Supporting Information 2 – Materials and Methods
Corneal tissue – A corneal flap consisting of epithelium, Bowman’s layer, and superficial
stroma was removed from GCD2 post-LASIK patients having LASIK surgery again as
treatment for protein aggregation. Control tissue consisting of superficial stroma was
excised from the cornea during ReLEx SMILE surgery of healthy individuals. Isolated
epithelium was obtained post-mortem from a 96-year-old male with no history of
corneal defects. All tissues were kept at -80°C until further use. The patient and control
groups consisted of six biological samples each, representing mixed genders and with an
average age of 34 years and 36 years, respectively (Supporting Information 1). The
tissue was obtained and used for scientific purpose and ethical issues have been
handled according to Danish and South Korean healthcare laws. All work was carried
out in accordance with the Declaration of Helsinki.
Sample preparation – GCD2 patient and control tissues were lyophilized for 2 h using a
speedvac concentrator and grinded into powder using liquid nitrogen. Approximately,
0.3mg of each tissue was subjected to 0.66 M CNBr in 70% trifluoroacetic acid at 23°C.
The following day the tissues were lyophilized and re-suspended in 8 M Urea in 0.2 M
Tris-HCl, pH 8.3, reduced in 5 mM dithiothreitol for 30 min, and alkylated in 15 mM
iodoacetamide for 30 min. Samples were then diluted 5 times using 0.1M Tris-HCl, pH
8.3, followed by overnight digestion with 1:50 w/w sequencing grade modified trypsin
(Sigma) at 37°C. The samples were desalted using POROS 50 R2 RP column material
(Applied Biosystems) packed in GELoader Tips (Eppendorf).
LC-MS/MS – LC-MS/MS analyses were performed on an EASY-nLC II system (Thermo
Fisher Scientific) connected to a TripleTOF 5600+ mass spectrometer (AB SCIEX)
equipped with a NanoSpray III source (AB SCIEX) and operated under Analyst TF 1.6.0
control. The CNBr and trypsin cleaved samples were dissolved in 0.1% formic acid,
injected and trapped on an in-house packed trap column (2 cm x 100 μm I.D) using RP
ReproSil-Pur C18-AQ 3 μm resin (Dr. Maisch GmbH). Peptides were eluted from the trap
column and separated on a 15 cm analytical column (75 μm i.d.) pulled and packed inhouse with RP ReproSil-Pur C18-AQ 3 μm resin (Dr. Maisch GmbH) and sprayed directly
into the mass spectrometer. Peptides were eluted at a flow rate of 250 nL/min using a
20 min gradient from 5% to 35% phase B (0.1% formic acid and 90% acetonitrile). The
acquisition method used for the area based extracted ion chromatogram (XIC)
quantification was set up as an information-dependent acquisition (IDA) experiment
collecting up to 25 MS/MS spectra in each 1.6 sec cycle using an exclusion window of 6
sec. The GCD2 patient and control samples were run in triplicates on the mass
spectrometer.
Data processing – For protein identification all raw MS files were processed using
Mascot Distiller 2.5.1.0 (Matrix Science) and MGF files generated were search in the
Swiss-Prot (v. 2015_03) Homo sapiens database using Mascot 2.5.0 (Matrix science,
London, UK). CNBr+Trypsin was selected as the digestion enzyme allowing one missed
cleavage. To address endogenous P1 cleavage sites a second search was performed
using semi-trypsin as specified enzyme. In both searches carbamidomethyl was entered
as a fixed modification, whereas hydroxylation of proline, oxidation of methionine, and
C-terminal conversion of methionine to homoserine and homoserine lactone by CNBr
were used as variable modifications. All data were searched with a mass tolerance of the
precursor and product ions of 10 ppm and 0.2 Da using ESI-QUAD-TOF as the
instrument setting and all searches was adjusted to a 1% FDR at the peptide level. All
data were imported and processed using MS Data Miner v. 1.3.0 [1]. All protein
identifications based on at least one peptide with an ion score cut-off of 45 or minimum
two peptides with an ion score cut-off of 30 was included for further data analysis
(Supporting Information 3). Protein identifications based on one peptide was subjected
to manual validation before accepted (Supporting Information 5).
