bovine eyes ( from 3-year - old animals ) were obtained from a local slaughterhouse within 3 hours of death .
the eye globe was dissected from its anterior aspect , the cornea and iris were removed , and the front face of the ciliary processes was exposed .
the lens , zonule , and ciliary body were released from the eye and transferred to a petri dish filled with ringer 's solution .
we divided the zonular fibers into two groups : the  equatorial zonule  ( blue fibers in fig .
other investigators have subdivided these fibers into anterior , equatorial , and posterior groupings , but here they were all included in a single sample , the equatorial zonule . using iridectomy scissors , fibers from the equatorial zonule
were first transected near the ciliary body and then grasped with fine forceps and cut close to the lens .
because the equatorial fibers are anatomically isolated within the eye , it was possible to remove them precisely , with little or no contamination from other tissues .
once the equatorial fibers had been removed , the  hyaloid zonule  was exposed .
the hyaloid zonule was defined as the set of fibers that were closely associated with the anterior face of the vitreous .
they were also significantly longer than the equatorial fibers . because they were intimately connected to the vitreous face
, it was not possible to dissect the hyaloid fibers as precisely as the equatorial fibers , and the hyaloid sample inevitably contained some elements of the anterior vitreous humor .
samples were centrifuged for 10 minutes at maximum speed , supernatants were removed , and the zonular pellet was frozen immediately . a third sample , taken from the center of the vitreous humor ,
a single bovine eye yielded sufficient tissue for a proteomic analysis of the equatorial zonule , hyaloid zonule , and vitreous humor .
human eyes ( from 10 donors 2366 years of age ; table ) were obtained from a local eye bank and dissected using a similar approach to that described for the bovine eye . because the vitreous of the aged human eye was often partially liquefied , it was not possible to collect a human hyaloid zonule sample .
thus , the human zonular sample was equivalent to the bovine equatorial zonule sample . description of human zonular samples to have sufficient material for analysis , human zonular samples were pooled ( table ) .
thus , each pool contained combined material from four eyes of both sexes , with mean ages ranging from 38.5 ( pool d ) to 60 years ( pool c ) .
human eyes were fixed for 1 week in 4% paraformaldehyde / pbs , dehydrated through graded ethanols and xylene , and embedded in paraffin wax .
sections ( 4 m thick ) were cut in the midsagittal plane and processed for immunofluorescence .
deparaffinized sections were antigen retrieved using a 60c overnight incubation in sodium citrate buffer ( 10 mm sodium citrate , 0.05% tween-20 ; ph 6.0 ) .
sections were blocked with 3% bsa for 2 hours and incubated overnight at 4c in a 1:501:100 dilution of primary antibody .
the following antibodies were used : adamtsl-6/thsd4 ( atlas antibodies , stockholm , sweden ) , emilin-1 ( santa cruz biotechnology , santa cruz , ca , usa ) , fbn1 ( millipore , temecula , ca , usa ) , fibulin-6/hemicentin-1 ( millipore ) , ltbp2 ( gift of tomoyuki nakamura , kansai medical university , osaka , japan ) , and mfap2 ( santa cruz biotechnology ) . after washing in pbs ( 3  10 minutes )
, sections were incubated for 2 hours in 1:300 dilution of appropriate secondary antibody ( alexa 488conjugated goat anti - mouse or anti - rabbit ; invitrogen , carlsbad , ca , usa ) containing methyl green ( a nuclear counterstain ) .
sections were then washed , coverslipped ( prolong gold ; invitrogen ) , and viewed using an olympus fv1000 confocal microscope ( center valley , pa , usa ) .
bovine and human zonules were suspended in 50 l buffer containing 4% sds , 0.2% deoxycholic acid ( dca ) , and 100 mm ammonium bicarbonate ( ph 8) and sonicated using a fisher scientific model 60 sonic dismembranator ( thermo fischer scientific , waltham , ma , usa ) , using three treatments of 10 seconds each .
samples were then shaken for 10 minutes at 600 rpm in a thermo - mixer at 90c .
five microliters was removed for a pierce bicinchoninic acid ( bca ) assay ( thermo fisher scientific ) using bsa as a standard .
five microliters of 0.5 m tris ( 2-carboxyethyl)phosphine ( tcep ) was then added , and the samples were heated at 90c for an additional 10 minutes and centrifuged at 14,000 g for 10 minutes , the supernatant was mixed with 200 l exchange buffer containing 8 m urea , 0.2% dca , and 100 mm ammonium bicarbonate , and the mixture was transferred to tween-20 passivated ultra-0.5 centrifuge filter units ( amicon ufc503008 ; emd millipore , billerica , ma , usa ) .
the remaining sample preparation and digestion used the enhanced filter - aided sample preparation ( efasp ) method of erde et al .
briefly , the sds containing lysis buffer was replaced with exchange buffer , proteins were alkylated with iodoacetamide , and exchange buffer was replaced with digestion buffer containing 0.2% dca and 50 mm ammonium bicarbonate .
a ratio of 1:50 of sequencing grade modified trypsin ( promega , madison , wi , usa ) to substrate was then added , and the filter units were shaken at approximately 100 rpm for 12 hours at 37c .
peptides were recovered by centrifugation and passage through the ultrafiltration membrane , and membranes were further washed with two 50-l portions of 50 mm ammonium bicarbonate .
the dca in the combined filtrates was then extracted using ethyl acetate as previously described , and peptides in the aqueous phase were dried by vacuum centrifugation .
bovine vitreous humor samples ( 200 l ) containing approximately 100 g protein were dried by vacuum centrifugation and dissolved in 50 l 8 m urea , 1.0 m tris ( ph 8.5 ) , 8 mm cacl2 , and 0.2 m methylamine .
samples were reduced by addition of 4 l 0.2 m dithiothreitol and incubation at 50c for 15 minutes , followed by alkylation through addition of 4 l 0.5 m iodoacetamide and incubation at room temperature ( rt ) for 30 minutes in the dark .
an additional 8 l 0.2 m dithiothreitol was then added , samples were incubated for an additional 15 minutes at rt , and 94 l water was added , followed by 40 l 0.1 g/l trypsin .
digestion proceeded overnight at 37c and then 10 l formic acid was added to stop the reaction .
peptides were solid phase extracted using sep - pak light c18 cartridges ( waters , milford , ma , usa ) , and the final eluate was dried by vacuum concentration .
samples were dissolved in water containing 5% formic acid and transferred to autosampler vials , and 4 g of each zonule peptide digest ( or 2 g of each vitreous humor digest ) was analyzed .
the samples were injected at a flow rate of 5 l / min onto an acclaim pepmap 100-m  2-cm nanoviper 5-m c18 trap ( thermo fisher scientific ) using mobile phase a containing water and 0.1% formic acid .
after 5 minutes , the trap was switched in - line with a pepmap rslc c18 , 2 m , 75-m  25-cm easyspray column fitted in an easyspray nano electrospray source ( thermo fisher scientific ) at 40c .
peptides were eluted with a 90-minute gradient of 7.5%30% mobile phase b containing acetonitrile and 0.1% formic acid at a flow rate of 0.3 l / min .
survey scans were performed in the orbitrap mass analyzer at a resolution of 120,000 with a scan range of 4001500 , maximum inject time of 50 ms , and automatic gain control ( acg ) target of 2  10 .
the quadrupole was used to isolate ions for mass spectroscopy ( ms)/ms scans with a 1.6-m / z isolation window , and fragmentation was performed by higher - energy collisional dissociation ( hcd ) with normalized collision energy of 35% .
top speed data - dependent ms / ms spectra were collected in parallel using the ion trap , with a maximum 35-ms inject time , single scan , signal - to - noise ration ( s / n ) threshold = 2 , minimum ion intensity threshold of 5000 , and a maximum interval of 3 seconds between survey scans .
dynamic exclusion was performed with the monoisotopic precursor selection ( mips ) filter on , exclusion of + 1 ions , 60-second exclusion time , exclusion mass tolerance of 10 ppm , and count = 1 .
