respectively, and ∑j∈AMD(SpC)j and ∑k∈AMD(SpC)k are the total numbers of
spectra. The G-value for the i-th protein was then calculated as follows:
A G-value higher than 3.841 was considered significant, with P < 0.05 according to the χ2distribution.{Zhang, 2006 #40} In addition, the rank-sum test (Mann-Whitney test) was used
to test the difference in protein expression between AMD patients and HCs. All statistical
analyses were performed using MedCalc software (version 9.6.2.0).
Pathway analysis
DEPs quantified by spectral counts were analyzed using the Ingenuity Pathways Analysis
(IPA) program (Ingenuity Systems, Redwood City, CA, USA). Proteins in the proteomic data
were mapped to corresponding gene objects in the Ingenuity Pathways Knowledge Base
(IPKB). Then, biological networks were generated using the knowledge base for interactions
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between the uploaded gene list and all other stored gene objects. Functional analysis of the
networks was performed to identify the biological functions and/or diseases that were most
significant to the genes in the network. For each network, a score was computed according to
the fit of the data set of significant genes by Fisher’s exact test. The score was derived from
the P-value of the test and indicates the likelihood of the mapped genes in a network being
found together due to random chance (score = -log10p).
Western blotting
Plasma samples were size-fractionated by sodium dodecyl sulfate polyacrylamide gel
electrophoresis (SDS-PAGE), transferred onto polyvinylidene difluoride (PVDF) membranes
(Amersham Bioscience, Piscataway, NJ, USA), blocked with 5 skim milk in TTBS (20 mM
Tris, pH 7.4, 150 mM NaCl, and 0.05% Tween 20 with 0.01% sodium azide), and incubated
with specific antibodies. The membranes were incubated at 4°C overnight with primary
antibodies specific for vinculin (VCL; Abcam Inc., Cambridge, MA, USA), complement
component C9 (C9), S100A8, S100A9, antithrombin-III (SERPINC1), and thrombospondin1 (THBS1; Santa Cruz Biotechnology, Santa Cruz, CA, USA). After washing, the
membranes were incubated at 25°C for 1 h with appropriate horseradish peroxidase (HRP)conjugated secondary antibodies, washed, and then developed with a chemiluminescence
reagent (ECL, ECL plus; GE Healthcare, Uppasla, Sweden). For multiblot western blotting
analysis for vinculin and S100A9 in plasma, 10 μg of each immunodepleted sample was
fractionated on mini gels (10–14 samples per each gel). After SDS-PAGE, lower parts below
20 kDa (S100A9, 14 kDa) and upper parts above 90 kDa (Vinculin, 117 kDa) were excised
from all the gels and arranged row by row on a single big PVDF membrane (18 × 16 cm).
Electroblotting was carried out using a Bio-Rad Trans-Blot Cell system (Bio-Rad, Hercules,
CA, USA). Equal loading for each sample was confirmed by Coomassie staining on the
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remaining portion of the SDS-PAGE gels. Band intensities in western blotting images were
quantified using TotalLab ID v11.5 (Core Bio, Seoul, Korea).
Enzyme-linked immunosorbent assay (ELISA)
The concentrations of triosephosphateisomerase, protein S100A8, tenascinX, protein zdependent protease inhibitor, apolipoprotein E, and C-X-C motif chemokine 7 in human
plasma of HCs and patients with AMD were measured using commercially available human
ELISA kits (Uscn Life Science Inc., Houston, TX, USA; Abcam, Cambridge, MA, USA).
Assays were performed according to the manufacturers’ instructions. The absorbance was
measured at 450 nm using an ELISA reader.
