proteomics - grade porcine trypsin , ethanol , high - performance liquid chromatography ( hplc)-grade acetonitrile , formic acid , urea , phosphate buffered saline ( pbs ) , sodium hydroxide , tris(hydroxymethyl)aminomethane ( tris ) , and xylenes were purchased from sigma - aldrich corp .
( st . louis , mo , usa ) . acetic acid , ammonium bicarbonate ,
dithiothreitol ( dtt ) , chloroacetamide ( claa ) , hplc - grade h2o with 0.1% formic acid , hplc - grade h2o , and hplc - grade acetonitrile with 0.1% formic acid were from fisher scientific ( pittsburgh , pa , usa ) .
fresh human lenses were obtained from the national disease research interchange ( philadelphia , pa , usa ) .
three lenses were analyzed in total : 21-year ( m ) , 22-year ( m ) , and 27-year ( f ) .
lenses were stored on wet ice during shipment and fixed immediately upon arrival . a solution of 75:25 ethanol : acetic acid
was used to lightly fix tissue while avoiding traditional formaldehyde crosslinking that can interfere with mass spectrometry experiments .
whole lenses were fixed for 12 hours , manually cut in half across the equator using a razor blade , and fixed for an additional 12 hours .
eight micron sections were cut and collected using a microm hm 325 microtome ( thermo scientific , waltham , ma , usa ) and allowed to dry on glass slides overnight at 37c .
sections were deparaffinized prior to labeling , using a series of xylene and alcohol washes ( xylenes  3 , 100% ethanol , 95% ethanol , 70% ethanol , pbs  5 minutes each ) . to visualize the rz
, lenses were incubated in wheat germ agglutinin - tritc ( life technologies , eugene , or , usa ) , using a 1 mg / ml stock solution at 1:100 dilution for 2 hours , followed by three , 5-minute pbs washes .
sections used for confocal imaging were covered with vectashield mounting medium ( vector laboratories , burlingame , ca , usa ) and sealed with a coverslip .
laser capture microdissection was performed on a palm microbeam system ( carl zeiss microscopy , thornwood , ny , usa ) .
uncovered sections were visualized at 20 magnification using the rhodamine fluorescence setting ( 540/600 nm excitation / emission ) .
regions of interest were manually selected in the software , based on cellular morphology and measured distance from lens capsule .
tissue was cut in brightfield mode and cells from each selected region were catapulted into an eppendorf cap containing 25-l hplc - grade h2o .
microdissection was carried out until 2  10 m tissue was collected for each region to ensure approximately equal starting material . in total , tissue from four regions was collected : df ( outer fiber cell layers , 0100 m from lens capsule ) ; rz ( 100140 m from lens capsule ) ; tz ( 140240 m from lens capsule ) ; ic ( 240 m and beyond ) .
tissue was then spun down into the tube and frozen for side - by - side membrane preparation of all four regions . for each lens ,
a total of three replicates of df , rz , tz , and ic were collected for initial experiments .
collected cells were washed to prepare membranes and to remove soluble proteins , as described previously .
samples were first reduced ( 25 mm dtt , 56c for 1 hour ) and alkylated ( 55 mm claa , room temperature , 45 minutes in dark ) before a series of washing and centrifugation steps to remove soluble proteins .
the sample was vortexed and incubated for 30 minutes in homogenizing buffer containing 8 m urea ( 50 mm ammonium bicarbonate , 5 mm edta , 10 mm naf , 1 mm dtt , ph 8) , followed by 20-minute centrifugation at 100,000 g ( sorvall mtx150 micro ultracentrifuge , thermo scientific ) .
the supernatant was removed and the pellet was saved and washed further using the following procedure : homogenizing buffer containing 8 m urea ( three times total ) , 0.1 m naoh ( one time ) , hplc - grade h2o ( one time ) , 95% ethanol ( one time ) , and hplc - grade h2o ( three times ) . prior to trypsin digestion ,
the pellet was resuspended in 50% tfe in 50 mm ammonium bicarbonate , then diluted to 5% tfe before addition of 1 l of 0.1 g/l trypsin ( pierce ) in a volume of 100 l .
digestion proceeded overnight at 37c and was stopped with the addition of 0.2 l neat formic acid .
for each tissue region , the entire sample was bomb - loaded onto a reverse - phase 360 m outer diameter ( o.d . )  100 m inner diameter ( i.d . )
capillary trap column ( 3 cm length/5 m jupiter c18 beads , 300  , phenomenex ) in - line with a 360 m o.d .