, is provided as supplementary materials s1 . the open source proteowizard toolkit was used to convert thermo raw files into compressed text files using msconvert via command line .
an in - house python program was used to convert information in the compressed text file into appropriately formatted ms2 files .
ms2 scans were extracted with a minimum ion count of 15 and minimum total absolute intensity of 100 .
databases were downloaded from ensembl ( www.ensembl.org , in the public domain ) for bovine samples or from uniprot ( www.uniprot.org , in the public domain ) for human samples and processed with utilities available from www.proteomicanalysisworkbench.com , in the public domain .
common contaminant sequences ( 179 ) were added before all sequences were reversed for peptide and protein error estimation using the target / decoy method .
the ensembl bovine database ( release 79 , march 2015 ) had 22,118 protein sequences .
the human database consisted of 20,207 uniprot reviewed canonical sequences ( swiss - prot ) downloaded july 2015 .
searches were performed using comet version 2015.02 rev . 1 ( human samples ) or 2016.01 rev .
2 ( bovine samples ) . monoisotopic mass parent ion and fragment ion tolerances of 1.25 and 1.0005 da were used , respectively .
other parameters were as follows : tryptic enzyme specificity , static modifications of + 57 da on cysteine residues , variable modifications of + 16 da on methionine residues ( maximum of 2 modifications ) , and use of y- and b - ions in scoring .
the paw analysis pipeline was used to control peptide sequencing errors , infer protein identities from observed peptides , and provide quantitative protein abundance estimates .
comet search scores were transformed into discriminant scores using functions similar to those used in peptideprophet .
target and decoy discriminant scores were displayed as histograms and overlaid for interactive setting of score thresholds to filter peptide - spectral matches at an overall false discovery rate ( fdr ) of 3% .
protein inference used basic parsimony logic where proteins having indistinguishable peptide sets were combined into protein groups and proteins having peptide sets that were subset of other protein 's peptide sets were removed .
protein inference was performed experiment - wide with additional requirements of a minimum of two distinct peptides per protein per biological sample applied after protein inference . in a final round of processing , straightforward extensions of parsimony principles
these results were used for quantitative analyses using ms2 fragment ion intensity  weighted spectral counting . to more confidently determine the composition of the zonule , contaminating proteins from surrounding tissues have to be identified and excluded .
it was assumed that the zonule would be composed predominantly of extracellular matrix ( ecm ) proteins .
comprehensive lists of ecm proteins for human and mouse have been compiled ( http://matrisomeproject.mit.edu , in the public domain ) , and the lists of core and ecm - associated proteins for human were used to extract the corresponding protein sequence from uniprot .
a fasta database file of all human ecm proteins was constructed . for both human and bovine samples ,
the lists of identified proteins were used to extract separate fasta database files for identified proteins .
an in - house python program was used to run a local installation of blast ( ftp://ftp.ncbi.nlm.nih.gov/blast/executables/blast+/latest/ , in the public domain ) to determine the reciprocal best matches between the identified proteins and the human ecm proteins .
analysis of alignment scores was used to categorize likely matches to matrisome proteins from poor alignments between matrisome proteins and contaminating proteins .
common contaminant proteins and proteins not matching to matrisome ecm proteins were excluded from quantitative analysis .
extracellular matrix proteins constituted 90% of the bovine zonule sample and 82% of the human zonule sample .
the total abundances of putative zonule proteins ( using ms2 intensity  weighted spectral counts ) were set to 100.00 , and the relative abundance of the proteins within each sample was determined .
average protein abundances and their standard deviations were computed using standard excel functions . unlike the human dataset , multiple proteins from the bovine dataset mapped to individual matrisome genes .
this was because the bovine ensemble protein database ( unlike the human swiss prot database ) contained multiple entries for protein isoforms of nearly identical sequence . to facilitate comparison to the human samples
, the ms2 intensity  weighted spectral counts for all proteins mapping to the same matrisome gene symbol were summed together .
full , annotated protein reports ( supplementary materials s2 , s3 ) and associated detailed peptide evidence files ( supplementary materials s4 , s5 ) for each experiment are included .
bovine eyes ( from 3-year - old animals ) were obtained from a local slaughterhouse within 3 hours of death .
the eye globe was dissected from its anterior aspect , the cornea and iris were removed , and the front face of the ciliary processes was exposed .
the lens , zonule , and ciliary body were released from the eye and transferred to a petri dish filled with ringer 's solution .
we divided the zonular fibers into two groups : the  equatorial zonule  ( blue fibers in fig .
other investigators have subdivided these fibers into anterior , equatorial , and posterior groupings , but here they were all included in a single sample , the equatorial zonule . using iridectomy scissors , fibers from the equatorial zonule
were first transected near the ciliary body and then grasped with fine forceps and cut close to the lens .
because the equatorial fibers are anatomically isolated within the eye , it was possible to remove them precisely , with little or no contamination from other tissues .
once the equatorial fibers had been removed , the  hyaloid zonule  was exposed .
the hyaloid zonule was defined as the set of fibers that were closely associated with the anterior face of the vitreous .
they were also significantly longer than the equatorial fibers . because they were intimately connected to the vitreous face
, it was not possible to dissect the hyaloid fibers as precisely as the equatorial fibers , and the hyaloid sample inevitably contained some elements of the anterior vitreous humor .
samples were centrifuged for 10 minutes at maximum speed , supernatants were removed , and the zonular pellet was frozen immediately . a third sample , taken from the center of the vitreous humor ,
a single bovine eye yielded sufficient tissue for a proteomic analysis of the equatorial zonule , hyaloid zonule , and vitreous humor .
human eyes ( from 10 donors 2366 years of age ; table ) were obtained from a local eye bank and dissected using a similar approach to that described for the bovine eye . because the vitreous of the aged human eye was often partially liquefied , it was not possible to collect a human hyaloid zonule sample .
thus , the human zonular sample was equivalent to the bovine equatorial zonule sample . description of human zonular samples to have sufficient material for analysis , human zonular samples were pooled ( table ) .
thus , each pool contained combined material from four eyes of both sexes , with mean ages ranging from 38.5 ( pool d ) to 60 years ( pool c ) .
human eyes were fixed for 1 week in 4% paraformaldehyde / pbs , dehydrated through graded ethanols and xylene , and embedded in paraffin wax . sections ( 4 m thick ) were cut in the midsagittal plane and processed for immunofluorescence .