To address endogenous P1 cleavage sites all TGFBIp peptide spectra with an ion score
cut-off of minimum 45 within the semi-tryptic search was extracted and the distribution
of P1 amino acid residues of the non-tryptic cleavage site was calculated and compared
between control and patient groups (Supporting Information 9 and 11).
For the area-based XIC quantification Mascot Distiller 2.5.10 was used and the mascot
search was performed using the same settings as for protein identification above except
that the default average [MD] quantitation protocol was selected using a 1% FDR
threshold at the peptide level, number of peptides used for quantitation was 3, matched
rho was 0.8, XIC threshold was 0.3 and isolated precursor threshold was set at 0.7.
The area-based XIC quantification was based on the average intensity of the 3 most
abundant peptides per protein and displayed as a molar percentage by dividing the
average intensity with the total intensity of the given MS analysis (Supporting
Information 4). Using Gene Ontology annotations all extracellular proteins were ranked
according to their molar fraction of a sample and the average molar percentage across
patient and control groups were compared if a protein was quantified in minimum 3 of
the 6 biological replicates in a group (Table 1, Supporting Information 8).
2D-PAGE – Corneal powder was dissolved in lysis buffer (7 M urea, 2 M thiourea, 4 %
(w/v) 3-[(3-cholamidopropyl)-dimethylammonio]-1-propanesulfonate (CHAPS), 10 mM
Tris pH 8.8, 5 mM EDTA, cOmplete EDTA-free protease inhibitor cocktail (Roche
Diagnostics, Basel, Switzerland), 0.5 % (v/v) IPG buffer of pH 3-10 or 4-7 and 10 mM
DTT) and incubated rotating for 1h at room temperature. The samples were centrifuged
and supernatants were assayed for protein content using the 2D Quant kit (GE
Healthcare Life Sciences, Buckinghamshire, England). The protein amount was verified
and adjusted on a 1D SDS-PAGE gel. For each sample 2 µg of total protein was loaded
onto 7-cm Immobiline DryStrips (GE Healthcare Life Sciences) with pH range 3-10 or 47. The strips were rehydrated overnight at room temperature and isoelectric focusing
was performed using the Ettan IPGphor System II (GE Healthcare Life Sciences) for 5.3
kVh. Following isoelectric focusing proteins were reduced in equilibration buffer (50
mM Tris-HCl pH 8.8, 6 M urea, 30 % (v/v) glycerol and 2 % SDS) with 6.5 mM DTT and
then alkylated in equilibration buffer containing 10 mM iodoacetamide. The strips were
placed on top of 10 % SDS-polyacrylamide gels and the second-dimension were run at
17.5 mA per gel in running buffer (25 mM Tris, 192 mM glycine and 0.1 % (w/v) SDS).
Immunoblotting of TGFBIp – Proteins separated by 2DE were electroblotted onto a
polyvinylidene difluoride (PVDF) membrane (Immobilon-P, Millipore, Billerica, MA) for
immunoblotting. The membranes were blocked in 40 ml 5 % dry milk solution in TBS-T
(20 mM Tris-HCl pH 7.6, 137 mM NaCl and 0.1 % tween) for 1 h at room temperature.
An antiserum from rabbit against rhTGFBIp [2] was added to the blocking solution in a
ratio of 1:10000 and membranes were incubated overnight at 4°C. The membranes
were washed thoroughly with TBS-T, before incubated for 2 h at room temperature in
TBS-T containing goat anti-rabbit IgG peroxidase conjugate (Sigma-Aldrich Co., St. Louis,
MO) in a ratio of 1:10000 in 5 % milk. Finally, the membranes were washed thoroughly
with TBS-T and developed for 0.25-1 min using the enhanced chemiluminescence (ECL)
Western blotting detection system and reagents (GE Healthcare Life Sciences).
References
[1]
Dyrlund, T.F., Poulsen, E.T., Scavenius, C., Sanggaard, K.W., Enghild, J.J., MS Data
Miner: a web-based software tool to analyze, compare, and share mass
spectrometry protein identifications. Proteomics 2012, 12, 2792–2796.
[2]
Runager, K., Klintworth, G.K., Karring, H., Enghild, J.J., The insoluble TGFBIp
fraction of the cornea is covalently linked via a disulfide bond to type XII collagen.
Biochemistry 2013, 52, 2821–2827.