reverse - phase analytical column packed with 20 cm jupiter c18 beads ( 3 m , 300  , phenomenex ) and equipped with a laser - pulled emitter tip . using an eksigent nanolc - ultra hplc system , peptides were eluted at a flow rate of 500 nl / min over a 120-minute gradient of 0.1% formic acid in water ( solvent a ) and 0.1% formic acid in acetonitrile ( solvent b ) . the gradient consisted of 2% to 10% b in 20 minutes , 10% to 30% b in 30 minutes , 30% to 95% b in 15 minutes , 95% b for 15 minutes , followed by equilibration at 2% b. gradient - eluted peptides were mass analyzed on an ltq velos pro linear ion trap mass spectrometer with a nanoelectrospray ionization source ( thermo scientific ) .
full scan ( m / z 3002000 ) spectra were acquired and the top 10 most abundant ions in each ms scan were selected for fragmentation via collision - induced dissociation ( cid ) .
tandem mass spectra were converted into dta files using scansifter and searched using a custom version of sequest ( thermo fisher scientific ) operating on the vanderbilt accre computing cluster .
tandem mass ( ms / ms ) spectra were searched against a concatenated forward and reverse ( decoy ) database containing the homo sapiens subset of uniprotkb sprot protein database ( www.uniprot.org in the public domain ) .
additional search parameters included : trypsin enzyme specificity , monoisotopic masses were used for searching product ions , and oxidation of methionine , carbamidomethylation of cysteine , and phosphorylation of serine , threonine and tyrosine were allowed as variable modifications .
scaffold 4.3.4 ( proteome software , portland , or , usa ) was used to summarize and validate search results , where a minimum probability threshold of 95% was required for peptide identifications and data were filtered to a false - discovery rate ( fdr ) of < 1% at the protein level .
peptide abundance in each region was compared using normalized spectral counts and a student 's t - test was used to determine whether differences between the df and rz were statistically significant ( p < 0.05 ) .
proteins with statistically significant changes near the rz were selected for further quantitation using mrm .
representative peptides for each protein were selected based on their appearance across multiple samples in the discovery phase of analysis described above .
heavy - labeled peptide standards were synthesized by jpt ( spiketides tql peptides , jpt , berlin , germany ) .
peptides contained isotopically labeled terminal arginine or lysine residues ( c and n ) and a trypsin - cleavable c - terminal tag .
peptide standards were used to generate a calibration curve , which confirmed a linear response between 1.5 and 100 fmol/l ( data not shown ) .
after membrane enrichment and before enzymatic digestion , isotopically labeled peptides were spiked into samples at approximately endogenous levels .
skyline software ( university of washington , maccoss lab ) was used to set up scheduled , targeted mrm methods and four to five ms / ms transitions were monitored per peptide .
for targeted proteomics , 6  10 m tissue was collected and membranes were prepared as described above .
prior to trypsin digestion , the pellet was resuspended in 8 m urea ( 100 mm tris buffer , ph 8) , then diluted with 100 mm tris to 2 m urea before addition of 1 l of 0.1 g/l trypsin ( mass spectrometry - grade , pierce ) in a total volume of 10 l .
digestion proceeded overnight at 37c and was stopped with the addition of a small amount of neat formic acid .
the 10 l digest was transferred to a reduced - volume autosampler vial and 2.5 l sample per run was injected via autosampler ( nanoacuity hplc system , waters ) onto a vented column setup utilizing a 40 mm by 0.1 mm ( jupiter 5 micron , 300a ) kasil fritted trap followed by a 200 mm by 0.1 mm ( jupiter 3 micron , 300a ) , self - packed analytical column coupled directly to a tsq - vantage ( thermo scientific ) via a nanoelectrospray source . after trapping and equilibration
, peptides were resolved using a 90-minute aqueous to organic gradient ( solvent a = 0.1% fa in water and b = 0.1% fa in acn ) operating at 400 nl / min .
a series of unscheduled runs determined retention times and the most useful transitions to monitor and then a scheduled instrument method encompassing a 6-minute window around the measured retention time along with calculated collision energies was created using skyline .
q1 peak width resolution was set to 0.7 , collision gas pressure was 1 mtorr , and utilized an ez method cycle time of 3 seconds .
the resulting raw instrument files were imported into skyline for peak - picking and quantitation .
transition or fragment ion peak areas were summed to represent the intensity of endogenous peptides , which were normalized to the internal standard . for statistical analysis ,
a 1-way anova was performed with a post hoc multiple comparison tukey test using spss software ( ibm , armonk , new york , usa ) .
proteomics - grade porcine trypsin , ethanol , high - performance liquid chromatography ( hplc)-grade acetonitrile , formic acid , urea , phosphate buffered saline ( pbs ) , sodium hydroxide , tris(hydroxymethyl)aminomethane ( tris ) , and xylenes were purchased from sigma - aldrich corp .