deparaffinized sections were antigen retrieved using a 60c overnight incubation in sodium citrate buffer ( 10 mm sodium citrate , 0.05% tween-20 ; ph 6.0 ) .
sections were blocked with 3% bsa for 2 hours and incubated overnight at 4c in a 1:501:100 dilution of primary antibody .
the following antibodies were used : adamtsl-6/thsd4 ( atlas antibodies , stockholm , sweden ) , emilin-1 ( santa cruz biotechnology , santa cruz , ca , usa ) , fbn1 ( millipore , temecula , ca , usa ) , fibulin-6/hemicentin-1 ( millipore ) , ltbp2 ( gift of tomoyuki nakamura , kansai medical university , osaka , japan ) , and mfap2 ( santa cruz biotechnology ) . after washing in pbs ( 3  10 minutes )
, sections were incubated for 2 hours in 1:300 dilution of appropriate secondary antibody ( alexa 488conjugated goat anti - mouse or anti - rabbit ; invitrogen , carlsbad , ca , usa ) containing methyl green ( a nuclear counterstain ) .
sections were then washed , coverslipped ( prolong gold ; invitrogen ) , and viewed using an olympus fv1000 confocal microscope ( center valley , pa , usa ) .
bovine and human zonules were suspended in 50 l buffer containing 4% sds , 0.2% deoxycholic acid ( dca ) , and 100 mm ammonium bicarbonate ( ph 8) and sonicated using a fisher scientific model 60 sonic dismembranator ( thermo fischer scientific , waltham , ma , usa ) , using three treatments of 10 seconds each .
samples were then shaken for 10 minutes at 600 rpm in a thermo - mixer at 90c .
five microliters was removed for a pierce bicinchoninic acid ( bca ) assay ( thermo fisher scientific ) using bsa as a standard .
five microliters of 0.5 m tris ( 2-carboxyethyl)phosphine ( tcep ) was then added , and the samples were heated at 90c for an additional 10 minutes and centrifuged at 14,000 g for 10 minutes , the supernatant was mixed with 200 l exchange buffer containing 8 m urea , 0.2% dca , and 100 mm ammonium bicarbonate , and the mixture was transferred to tween-20 passivated ultra-0.5 centrifuge filter units ( amicon ufc503008 ; emd millipore , billerica , ma , usa ) .
the remaining sample preparation and digestion used the enhanced filter - aided sample preparation ( efasp ) method of erde et al .
briefly , the sds containing lysis buffer was replaced with exchange buffer , proteins were alkylated with iodoacetamide , and exchange buffer was replaced with digestion buffer containing 0.2% dca and 50 mm ammonium bicarbonate . a ratio of 1:50 of sequencing grade modified trypsin ( promega , madison , wi , usa ) to substrate
was then added , and the filter units were shaken at approximately 100 rpm for 12 hours at 37c .
peptides were recovered by centrifugation and passage through the ultrafiltration membrane , and membranes were further washed with two 50-l portions of 50 mm ammonium bicarbonate .
the dca in the combined filtrates was then extracted using ethyl acetate as previously described , and peptides in the aqueous phase were dried by vacuum centrifugation .
bovine vitreous humor samples ( 200 l ) containing approximately 100 g protein were dried by vacuum centrifugation and dissolved in 50 l 8 m urea , 1.0 m tris ( ph 8.5 ) , 8 mm cacl2 , and 0.2 m methylamine .
samples were reduced by addition of 4 l 0.2 m dithiothreitol and incubation at 50c for 15 minutes , followed by alkylation through addition of 4 l 0.5 m iodoacetamide and incubation at room temperature ( rt ) for 30 minutes in the dark .
an additional 8 l 0.2 m dithiothreitol was then added , samples were incubated for an additional 15 minutes at rt , and 94 l water was added , followed by 40 l 0.1 g/l trypsin .
digestion proceeded overnight at 37c and then 10 l formic acid was added to stop the reaction .
peptides were solid phase extracted using sep - pak light c18 cartridges ( waters , milford , ma , usa ) , and the final eluate was dried by vacuum concentration .
samples were dissolved in water containing 5% formic acid and transferred to autosampler vials , and 4 g of each zonule peptide digest ( or 2 g of each vitreous humor digest ) was analyzed .
the samples were injected at a flow rate of 5 l / min onto an acclaim pepmap 100-m  2-cm nanoviper 5-m c18 trap ( thermo fisher scientific ) using mobile phase a containing water and 0.1% formic acid .
after 5 minutes , the trap was switched in - line with a pepmap rslc c18 , 2 m , 75-m  25-cm easyspray column fitted in an easyspray nano electrospray source ( thermo fisher scientific ) at 40c .
peptides were eluted with a 90-minute gradient of 7.5%30% mobile phase b containing acetonitrile and 0.1% formic acid at a flow rate of 0.3 l / min .
survey scans were performed in the orbitrap mass analyzer at a resolution of 120,000 with a scan range of 4001500 , maximum inject time of 50 ms , and automatic gain control ( acg ) target of 2  10 .
the quadrupole was used to isolate ions for mass spectroscopy ( ms)/ms scans with a 1.6-m / z isolation window , and fragmentation was performed by higher - energy collisional dissociation ( hcd ) with normalized collision energy of 35% .
top speed data - dependent ms / ms spectra were collected in parallel using the ion trap , with a maximum 35-ms inject time , single scan , signal - to - noise ration ( s / n ) threshold = 2 , minimum ion intensity threshold of 5000 , and a maximum interval of 3 seconds between survey scans .
dynamic exclusion was performed with the monoisotopic precursor selection ( mips ) filter on , exclusion of + 1 ions , 60-second exclusion time , exclusion mass tolerance of 10 ppm , and count = 1 .
the open source proteowizard toolkit was used to convert thermo raw files into compressed text files using msconvert via command line .
an in - house python program was used to convert information in the compressed text file into appropriately formatted ms2 files .
ms2 scans were extracted with a minimum ion count of 15 and minimum total absolute intensity of 100 .
databases were downloaded from ensembl ( www.ensembl.org , in the public domain ) for bovine samples or from uniprot ( www.uniprot.org , in the public domain ) for human samples and processed with utilities available from www.proteomicanalysisworkbench.com , in the public domain .
common contaminant sequences ( 179 ) were added before all sequences were reversed for peptide and protein error estimation using the target / decoy method .
the ensembl bovine database ( release 79 , march 2015 ) had 22,118 protein sequences .
the human database consisted of 20,207 uniprot reviewed canonical sequences ( swiss - prot ) downloaded july 2015 .
searches were performed using comet version 2015.02 rev . 1 ( human samples ) or 2016.01 rev .