Immunofluorescence staining
Tissue sections were rinsed in phosphate buffered saline (PBS, containing 137 mM NaCl,
2.68 mM KCl, 8.1 mM Na2HPO4, and 1.47 mM KH2PO4) and blocked in 5% fetal bovine
serum (FBS), 0.3% bovine serum albumin (BSA), and 0.1% Triton X-100 in PBS. For
antigen unmasking, slides were boiled in 10 mM sodium citrate buffer (pH 6.0) and then
maintained at a subboiling temperature for 40 min using a steamer. Slides were cooled on the
bench top for 20 min. Anti-vinculin antibodies (1:200, Abcam) were diluted in blocking
solution and incubated overnight at 4°C. The following day, slides were rinsed with PBS and
incubated with appropriate secondary antibodies conjugated to Alexa 596 anti-mouse IgG
(1:500, Molecular Probes) and fluorescein isothiocyanate (FITC)-BS-lectin (1:500, Sigma,
St. Louis, MO, USA) for 1 h at room temperature. Slides were then rinsed and mounted using
Vectashield mounting media (Vector Labs, Burlingame, CA, USA). Negative controls were
performed in parallel by omission of the primary antibody. Analysis of double-labeled
sections was performed on a confocal laser-scanning microscope (LSM710, Carl Zeiss, AG
Oberkochen, Germany). Figures were prepared using Zen2011 software (Carl Zeiss).
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Exposure of ARPE-19 cells to hypoxic stress
Cell culture
Oxidative stress experiments were performed using ARPE-19, a human diploid RPE cell
line (ATCC, Cat. No. CRL-2302), which is in many ways similar to RPE cells in
vivo.{Dunn, 1996 #77} All cell culture media were obtained from Life Technologies
(Paisley, UK), and other reagents were obtained from Sigma-Aldrich (Poole, UK) unless
otherwise stated. Before experimentation, cells were seeded at approximately 0.5 × 105
cells/cm2 and were maintained in culture for 1 or 5 weeks after seeding, unless otherwise
stated. All cultures were fed twice weekly with Dulbecco’s modified Eagle medium
(DMEM)/nutrient mixture F12, plus 10% FBS, 2 mM
L-glutamine,
and 100 IU/mL
penicillin-streptomycin. Cultures were maintained at 37°C in 5% CO2.
Experimental conditions
ARPE-19 cells (5 × 105 cells/well) were seeded in 10 mL medium on 100-mm culture
dishes and incubated with DMEM containing 10% FBS for 3 days. The medium was
discarded, and the cells were washed with PBS. Hydrogen peroxide (H2O2) was added to the
fresh culture medium, and cells were exposed to graded oxidative stress (0, 10, 50, 100, or
300 μM H2O2) with chronic treatment (24 h) in medium containing serum. Three dishes were
used for each condition.
Western blotting analysis of cellular protein extracts
Cells were lysed in RIPA buffer (Piece Biotechnology, Rockford, IL, USA) containing
ProteaseArrest (G-Biosciences, USA), according to a previously published method.{Song,
2005 #78} Conditioned media supernatants were concentrated using Ultracel-10K cellulose
filters (10000 MWCO; Millipore). Protein content was quantified with protein assays (BioRad), using BSA as a standard. Electrophoresis by SDS-PAGE was followed by transfer to
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PVDF membranes (GE Healthcare Limited, Buckinghamshire, UK). Membranes were
blocked in 5% (w/v) Difco Skim milk (Becton Dickinson and Company, Le Pont de Claix,
France) in PBS plus 0.5% (v/v) Tween 20 (PBST; Sigma-Aldrich). Membranes were then
incubated with anti-vinculin antibodies (1:500, Abcam) in blocking solution for 1 h at room
temperature or overnight at 4°C. Secondary antibodies were also diluted in blocking solution.
PBST was used for washing steps. Detection was facilitated with chemiluminescence
reagents (ECL plus; Amersham Biosciences, Little Chalfont, UK). Immunoreactive bands
were quantified, and relative sum intensities of bands were compared using image analysis
software (Kodak Digital Science, 1D Image Analysis Software, Eastman Kodak Co.,
Rochester, NY).
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