( st . louis , mo , usa ) . acetic acid , ammonium bicarbonate ,
dithiothreitol ( dtt ) , chloroacetamide ( claa ) , hplc - grade h2o with 0.1% formic acid , hplc - grade h2o , and hplc - grade acetonitrile with 0.1% formic acid were from fisher scientific ( pittsburgh , pa , usa ) .
fresh human lenses were obtained from the national disease research interchange ( philadelphia , pa , usa ) .
three lenses were analyzed in total : 21-year ( m ) , 22-year ( m ) , and 27-year ( f ) .
lenses were stored on wet ice during shipment and fixed immediately upon arrival . a solution of 75:25 ethanol : acetic acid
was used to lightly fix tissue while avoiding traditional formaldehyde crosslinking that can interfere with mass spectrometry experiments .
whole lenses were fixed for 12 hours , manually cut in half across the equator using a razor blade , and fixed for an additional 12 hours .
eight micron sections were cut and collected using a microm hm 325 microtome ( thermo scientific , waltham , ma , usa ) and allowed to dry on glass slides overnight at 37c .
sections were deparaffinized prior to labeling , using a series of xylene and alcohol washes ( xylenes  3 , 100% ethanol , 95% ethanol , 70% ethanol , pbs  5 minutes each ) . to visualize the rz
, lenses were incubated in wheat germ agglutinin - tritc ( life technologies , eugene , or , usa ) , using a 1 mg / ml stock solution at 1:100 dilution for 2 hours , followed by three , 5-minute pbs washes .
sections used for confocal imaging were covered with vectashield mounting medium ( vector laboratories , burlingame , ca , usa ) and sealed with a coverslip .
laser capture microdissection was performed on a palm microbeam system ( carl zeiss microscopy , thornwood , ny , usa ) .
uncovered sections were visualized at 20 magnification using the rhodamine fluorescence setting ( 540/600 nm excitation / emission ) .
regions of interest were manually selected in the software , based on cellular morphology and measured distance from lens capsule .
tissue was cut in brightfield mode and cells from each selected region were catapulted into an eppendorf cap containing 25-l hplc - grade h2o .
microdissection was carried out until 2  10 m tissue was collected for each region to ensure approximately equal starting material . in total , tissue from four regions was collected : df ( outer fiber cell layers , 0100 m from lens capsule ) ; rz ( 100140 m from lens capsule ) ; tz ( 140240 m from lens capsule ) ; ic ( 240 m and beyond ) .
tissue was then spun down into the tube and frozen for side - by - side membrane preparation of all four regions . for each lens , a total of three replicates of df , rz , tz , and ic were collected for initial experiments .
collected cells were washed to prepare membranes and to remove soluble proteins , as described previously .
samples were first reduced ( 25 mm dtt , 56c for 1 hour ) and alkylated ( 55 mm claa , room temperature , 45 minutes in dark ) before a series of washing and centrifugation steps to remove soluble proteins .
the sample was vortexed and incubated for 30 minutes in homogenizing buffer containing 8 m urea ( 50 mm ammonium bicarbonate , 5 mm edta , 10 mm naf , 1 mm dtt , ph 8) , followed by 20-minute centrifugation at 100,000 g ( sorvall mtx150 micro ultracentrifuge , thermo scientific ) . the supernatant was removed and the pellet was saved and washed further using the following procedure : homogenizing buffer containing 8 m urea ( three times total ) , 0.1 m naoh ( one time ) , hplc - grade h2o ( one time ) , 95% ethanol ( one time ) , and hplc - grade h2o ( three times ) .
prior to trypsin digestion , the pellet was resuspended in 50% tfe in 50 mm ammonium bicarbonate , then diluted to 5% tfe before addition of 1 l of 0.1 g/l trypsin ( pierce ) in a volume of 100 l .
digestion proceeded overnight at 37c and was stopped with the addition of 0.2 l neat formic acid .
for each tissue region , the entire sample was bomb - loaded onto a reverse - phase 360 m outer diameter ( o.d . )  100 m inner diameter ( i.d . )
capillary trap column ( 3 cm length/5 m jupiter c18 beads , 300  , phenomenex ) in - line with a 360 m o.d .