2 ( bovine samples ) . monoisotopic mass parent ion and fragment ion tolerances of 1.25 and 1.0005 da were used , respectively .
other parameters were as follows : tryptic enzyme specificity , static modifications of + 57 da on cysteine residues , variable modifications of + 16 da on methionine residues ( maximum of 2 modifications ) , and use of y- and b - ions in scoring .
the paw analysis pipeline was used to control peptide sequencing errors , infer protein identities from observed peptides , and provide quantitative protein abundance estimates .
comet search scores were transformed into discriminant scores using functions similar to those used in peptideprophet .
target and decoy discriminant scores were displayed as histograms and overlaid for interactive setting of score thresholds to filter peptide - spectral matches at an overall false discovery rate ( fdr ) of 3% .
protein inference used basic parsimony logic where proteins having indistinguishable peptide sets were combined into protein groups and proteins having peptide sets that were subset of other protein 's peptide sets were removed .
protein inference was performed experiment - wide with additional requirements of a minimum of two distinct peptides per protein per biological sample applied after protein inference . in a final round of processing , straightforward extensions of parsimony principles
these results were used for quantitative analyses using ms2 fragment ion intensity  weighted spectral counting .
the open source proteowizard toolkit was used to convert thermo raw files into compressed text files using msconvert via command line .
an in - house python program was used to convert information in the compressed text file into appropriately formatted ms2 files .
ms2 scans were extracted with a minimum ion count of 15 and minimum total absolute intensity of 100 .
databases were downloaded from ensembl ( www.ensembl.org , in the public domain ) for bovine samples or from uniprot ( www.uniprot.org , in the public domain ) for human samples and processed with utilities available from www.proteomicanalysisworkbench.com , in the public domain .
common contaminant sequences ( 179 ) were added before all sequences were reversed for peptide and protein error estimation using the target / decoy method .
the ensembl bovine database ( release 79 , march 2015 ) had 22,118 protein sequences .
the human database consisted of 20,207 uniprot reviewed canonical sequences ( swiss - prot ) downloaded july 2015 .
searches were performed using comet version 2015.02 rev . 1 ( human samples ) or 2016.01 rev .
2 ( bovine samples ) . monoisotopic mass parent ion and fragment ion tolerances of 1.25 and 1.0005 da were used , respectively .
other parameters were as follows : tryptic enzyme specificity , static modifications of + 57 da on cysteine residues , variable modifications of + 16 da on methionine residues ( maximum of 2 modifications ) , and use of y- and b - ions in scoring .
the paw analysis pipeline was used to control peptide sequencing errors , infer protein identities from observed peptides , and provide quantitative protein abundance estimates .
comet search scores were transformed into discriminant scores using functions similar to those used in peptideprophet .
target and decoy discriminant scores were displayed as histograms and overlaid for interactive setting of score thresholds to filter peptide - spectral matches at an overall false discovery rate ( fdr ) of 3% .
protein inference used basic parsimony logic where proteins having indistinguishable peptide sets were combined into protein groups and proteins having peptide sets that were subset of other protein 's peptide sets were removed .
protein inference was performed experiment - wide with additional requirements of a minimum of two distinct peptides per protein per biological sample applied after protein inference . in a final round of processing , straightforward extensions of parsimony principles
these results were used for quantitative analyses using ms2 fragment ion intensity  weighted spectral counting .
to more confidently determine the composition of the zonule , contaminating proteins from surrounding tissues have to be identified and excluded .
it was assumed that the zonule would be composed predominantly of extracellular matrix ( ecm ) proteins .
comprehensive lists of ecm proteins for human and mouse have been compiled ( http://matrisomeproject.mit.edu , in the public domain ) , and the lists of core and ecm - associated proteins for human were used to extract the corresponding protein sequence from uniprot .
a fasta database file of all human ecm proteins was constructed . for both human and bovine samples ,
the lists of identified proteins were used to extract separate fasta database files for identified proteins .
an in - house python program was used to run a local installation of blast ( ftp://ftp.ncbi.nlm.nih.gov/blast/executables/blast+/latest/ , in the public domain ) to determine the reciprocal best matches between the identified proteins and the human ecm proteins .
analysis of alignment scores was used to categorize likely matches to matrisome proteins from poor alignments between matrisome proteins and contaminating proteins . once matrisome proteins were identified
common contaminant proteins and proteins not matching to matrisome ecm proteins were excluded from quantitative analysis .
extracellular matrix proteins constituted 90% of the bovine zonule sample and 82% of the human zonule sample .
the total abundances of putative zonule proteins ( using ms2 intensity  weighted spectral counts ) were set to 100.00 , and the relative abundance of the proteins within each sample was determined .
average protein abundances and their standard deviations were computed using standard excel functions . unlike the human dataset , multiple proteins from the bovine dataset mapped to individual matrisome genes .
this was because the bovine ensemble protein database ( unlike the human swiss prot database ) contained multiple entries for protein isoforms of nearly identical sequence . to facilitate comparison to the human samples
, the ms2 intensity  weighted spectral counts for all proteins mapping to the same matrisome gene symbol were summed together .
relative abundances within each sample by gene were computed as described above . full , annotated protein reports ( supplementary materials s2 , s3 ) and associated detailed peptide evidence files ( supplementary materials s4 ,
we began by comparing the protein composition of the human and bovine ciliary zonule ( the zonulome ) .
for this purpose , the human zonular sample was compared with the bovine equatorial zonule sample ( see materials and methods ) .
our initial goal was to identify those proteins common to both , reasoning that protein conservation may imply important structural or signaling roles for the set of conserved proteins .
liquid chromatography  ms analysis of tryptic digests identified > 1000 proteins , but , following removal of common contaminants and filtering against the matrisome database , this number was reduced to 268 proteins ( 176 proteins in the bovine zonulome and 144 in the human ) .
3 ) . in terms of mass , > 95% of the human and bovine zonules consisted of the proteins that were common to both species .
two main classes of matrisomal proteins are defined : those constituting the core matrisome and those belonging to the group of matrisome - associated proteins .
the core matrisome is further divided into three subcategories : ecm glycoproteins , collagens , and proteoglycans .
the matrisome - associated protein category is also divided into three subcategories : ecm - affiliated proteins , ecm regulators , and secreted factors .
of the six classes of matrisomal proteins , the two best represented by the number of identifications in the zonulome were glycoproteins ( 28 proteins in the human zonule and 53 in the bovine zonule ) and ecm regulators ( 23 in humans and 26 in cows ; ( figs .
however , when the data were weighted according to protein abundance , based on ms2 intensity  weighted spectral counts , it was clear that in both cases the zonulome was dominated by glycoproteins ( figs . 4c , 4d ) .
the nine most abundant glycoproteins in the human and bovine zonulome are ranked in figure 5 .
the five most abundant proteins in either case were fibrillin-1 ( fbn1 ) , microfibrillar - associated protein 2 ( mfap2 ) , latent - transforming growth factor -binding protein-2 ( ltbp2 ) , emilin-1 , and thrombospondin type-1 domain - containing protein 4 ( thsd4 , also known as adamtsl-6 ) .
overview of zonule composition in bovine ( a , c ) and human ( b , d ) samples . in terms of the number of proteins present ( a , b ) , all six categories of matrisomal proteins are well represented in the zonulome .
however , in terms of relative protein abundance , both zonulomes are composed predominantly of glycoproteins ( c , d ) .
shown here is a ranking of the nine most - abundant glycoproteins , which together account for 95% of the bovine zonule and 89% of the human zonule .
we used an orthogonal technique , confocal immunofluorescence , to verify the distribution of the four most abundant glycoproteins detected in the lc - ms analysis ( fbn1 , ltbp2 , mfap2 , and emilin-1 ; fig .