reverse - phase analytical column packed with 20 cm jupiter c18 beads ( 3 m , 300  , phenomenex ) and equipped with a laser - pulled emitter tip . using an eksigent nanolc - ultra hplc system , peptides were eluted at a flow rate of 500 nl / min over a 120-minute gradient of 0.1% formic acid in water ( solvent a ) and 0.1% formic acid in acetonitrile ( solvent b ) .
the gradient consisted of 2% to 10% b in 20 minutes , 10% to 30% b in 30 minutes , 30% to 95% b in 15 minutes , 95% b for 15 minutes , followed by equilibration at 2% b. gradient - eluted peptides were mass analyzed on an ltq velos pro linear ion trap mass spectrometer with a nanoelectrospray ionization source ( thermo scientific ) .
the instrument was operated using a data - dependent method with dynamic exclusion enabled . full scan ( m / z 3002000 ) spectra were acquired and the top 10 most abundant ions in each ms scan were selected for fragmentation via collision - induced dissociation ( cid ) .
tandem mass spectra were converted into dta files using scansifter and searched using a custom version of sequest ( thermo fisher scientific ) operating on the vanderbilt accre computing cluster .
tandem mass ( ms / ms ) spectra were searched against a concatenated forward and reverse ( decoy ) database containing the homo sapiens subset of uniprotkb sprot protein database ( www.uniprot.org in the public domain ) .
additional search parameters included : trypsin enzyme specificity , monoisotopic masses were used for searching product ions , and oxidation of methionine , carbamidomethylation of cysteine , and phosphorylation of serine , threonine and tyrosine were allowed as variable modifications .
scaffold 4.3.4 ( proteome software , portland , or , usa ) was used to summarize and validate search results , where a minimum probability threshold of 95% was required for peptide identifications and data were filtered to a false - discovery rate ( fdr ) of < 1% at the protein level .
peptide abundance in each region was compared using normalized spectral counts and a student 's t - test was used to determine whether differences between the df and rz were statistically significant ( p < 0.05 ) .
proteins with statistically significant changes near the rz were selected for further quantitation using mrm .
representative peptides for each protein were selected based on their appearance across multiple samples in the discovery phase of analysis described above .
heavy - labeled peptide standards were synthesized by jpt ( spiketides tql peptides , jpt , berlin , germany ) .
peptides contained isotopically labeled terminal arginine or lysine residues ( c and n ) and a trypsin - cleavable c - terminal tag .
peptide standards were used to generate a calibration curve , which confirmed a linear response between 1.5 and 100 fmol/l ( data not shown ) .
after membrane enrichment and before enzymatic digestion , isotopically labeled peptides were spiked into samples at approximately endogenous levels .
skyline software ( university of washington , maccoss lab ) was used to set up scheduled , targeted mrm methods and four to five ms / ms transitions were monitored per peptide . for targeted proteomics ,
prior to trypsin digestion , the pellet was resuspended in 8 m urea ( 100 mm tris buffer , ph 8) , then diluted with 100 mm tris to 2 m urea before addition of 1 l of 0.1 g/l trypsin ( mass spectrometry - grade , pierce ) in a total volume of 10 l .
digestion proceeded overnight at 37c and was stopped with the addition of a small amount of neat formic acid .
the 10 l digest was transferred to a reduced - volume autosampler vial and 2.5 l sample per run was injected via autosampler ( nanoacuity hplc system , waters ) onto a vented column setup utilizing a 40 mm by 0.1 mm ( jupiter 5 micron , 300a ) kasil fritted trap followed by a 200 mm by 0.1 mm ( jupiter 3 micron , 300a ) , self - packed analytical column coupled directly to a tsq - vantage ( thermo scientific ) via a nanoelectrospray source . after trapping and equilibration , peptides were resolved using a 90-minute aqueous to organic gradient ( solvent a = 0.1% fa in water and b = 0.1% fa in acn ) operating at 400 nl / min .
a series of unscheduled runs determined retention times and the most useful transitions to monitor and then a scheduled instrument method encompassing a 6-minute window around the measured retention time along with calculated collision energies was created using skyline .
q1 peak width resolution was set to 0.7 , collision gas pressure was 1 mtorr , and utilized an ez method cycle time of 3 seconds .
the resulting raw instrument files were imported into skyline for peak - picking and quantitation . transition or fragment ion peak areas were summed to represent the intensity of endogenous peptides , which were normalized to the internal standard . for statistical analysis ,
a 1-way anova was performed with a post hoc multiple comparison tukey test using spss software ( ibm , armonk , new york , usa ) .
although traditional fixation enables excellent antibody and lectin labeling of lens fiber cells , it causes protein - protein crosslinking that can interfere with traditional mass spectrometry proteomic analysis that uses trypsin to cleave at basic residues .
thus , we attempted alternative mass spectrometry - compatible fixation methods that could preserve cellular structure , including the lens capsule and outer cell layers .
fresh human lenses were lightly fixed in 75:25 ethanol : acetic acid for a total of 24 hours .
wheat germ agglutinin - labeled paraffin sections showed the outer cell layers remained intact and cellular morphology could be visualized .
confocal fluorescence microscopy images confirmed the presence of the rz approximately 100 m beneath the lens capsule ( fig .
a return to more ordered cellular organization is visible in the tz ( 140240 m from lens capsule ) where cellular compaction begins .
a test section was processed and analyzed by lc - ms / ms to ensure this fixation did not interfere with trypsin digestion and mass spectrometry ( data not shown ) .
lens was ethanol - fixed and sectioned , followed by labeling with wga - tritc to highlight cell membranes .
the ordered differentiating fiber cells ( df ) rapidly become morphologically more complex in the rz before radial cell columns are again visible in the tz . after
light fixation and wga labeling , lens sections were visualized on the lcm instrument using the fluorescence microscope setting .
regions and approximate measurements included differentiating fiber cells ( df , outer fiber cell layers , 0100 m from lens capsule ) ; rz ( 100140 m from lens capsule ) ; tz ( 140240 m from lens capsule ) ; ic ( 240 m and beyond ) ; 2  10 m of tissue was collected per region , which is equivalent to approximately 80,000 fiber cell cross - sections based on average fiber cell diameter .
after each sample was prepared to isolate integral membrane and membrane - associated proteins , the proteins were digested with trypsin and were analyzed by lc - ms / ms .
shotgun proteomics results identified expected lens proteins , ranging from crystallins to cytoskeletal proteins ( full protein list for 22y lens in supplementary table s1 ) .
compared to conventional tissue homogenization proteomics experiments , total spectral counts from our lcm samples are quite low due to the very small amount of tissue captured and prepared for proteomic analysis .
the small amount of tissue is also likely why naturally abundant lens fiber major intrinsic protein ( aqp0 ) only appears in the rz region at low spectral counts given that trypsin efficiency is likely low for this integral membrane protein . to identify proteins that were changing in the rz
, we initially performed the student 's t - test to compare peptide spectral counts between the df and rz regions .
statistically significant changes were detected for the if protein vimentin , which decreases by approximately 80% from the df to the rz ( fig .
2 ) and decreases further in the tz and ic . a concurrent increase in the beaded filament proteins filensin and phakinin is observed at the rz , suggesting a switch of ifs occurs near the rz . other cytoskeletal proteins including periplakin also change significantly , while spectrins and several crystallins remained fairly constant across the regions analyzed .
these results were used to develop a targeted quantitative mass spectrometry assay for more accurate quantitation of proteins of interest with significant changes at the rz .
* indicates a statistically significant difference detected between the df and rz regions ( p < 0.05 ) .
to more accurately quantify protein changes around the rz , we adopted a targeted proteomics strategy , using mrm for protein quantitation . in this experiment , known concentrations of heavy - labeled isotopes for each peptide of interest that differ in mass from the endogenous peptide but share physiochemical properties and behave similarly in the instrument were spiked into samples .
absolute endogenous peptide concentrations can be calculated from the measured ratio of the endogenous peptide to its heavy - labeled counterpart and the known concentration of the heavy internal standard .
using shotgun proteomics data , one to three representative peptides were chosen for each protein of interest ( supplementary table s2 ) .
peptides were selected based on several criteria , including identification across several mass spectrometry runs and biological samples , absence of labile or modifiable residues like methionine or cysteine , and ideal peptide length ( 720 amino acids ) for detection by mass spectrometry .
standard curves were measured to determine the quantifiable range of the internal standards ( data not shown ) and the concentration of each internal peptide standard was optimized to approximate endogenous peptide levels . for the final analysis
, the 4 regions of interest were isolated from 3 separate lenses ( 21- , 22- , and 27-year )
. a larger amount of tissue ( 6  10 m ) was pooled for each region so technical replicates could be run for each sample .
an example of the mrm signals observed for an endogenous and heavy - labeled filensin peptide is shown in figure 3 .
transitions , or peptide fragmentation patterns , selected for mrm of a filensin tryptic peptide ( 77lgelagpedalar89 ) .
precursor masses for endogenous ( m / z 656.35 , + 2 ) and heavy ( m / z 661.35 , + 2 ) peptides were monitored along with characteristic fragment transitions shown above .