, the zonule appeared as a system of fibers projecting from the pars plana region of the nonpigmented ciliary epithelium , fanning out as they approached the lens . at their midpoint , the fibers had a relatively large diameter but near the lens surface , the large fibers divided to form smaller diameter fibers that inserted into the lens capsule ( see insets in fig .
the three most abundant glycoproteins in the zonulome ( fbn1 , ltbp2 , and mfap2 ) labeled all zonular elements uniformly ( figs .
fbn1 , ltbp2 , and mfap2 are distributed throughout the system of zonular fibers , including the small diameter fibers near the lens surface ( inset a in b ) and large diameter fibers at the midpoint of the zonular span ( inset b in b ) .
emilin-1 was absent from zonular fibers near the lens surface ( arrow in d ) .
fibrillins and ltbps comprise a superfamily of seven structurally related glycoproteins ( fbn1 - 3 and ltbp1 - 4 ) .
all seven superfamily members were detected in the bovine zonule and only ltbp4 was absent from the human zonule ( fig .
the fbn / ltbp superfamily constituted 83.4% of the bovine zonule and 76.0% of the human zonule and represented the most abundant protein family in either zonulome .
fbn1 was the most abundant individual protein , accounting for 63.49% and 75.57% of the protein in the human and bovine ciliary zonule , respectively .
all family members are present ( with the exception of ltbp4 in the human zonule ) .
the high representation of fbn / ltbp superfamily members in the zonule is consistent with the ocular phenotypes of syndromes resulting from mutations in those proteins .
mutations in fbn1 underlie marfan syndrome , a condition in which progressive lysis of the zonular fibers leads to ectopia lentis in most ( 71% ) patients .
mutations in fbn1 and ltbp2 have also been shown to cause weill - marchesani syndrome ( types 2 and 3 , respectively ) , which is a disease characterized by microspherophakia and ectopia lentis , in addition to systemic symptoms .
together , these observations support the notion that fbn1 and ltbp2 play indispensable structural roles in the ciliary zonule .
ltbps , as their name implies , have the ability to bind and sequester latent forms of tgf in the ecm .
both the human and the bovine zonule contained relatively high levels of tgf-2 ( lesser amounts of tgf-3 were also detected in the human sample ) .
curiously , ltbp2 , by far the most abundant of the zonular ltbps , is the only family member incapable of binding tgf , so its role in the zonule and elsewhere is unresolved .
another class of proteins linked with inherited ectopia lentis ( and the closely related condition ectopia lentis et pupillae , in which both the lens and the pupil are displaced ) is the adamtsl family .
adamtsls share significant sequence similarity with the adamts family of metalloproteases but lack the protease domain found in the latter . mutations in adamtsl-4 cause isolated ectopia lentis and ectopia lentis et pupillae .
adamtsl-4 was detected in both the bovine and human zonules ( at somewhat higher levels in the latter ) , as were adamtsl-1,-2 and -5 .
unexpectedly , however , the most abundant member of the family was adamtsl-6 ( also known as thsd4 ) , which , in the human zonule , accounted for > 2% of the total protein ( figs . 5 , 8) .
we used immunofluorescence to verify the expression of adamtsl-6 in the human eye ( fig .
adamtsl-6 was strongly expressed in the large diameter fibers in a similar fashion to emilin-1 ( fig .
in vitro studies suggest that adamtsl-6 can bind directly to fbn1 , promoting the assembly of fibrillin - rich ecm .
immunofluorescence is largely restricted to the large diameter fibers in the center of the zonular span and absent from small fibers at the lens surface ( see fig .
a number of protease inhibitors were detected in the zonule , including several members of the serpin family , but the most abundant inhibitor , particularly in the human sample , was timp3 ( fig .
the only protease detected in both the human and bovine zonule was cathepsin d , a lysosomal aspartic endopeptidase that may also be secreted from cells .
work on model systems suggest that the material properties of microfibrillar networks are shaped by both nonreducible crosslinks and disulfide bonds .
we examined human and bovine zonules for the presence of known crosslinking enzymes that might play a role in bundling of microfibrils .
several transglutaminases ( tgm1 - 3 ) were detected , but the most abundant crosslinker detected was lysyl oxidase - like 1 ( loxl1 ; fig .
transglutaminase - derived crosslinks have been identified previously in fibrillin microfibrils and may be important in the zonule .
the presence of loxl1 is intriguing because variants in the loxl1 gene have been shown to predispose to exfoliation syndrome .
exfoliation syndrome is a condition in which disintegration of the zonular fibers and unscheduled production of matrix components results in formation of fibrillar aggregates that can clog the outflow pathways of the eye , leading to exfoliation glaucoma .
given its demonstrated role in elastin polymerization , it is curious that loxl1 is such a prominent component of the elastin - free ciliary zonule .
one explanation may be that fibrillin or other zonule components serve as substrates for loxl1 .
the availability of mice deficient in loxl1 should allow us to test whether loxl1 has a role in crosslinking microfibrils .
expression of crosslinking enzymes ( transglutaminases and lysyloxidase - like 1 ) in the ciliary zonule .
the expression of collagens , secreted factors , and proteoglycans was compared in the human and bovine zonular samples ( supplementary tables s1 , s2 ) .
only trace amounts of collagen were detected , of which the most abundant were col2a1 and col9a2 .
col4 and col18 , components of basement membranes , were also detected and may represent contamination from the lens capsule or the inner limiting membrane covering the basal surface of the nonpigmented ciliary epithelium .
megf6 , shown to interact via its egf domains with mfap5 , was one of the most abundant .
immune components , such as cxcl14 and egfl7 , were identified in the human but not bovine zonule .
this may reflect the fact that the human samples were largely from middle - aged individuals ( table ) , whereas the bovine samples were from young animals .
similarly , the presence of il17d in human samples may indicate ongoing inflammatory reactions in the aged human eyes .
cxcl14 is a chemokine implicated in the recruitment of immune cells in all types of inflammatory conditions .
proteoglycans were not prominent zonule components in either species , although in the bovine zonule , opticin ( opt ) was detected at moderate levels .
unlike the bovine zonule ( where members of a single glycoprotein family , the fbn / ltbp family , accounted for > 80% of the protein ) , the composition of the vitreous was not dominated by a restricted set of super abundant proteins ( supplementary material s3 ) . among the more abundant vitreous proteins were broad spectrum protease inhibitors ( a2ml1 , a2 m , cpmd8 , cst3 , and serping1 ) , collagens ( col9a2 , col18a1 , and col2a1 ) , fibulins ( fbln1 , fbln2 , and fbnl3 ) , the proteoglycans opt and hspg2 , and wnt signaling components sfrp2 and sfrp3 .
fbn1 , the most abundant single component in the equatorial zonule , accounted for only 2.5% of the protein in the vitreous sample .
recent proteomic analyses of human vitreous humor have identified many hundreds of protein components , of which a core set of 231 proteins were detected in all studies . in the present study , after filtering against the matrisome database , we identified 200 components of the bovine vitreous .
comparing the bovine vitreous proteome with that of the equatorial and hyaloid zonular samples , it was evident that the zonular samples were quite distinct from the vitreous sample ( figs .
proteins such as mfap2 and fbn2 , for example , were almost undetectable in the vitreous but relatively abundant in the zonular samples . overlapping distribution of matrisome proteins in samples from the bovine equatorial zonule , hyaloid zonule , and vitreous humor .
protein composition of samples from the bovine vitreous humor ( gray ) , equatorial zonule ( light blue ) , and hyaloid zonule ( dark blue ) .
some proteins are relatively abundant in all three samples ; others , such as thsd4 , are abundant in the zonular samples but barely detectable in the vitreous
three classes of fibrillary collagen ( type 2a , 11a , and 5a ; circled in yellow ) are significantly overrepresented in the hyaloid zonule sample compared with the equatorial zonule or vitreous .
note that opticin ( opt ) accounts for 30% of the hyaloid zonule but was barely detectable in the equatorial zonule .
the hyaloid zonule is associated with the vitreous face , and it is possible that during dissection , the hyaloid sample might have been contaminated with proteins emanating from the vitreous .
if we assume that the hyaloid zonule has a similar basic composition to the equatorial zonule , then vitreous contamination should lead to values for relative protein abundance that were intermediate between the vitreous and equatorial zonule values .
although such instances were certainly observed , there were also many examples where the abundance of a protein in the hyaloid zonule significantly exceeded ( by orders of magnitude ) that of the vitreous or the equatorial zonule .
such proteins are likely to be unique to , or significantly enriched in , the hyaloid zonule sample .
the set of hyaloid - enriched proteins included opt , a3i3bp , cochlin , and the collagens col2a1 , col11a1 , col5a3 , and col5a2 ( fig .
histograms showing relative abundance of each class of matrisomal proteins in the bovine samples are included as supplementary figures s6s12 .
we began by comparing the protein composition of the human and bovine ciliary zonule ( the zonulome ) .
for this purpose , the human zonular sample was compared with the bovine equatorial zonule sample ( see materials and methods ) .
our initial goal was to identify those proteins common to both , reasoning that protein conservation may imply important structural or signaling roles for the set of conserved proteins .
liquid chromatography  ms analysis of tryptic digests identified > 1000 proteins , but , following removal of common contaminants and filtering against the matrisome database , this number was reduced to 268 proteins ( 176 proteins in the bovine zonulome and 144 in the human ) .
3 ) . in terms of mass , > 95% of the human and bovine zonules consisted of the proteins that were common to both species .
two main classes of matrisomal proteins are defined : those constituting the core matrisome and those belonging to the group of matrisome - associated proteins .
the core matrisome is further divided into three subcategories : ecm glycoproteins , collagens , and proteoglycans .
the matrisome - associated protein category is also divided into three subcategories : ecm - affiliated proteins , ecm regulators , and secreted factors .
of the six classes of matrisomal proteins , the two best represented by the number of identifications in the zonulome were glycoproteins ( 28 proteins in the human zonule and 53 in the bovine zonule ) and ecm regulators ( 23 in humans and 26 in cows ; ( figs .
however , when the data were weighted according to protein abundance , based on ms2 intensity  weighted spectral counts , it was clear that in both cases the zonulome was dominated by glycoproteins ( figs . 4c , 4d ) .
the nine most abundant glycoproteins in the human and bovine zonulome are ranked in figure 5 .
the five most abundant proteins in either case were fibrillin-1 ( fbn1 ) , microfibrillar - associated protein 2 ( mfap2 ) , latent - transforming growth factor -binding protein-2 ( ltbp2 ) , emilin-1 , and thrombospondin type-1 domain - containing protein 4 ( thsd4 , also known as adamtsl-6 ) .
overview of zonule composition in bovine ( a , c ) and human ( b , d ) samples . in terms of the number of proteins present ( a , b ) , all six categories of matrisomal proteins are well represented in the zonulome .
however , in terms of relative protein abundance , both zonulomes are composed predominantly of glycoproteins ( c , d ) .
shown here is a ranking of the nine most - abundant glycoproteins , which together account for 95% of the bovine zonule and 89% of the human zonule .
we used an orthogonal technique , confocal immunofluorescence , to verify the distribution of the four most abundant glycoproteins detected in the lc - ms analysis ( fbn1 , ltbp2 , mfap2 , and emilin-1 ; fig .
, the zonule appeared as a system of fibers projecting from the pars plana region of the nonpigmented ciliary epithelium , fanning out as they approached the lens . at their midpoint , the fibers had a relatively large diameter but near the lens surface , the large fibers divided to form smaller diameter fibers that inserted into the lens capsule ( see insets in fig .
the three most abundant glycoproteins in the zonulome ( fbn1 , ltbp2 , and mfap2 ) labeled all zonular elements uniformly ( figs .
fbn1 , ltbp2 , and mfap2 are distributed throughout the system of zonular fibers , including the small diameter fibers near the lens surface ( inset a in b ) and large diameter fibers at the midpoint of the zonular span ( inset b in b ) .
emilin-1 was absent from zonular fibers near the lens surface ( arrow in d ) .
fibrillins and ltbps comprise a superfamily of seven structurally related glycoproteins ( fbn1 - 3 and ltbp1 - 4 ) .
all seven superfamily members were detected in the bovine zonule and only ltbp4 was absent from the human zonule ( fig .
the fbn / ltbp superfamily constituted 83.4% of the bovine zonule and 76.0% of the human zonule and represented the most abundant protein family in either zonulome .
fbn1 was the most abundant individual protein , accounting for 63.49% and 75.57% of the protein in the human and bovine ciliary zonule , respectively .
all family members are present ( with the exception of ltbp4 in the human zonule ) .
the high representation of fbn / ltbp superfamily members in the zonule is consistent with the ocular phenotypes of syndromes resulting from mutations in those proteins .
mutations in fbn1 underlie marfan syndrome , a condition in which progressive lysis of the zonular fibers leads to ectopia lentis in most ( 71% ) patients .
mutations in fbn1 and ltbp2 have also been shown to cause weill - marchesani syndrome ( types 2 and 3 , respectively ) , which is a disease characterized by microspherophakia and ectopia lentis , in addition to systemic symptoms .
together , these observations support the notion that fbn1 and ltbp2 play indispensable structural roles in the ciliary zonule .
ltbps , as their name implies , have the ability to bind and sequester latent forms of tgf in the ecm .
both the human and the bovine zonule contained relatively high levels of tgf-2 ( lesser amounts of tgf-3 were also detected in the human sample ) .
curiously , ltbp2 , by far the most abundant of the zonular ltbps , is the only family member incapable of binding tgf , so its role in the zonule and elsewhere is unresolved .
another class of proteins linked with inherited ectopia lentis ( and the closely related condition ectopia lentis et pupillae , in which both the lens and the pupil are displaced ) is the adamtsl family .
adamtsls share significant sequence similarity with the adamts family of metalloproteases but lack the protease domain found in the latter . mutations in adamtsl-4 cause isolated ectopia lentis and ectopia lentis et pupillae .
adamtsl-4 was detected in both the bovine and human zonules ( at somewhat higher levels in the latter ) , as were adamtsl-1,-2 and -5 .
unexpectedly , however , the most abundant member of the family was adamtsl-6 ( also known as thsd4 ) , which , in the human zonule , accounted for > 2% of the total protein ( figs . 5 , 8) .
we used immunofluorescence to verify the expression of adamtsl-6 in the human eye ( fig .