relative intensity and retention time of heavy peptide standard transitions ( a ) are consistent with endogenous peptide ( b ) . targeted ,
quantitative mrm results generally agreed with shotgun proteomics results , providing more confidence and better quantitation of small abundance changes between the four regions .
figure 4 shows the relative intensity of peptides quantified from 3 different lenses ( two technical replicates per lens ) .
intensities are normalized to the region with the highest peptide concentration ; other regions are expressed as a percentage of the highest value .
statistically significant changes were determined using a 1-way anova with tukey test independently for each peptide .
both vimentin peptides decreased 80% from the df to the rz , in agreement with shotgun proteomics data , and continued to decrease in the tz and ic ( fig .
4a ) . periplakin and periaxin follow similar trends , decreasing by 30% to 40% from df to rz and even further in the tz ( figs . 4b , 4c ) .
filensin and phakinin , which make up the beaded filament of the lens , increase at least 2-fold at the rz and show slightly variable but decreasing abundance in the tz with a trend toward increasing abundance in the ic ( figs .
brain acid soluble protein 1 ( basp1 ) follows a similar trend where increasing abundance was observed at the rz compared to the df zone with slightly less protein detected in the tz and ic .
supplementary table s3 shows the absolute quantitation of peptides in each region from three separate lenses , where peptide abundances ( fmol ) calculated represent the peptide quantity in approximately 2  10 m tissue collected . despite some discrepancies in the absolute fmol calculated per lens , which could be due to differences in tissue thickness
differences in the absolute amount of peptides from the same protein could be due to differing digestion efficiency , ionization efficiency , peptide solubility , or the presence of posttranslational modifications that affect peptide m / z and therefore signal for targeted peptide analysis .
each bar represents six measurements ( two technical replicates per lens , three lenses total : 21- , 22- , and 27-year ) . after calculating fmol values for each representative peptide , the region with the highest signal
was set to  1  and other regions expressed as a fraction of that value . a 1-way anova with tukey test was used to determine statistically significant differences between regions .
although traditional fixation enables excellent antibody and lectin labeling of lens fiber cells , it causes protein - protein crosslinking that can interfere with traditional mass spectrometry proteomic analysis that uses trypsin to cleave at basic residues .
thus , we attempted alternative mass spectrometry - compatible fixation methods that could preserve cellular structure , including the lens capsule and outer cell layers .
fresh human lenses were lightly fixed in 75:25 ethanol : acetic acid for a total of 24 hours .
wheat germ agglutinin - labeled paraffin sections showed the outer cell layers remained intact and cellular morphology could be visualized .
confocal fluorescence microscopy images confirmed the presence of the rz approximately 100 m beneath the lens capsule ( fig .
a return to more ordered cellular organization is visible in the tz ( 140240 m from lens capsule ) where cellular compaction begins .
a test section was processed and analyzed by lc - ms / ms to ensure this fixation did not interfere with trypsin digestion and mass spectrometry ( data not shown ) .
lens was ethanol - fixed and sectioned , followed by labeling with wga - tritc to highlight cell membranes .
the ordered differentiating fiber cells ( df ) rapidly become morphologically more complex in the rz before radial cell columns are again visible in the tz .
after light fixation and wga labeling , lens sections were visualized on the lcm instrument using the fluorescence microscope setting .
regions and approximate measurements included differentiating fiber cells ( df , outer fiber cell layers , 0100 m from lens capsule ) ; rz ( 100140 m from lens capsule ) ; tz ( 140240 m from lens capsule ) ; ic ( 240 m and beyond ) ; 2  10 m of tissue was collected per region , which is equivalent to approximately 80,000 fiber cell cross - sections based on average fiber cell diameter .
after each sample was prepared to isolate integral membrane and membrane - associated proteins , the proteins were digested with trypsin and were analyzed by lc - ms / ms .
shotgun proteomics results identified expected lens proteins , ranging from crystallins to cytoskeletal proteins ( full protein list for 22y lens in supplementary table s1 ) .
compared to conventional tissue homogenization proteomics experiments , total spectral counts from our lcm samples are quite low due to the very small amount of tissue captured and prepared for proteomic analysis .
the small amount of tissue is also likely why naturally abundant lens fiber major intrinsic protein ( aqp0 ) only appears in the rz region at low spectral counts given that trypsin efficiency is likely low for this integral membrane protein . to identify proteins that were changing in the rz
, we initially performed the student 's t - test to compare peptide spectral counts between the df and rz regions .
statistically significant changes were detected for the if protein vimentin , which decreases by approximately 80% from the df to the rz ( fig .
a concurrent increase in the beaded filament proteins filensin and phakinin is observed at the rz , suggesting a switch of ifs occurs near the rz . other cytoskeletal proteins including periplakin also change significantly , while spectrins and several crystallins remained fairly constant across the regions analyzed .
these results were used to develop a targeted quantitative mass spectrometry assay for more accurate quantitation of proteins of interest with significant changes at the rz .