adamtsl-6 was strongly expressed in the large diameter fibers in a similar fashion to emilin-1 ( fig .
in vitro studies suggest that adamtsl-6 can bind directly to fbn1 , promoting the assembly of fibrillin - rich ecm .
immunofluorescence is largely restricted to the large diameter fibers in the center of the zonular span and absent from small fibers at the lens surface ( see fig .
a number of protease inhibitors were detected in the zonule , including several members of the serpin family , but the most abundant inhibitor , particularly in the human sample , was timp3 ( fig .
the only protease detected in both the human and bovine zonule was cathepsin d , a lysosomal aspartic endopeptidase that may also be secreted from cells .
work on model systems suggest that the material properties of microfibrillar networks are shaped by both nonreducible crosslinks and disulfide bonds .
we examined human and bovine zonules for the presence of known crosslinking enzymes that might play a role in bundling of microfibrils .
several transglutaminases ( tgm1 - 3 ) were detected , but the most abundant crosslinker detected was lysyl oxidase - like 1 ( loxl1 ; fig .
transglutaminase - derived crosslinks have been identified previously in fibrillin microfibrils and may be important in the zonule .
the presence of loxl1 is intriguing because variants in the loxl1 gene have been shown to predispose to exfoliation syndrome .
exfoliation syndrome is a condition in which disintegration of the zonular fibers and unscheduled production of matrix components results in formation of fibrillar aggregates that can clog the outflow pathways of the eye , leading to exfoliation glaucoma .
given its demonstrated role in elastin polymerization , it is curious that loxl1 is such a prominent component of the elastin - free ciliary zonule .
one explanation may be that fibrillin or other zonule components serve as substrates for loxl1 .
the availability of mice deficient in loxl1 should allow us to test whether loxl1 has a role in crosslinking microfibrils .
expression of crosslinking enzymes ( transglutaminases and lysyloxidase - like 1 ) in the ciliary zonule .
the expression of collagens , secreted factors , and proteoglycans was compared in the human and bovine zonular samples ( supplementary tables s1 , s2 ) .
only trace amounts of collagen were detected , of which the most abundant were col2a1 and col9a2 .
col4 and col18 , components of basement membranes , were also detected and may represent contamination from the lens capsule or the inner limiting membrane covering the basal surface of the nonpigmented ciliary epithelium .
megf6 , shown to interact via its egf domains with mfap5 , was one of the most abundant .
immune components , such as cxcl14 and egfl7 , were identified in the human but not bovine zonule .
this may reflect the fact that the human samples were largely from middle - aged individuals ( table ) , whereas the bovine samples were from young animals .
similarly , the presence of il17d in human samples may indicate ongoing inflammatory reactions in the aged human eyes .
cxcl14 is a chemokine implicated in the recruitment of immune cells in all types of inflammatory conditions .
proteoglycans were not prominent zonule components in either species , although in the bovine zonule , opticin ( opt ) was detected at moderate levels .
unlike the bovine zonule ( where members of a single glycoprotein family , the fbn / ltbp family , accounted for > 80% of the protein ) , the composition of the vitreous was not dominated by a restricted set of super abundant proteins ( supplementary material s3 ) . among the more abundant vitreous proteins were broad spectrum protease inhibitors ( a2ml1 , a2 m , cpmd8 , cst3 , and serping1 ) , collagens ( col9a2 , col18a1 , and col2a1 ) , fibulins ( fbln1 , fbln2 , and fbnl3 ) , the proteoglycans opt and hspg2 , and wnt signaling components sfrp2 and sfrp3 .
fbn1 , the most abundant single component in the equatorial zonule , accounted for only 2.5% of the protein in the vitreous sample .
recent proteomic analyses of human vitreous humor have identified many hundreds of protein components , of which a core set of 231 proteins were detected in all studies . in the present study , after filtering against the matrisome database , we identified 200 components of the bovine vitreous . comparing the bovine vitreous proteome with that of the equatorial and hyaloid zonular samples , it was evident that the zonular samples were quite distinct from the vitreous sample ( figs .
proteins such as mfap2 and fbn2 , for example , were almost undetectable in the vitreous but relatively abundant in the zonular samples . overlapping distribution of matrisome proteins in samples from the bovine equatorial zonule , hyaloid zonule , and vitreous humor .
protein composition of samples from the bovine vitreous humor ( gray ) , equatorial zonule ( light blue ) , and hyaloid zonule ( dark blue ) .
some proteins are relatively abundant in all three samples ; others , such as thsd4 , are abundant in the zonular samples but barely detectable in the vitreous .
three classes of fibrillary collagen ( type 2a , 11a , and 5a ; circled in yellow ) are significantly overrepresented in the hyaloid zonule sample compared with the equatorial zonule or vitreous .
note that opticin ( opt ) accounts for 30% of the hyaloid zonule but was barely detectable in the equatorial zonule .
the hyaloid zonule is associated with the vitreous face , and it is possible that during dissection , the hyaloid sample might have been contaminated with proteins emanating from the vitreous .
if we assume that the hyaloid zonule has a similar basic composition to the equatorial zonule , then vitreous contamination should lead to values for relative protein abundance that were intermediate between the vitreous and equatorial zonule values .
although such instances were certainly observed , there were also many examples where the abundance of a protein in the hyaloid zonule significantly exceeded ( by orders of magnitude ) that of the vitreous or the equatorial zonule .
such proteins are likely to be unique to , or significantly enriched in , the hyaloid zonule sample .
the set of hyaloid - enriched proteins included opt , a3i3bp , cochlin , and the collagens col2a1 , col11a1 , col5a3 , and col5a2 ( fig .
histograms showing relative abundance of each class of matrisomal proteins in the bovine samples are included as supplementary figures s6s12 .
the ciliary zonule plays a key role in ocular function , centering the lens on the optical axis and transmitting the forces that mold its shape during accommodation .
the zonule is adversely affected in several pathologic conditions , including exfoliation syndrome , homocystinuria , and marfan syndrome . to understand how and why the zonule is compromised in these diseases , a detailed analysis of its composition
microfibrils ( the main structural elements of the zonule ) are ubiquitous components of the ecm but questions remain regarding their organization , synthesis , and role as signaling hubs .
the ciliary zonule , a readily accessible , cell - free system composed of bundled microfibrils , represents a useful model system in which to gain insights into microfibril organization .
the composition of the equatorial zonule was broadly similar in humans and cows , and the set of 52 proteins identified in both samples accounted for > 95% of the mass in either case .
the fact that the human samples were from middle - aged individuals , whereas the bovine samples were from young animals , might in part explain why more proteins were identified in the bovine zonulome ( 176 vs. 144 ) .
the bovine samples were also obtained shortly after death , whereas the human samples were obtained 2448 hours postmortem .
it is possible that labile proteins were lost from the human samples during the postmortem period . in both humans and cows , the zonule consisted of a microfibril backbone comprised primarily of fibrillin and a relatively restricted set of glycoproteins .
ltbp2 , a fibrillin - like protein implicated in autosomal recessive weill - marchesani syndrome type 3 ( wms3 : mim#614819 ) , was a surprisingly abundant component , comprising 10% of the total zonular protein .