* indicates a statistically significant difference detected between the df and rz regions ( p < 0.05 ) .
to more accurately quantify protein changes around the rz , we adopted a targeted proteomics strategy , using mrm for protein quantitation . in this experiment ,
known concentrations of heavy - labeled isotopes for each peptide of interest that differ in mass from the endogenous peptide but share physiochemical properties and behave similarly in the instrument were spiked into samples .
absolute endogenous peptide concentrations can be calculated from the measured ratio of the endogenous peptide to its heavy - labeled counterpart and the known concentration of the heavy internal standard .
using shotgun proteomics data , one to three representative peptides were chosen for each protein of interest ( supplementary table s2 ) .
peptides were selected based on several criteria , including identification across several mass spectrometry runs and biological samples , absence of labile or modifiable residues like methionine or cysteine , and ideal peptide length ( 720 amino acids ) for detection by mass spectrometry .
standard curves were measured to determine the quantifiable range of the internal standards ( data not shown ) and the concentration of each internal peptide standard was optimized to approximate endogenous peptide levels . for the final analysis
, the 4 regions of interest were isolated from 3 separate lenses ( 21- , 22- , and 27-year )
. a larger amount of tissue ( 6  10 m ) was pooled for each region so technical replicates could be run for each sample .
an example of the mrm signals observed for an endogenous and heavy - labeled filensin peptide is shown in figure 3 .
transitions , or peptide fragmentation patterns , selected for mrm of a filensin tryptic peptide ( 77lgelagpedalar89 ) .
precursor masses for endogenous ( m / z 656.35 , + 2 ) and heavy ( m / z 661.35 , + 2 ) peptides were monitored along with characteristic fragment transitions shown above .
relative intensity and retention time of heavy peptide standard transitions ( a ) are consistent with endogenous peptide ( b ) .
targeted , quantitative mrm results generally agreed with shotgun proteomics results , providing more confidence and better quantitation of small abundance changes between the four regions .
figure 4 shows the relative intensity of peptides quantified from 3 different lenses ( two technical replicates per lens ) .
intensities are normalized to the region with the highest peptide concentration ; other regions are expressed as a percentage of the highest value .
statistically significant changes were determined using a 1-way anova with tukey test independently for each peptide .
both vimentin peptides decreased 80% from the df to the rz , in agreement with shotgun proteomics data , and continued to decrease in the tz and ic ( fig .
4a ) . periplakin and periaxin follow similar trends , decreasing by 30% to 40% from df to rz and even further in the tz ( figs . 4b , 4c ) .
filensin and phakinin , which make up the beaded filament of the lens , increase at least 2-fold at the rz and show slightly variable but decreasing abundance in the tz with a trend toward increasing abundance in the ic ( figs .
, brain acid soluble protein 1 ( basp1 ) follows a similar trend where increasing abundance was observed at the rz compared to the df zone with slightly less protein detected in the tz and ic .
supplementary table s3 shows the absolute quantitation of peptides in each region from three separate lenses , where peptide abundances ( fmol ) calculated represent the peptide quantity in approximately 2  10 m tissue collected . despite some discrepancies in the absolute fmol calculated per lens , which could be due to differences in tissue thickness , the trends match very well among the three ages studied .
differences in the absolute amount of peptides from the same protein could be due to differing digestion efficiency , ionization efficiency , peptide solubility , or the presence of posttranslational modifications that affect peptide m / z and therefore signal for targeted peptide analysis .
each bar represents six measurements ( two technical replicates per lens , three lenses total : 21- , 22- , and 27-year ) . after calculating fmol values for each representative peptide , the region with the highest signal
was set to  1  and other regions expressed as a fraction of that value . a 1-way anova with tukey test was used to determine statistically significant differences between regions .
several different methods have been used to study changes in membrane - associated proteins in different lens fiber cell populations .
after gross dissection of whole human lenses , the quantitative proteomics method of itraq was used to assess binding of cytosolic proteins to the plasma membrane with age .
the combination of lcm and lc - ms / ms has also been used previously to analyze differences in the bovine cortical and nuclear lens membrane proteome . here , we isolated extremely narrow regions ( 2030 m ) of tissue for quantitative proteomic analysis of the human lens outer cortex to determine molecular changes associated with the extreme morphologic changes of the plasma membrane in the rz .