there are three fibrillin genes in humans and the presence of all three proteins was confirmed in the zonulome , consistent with earlier observations .
fbn2 , the second most abundant fibrillin in the zonule , is synthesized early in development and is essential for normal zonule formation in mice .
immunolocalization studies have previously failed to detect fbn2 in the adult zonule , but its presence in the human and bovine zonulome suggests that fbn2 epitopes may become inaccessible to antibody staining during postnatal development , as reported in other systems .
several proteins implicated in tgf signaling were detected in the zonule . despite its name , latent tgf binding protein 2 ( ltbp2 ) is not a bona fide tgf-binding protein , but two other family members , ltbp1 and ltbp3 , were detected in the zonulome .
in other systems , ltbp1 and ltbp3 facilitate the sequestration ( and release ) of tgf in the ecm through interaction with fbn1 and other components . of note
another protein with a potential role in modulating tgf signaling was emilin-1 , a prominent component of the human and bovine zonulome .
emilin-1 has been shown to inhibit tgf signaling by binding to pro - tgf precursor and preventing its furin - mediated maturation .
emilins were originally identified as elastic fiber components located at the interface between microfibrils and the underlying elastin core .
more recent studies have suggested that they associate directly with microfibrils . because zonular fibers insert into the lens capsule directly above the lens germinative zone ( the region of the lens epithelium containing the mitotically active cell population ) , tgf or other growth factors sequestered in the zonule are well placed to influence the growth of the lens directly .
it is also possible that the cyclical stretching of the zonule during accommodation might serve to release bound factors from the surface of zonular fibers .
one of the novel ( and abundant ) proteins detected in our study was adamtsl-6 ( thsd4 ) .
adamts proteins and adamtsl proteins have well - documented roles in ocular biology and pathology .
adamtsl-6 has not been identified previously in the zonule but , in other systems , is known to bind to the n terminus of fbn1 , where it may promote microfibril formation . among the proteins that we expected to detect in the zonulome , but did not , was adamts10 ; adamts10 mutations underlie weill - marchesani syndrome type 1 ( wms1 ) .
ectopia lentis is commonly observed in wms1 , implying a mechanical defect in the ciliary zonule .
clinically , wms1 is indistinguishable from wms2 and wms3 , syndromes caused by mutations in fbn1 and ltbp2 respectively , genes encoding two of the most abundant zonular proteins .
immunoelectron microscopy studies have previously suggested that adamts10 is abundant in the human ciliary zonule so its absence from the proteome is perplexing . a striking feature of the zonulome in human and cows was the presence of a diverse set of protease inhibitors , the most abundant in humans being timp3 .
the zonule is known to be exquisitely sensitive to proteolysis . before the introduction of modern cataract surgery
, ophthalmologists removed the cataractous lens in a single piece , a procedure facilitated by brief exposure to dilute solutions of -chymotrypsin , which served to lyse the zonule and release the lens from the eye .
the presence of timp3 and other protease inhibitors may help explain the evident durability of the zonule , despite lifelong exposure to matrix metalloproteinases and other proteases present in the ocular humors .
the hyaloid zonule is intimately associated with the anterior face of the vitreous . during ophthalmic surgery ,
the vitreous face behaves , physically , as if it were a distinct membrane ( the  hyaloid membrane  ) .
ophthalmologists take great care not to damage the hyaloid membrane during surgical procedures , lest the contents of the vitreous leak .
the current study may provide some clues as to the nature of the hyaloid membrane .
first , it is apparent that the hyaloid membrane is reinforced by the presence of a meshwork of fibrillin - rich microfibrils , the hyaloid zonule . during dissection of the hyaloid zonule
it is likely that proteins from the anterior - most region of the vitreous were coisolated .
the hyaloid zonule sample was characterized by the presence of high concentrations of opticin , consistent with previous immunolocalization studies of opticin in human eyes .
our analysis also suggested that the hyaloid sample was enriched in fibrillar collagens ( especially col2a1 , col5a2 , col5a3 , and col11a1 ) , which were detected elsewhere in the vitreous but at much lower concentrations .
interestingly , mutations in col2a1 and col11a1 result in stickler syndrome , a condition characterized by vitreous pathology . in stickler syndrome type 1 ( caused by col2a1 mutations ) , a  membranous vitreous  is often observed ; whereas in stickler syndrome type 2 ( col11a1 ) a  beaded vitreous  is observed . in each case the anterior face of the vitreous appears to be compromised .
thus , the phenotypes in stickler syndrome are consistent with a model in which fibrillar collagens form part of a meshwork ( along with fibrillin - rich microfibrils and opticin ) that defines and supports the vitreous face .
one question arising from the current study is why such an unassuming structure as the ciliary zonule should require a proteome with fifty or more components ?
in other settings , it is evident that microfibril scaffolds , decorated with latent growth factors , serve as both mechanosensors and signaling hubs .
the pleitropic ocular effects of mutations in zonular genes ( which include congenital glaucoma , cataract , ectopia lentis , and microspherophakia ) are consistent with the notion that the role of the ciliary zonule is not solely to secure the lens in place .
 purposethe zonule of zinn ( ciliary zonule ) is a system of fibers that centers the crystalline lens on the optical axis of the eye . 
 mutations in zonule components underlie syndromic conditions associated with a broad range of ocular pathologies , including microspherophakia and ectopia lentis . here 
 , we used hplc  mass spectrometry to determine the molecular composition of the zonule.methodstryptic digests of human and bovine zonular samples were analyzed by hplc  mass spectrometry . 
 the distribution of selected components was confirmed by immunofluorescence confocal microscopy . in bovine samples , 
 the composition of the equatorial zonule was compared to that of the hyaloid zonule and vitreous humor.resultsthe 52 proteins common to the zonules of both species accounted for > 95% of the zonular protein . 
 glycoproteins constituted the main structural components , with two proteins , fbn1 and ltbp2 , constituting 70%80% of the protein . 
 other abundant components were mfap2 , emilin-1 , and adamtsl-6 . 
 lysyl oxidase - like 1 , a crosslinking enzyme implicated in collagen and elastin biogenesis , was detected at significant levels . 
 the equatorial and hyaloid zonular samples were compositionally similar to each other , although the hyaloid sample was relatively enriched in the proteoglycan opticin and the fibrillar collagens col2a1 , col11a1 , col5a2 , and col5a3.conclusionsthe zonular proteome was surprisingly complex . 
 in addition to structural components , it contained signaling proteins , protease inhibitors , and crosslinking enzymes . 
 the equatorial and hyaloid zonules were similar in composition , but the latter may form part of a composite structure , the hyaloid membrane , that stabilizes the vitreous face . 
Materials and Methods
Zonule Dissection
Histology and Immunofluorescence
Zonule Digestion
Vitreous Humor Digestion
Liquid ChromatographyMass Spectroscopy Analysis of Zonule and Vitreous Humor Digests
Bioinformatics Analysis Pipeline
Conversion of RAW Files to MS2 Files.
Database Searching.
Search Results Processing.
Determination of Contaminating Proteins
Results
Comparison Between Human and Bovine Zonular Fibers
Comparison Between the Bovine Equatorial Zonule, Hyaloid Zonule, and Vitreous Humor
Discussion
Supplementary Material