these techniques enable spatially resolved proteomics of regions that can not be separated by manual dissection . although we enriched for the plasma membrane fraction , the majority of proteins detected by proteomic analysis were crystallins and cytoskeletal proteins , likely due to their high abundance and reported association with the plasma membrane , even after treatment with urea .
based on these results , we could not assess membrane protein changes and instead focused on cytoskeletal proteins .
targeted proteomics analysis enabled relative quantitation of peptides between different lens regions , even those present at low levels .
intermediate filaments are a highly conserved family of cytoskeletal proteins that can be organized into several classes .
immunohistochemical studies indicate vimentin , a major type iii if protein , is expressed in the lens epithelium and fiber cells , although the expression pattern changes based on lens age and region .
our results indicate vimentin is most abundant in the very outer fiber cells of the df region and its abundance decreases in the rz and further into the lens .
because we analyzed the plasma membrane fraction , this decrease in vimentin signal at the rz could be due to either reduced vimentin expression , increased solubility of the protein , or decreased association with the plasma membrane .
it is possible that loss of vimentin in the rz is due to a shift from membrane to cytoplasm , which has been documented in the literature .
studies of adult mouse lens show membrane - specific vimentin localization in outer cortical fibers , while vimentin antibody labeling is both cytoplasmic and membranous further into the lens before signal is lost around the region of nuclei loss .
similar labeling patterns have also been documented in bovine lens . furthermore , loss of vimentin antibody labeling has been correlated with nuclei loss in mouse lens .
however , we observe a decrease in vimentin signal in the rz , before nuclei are completely lost .
it is possible that the drop in vimentin signal in our experiments could be due to changes in vimentin solubility or membrane association .
deeper into the lens cortex , vimentin is known to be cleaved , which could affect solubility and detection by both immunohistochemistry and mass spectrometry .
phosphorylation of vimentin is known to induce depolymerization of ifs , although this modification was not detected in our studies .
although vimentin knockout does not result in any notable phenotype , vimentin overexpression causes cataract , suggesting that proper expression and regulation of vimentin is critical for lens clarity .
filensin and phakinin are lens - specific proteins that assemble to make 6 to 8 nm beaded filaments .
our results indicate that filensin and phakinin levels increase significantly from the df to the rz , while vimentin decreases .
the switch from vimentin ifs to lens - specific beaded filaments containing filensin and phakinin has been documented in several species including bovine and mouse .
the control of these two if systems appears to be independently modulated during fiber cell differentiation , as beaded filament expression is detected before vimentin labeling is lost .
adult mouse lenses labeled with anti - filensin antibody reveal membraneous labeling in the outer cortex , followed by membranous and cytoplasmic labeling further into the nucleated lens fiber cells . however , filensin is not present in developing embryonic fiber cells until after elongation has progressed , suggesting it plays a role in lens organization after cells have begun differentiation .
the decrease in signal from rz to tz could be due to several factors , including altered solubility or membrane association .
the functional consequences of beaded filament increase in the rz have not been established . however , tem studies indicate ball and socket joints arise in the rz , and knockout studies indicate beaded filaments are critical for maintaining interdigitations and long - range order of lens fiber cells .
therefore , filensin and phakinin may become more associated with the membrane to establish interlocking domains and a beaded filament system that is maintained further into the lens cortex .
the switch from ifs to beaded filaments at the rz suggests an important role for lens - specific ifs .
periaxin is localized to the plasma membrane , concentrated mostly at tricellular junctions where beaded filaments and vimentin labeling also occur .
periaxin is critical for hexagonal geometry and membrane stabilization , perhaps functioning as a scaffolding protein in lens .
periplakin , an if - associated protein ( ifap ) , has been shown to bind both ifs and beaded filaments , directly interacting with vimentin and phakinin .
periplakin and periaxin are also part of the eppd ( ezrin , periaxin , periplakin , desmoyokin ) junction complex in lens . although their roles are not well understood , both periaxin and periplakin could anchor ifs to the plasma membrane through protein - protein interactions .
aqp0 has been demonstrated to interact with beaded filaments , but aqp0 did not pull down with periplakin - bf complexes in previous studies , suggesting there may be another protein anchor connecting periplakin and bf to the plasma membrane .
brain acid soluble protein 1 has been detected in lens but little is known about its function in this tissue . in the brain , the growth - associated protein basp1 is localized to the plasma membrane at the tip of elongating axons .
although the role of basp1 in lens has not been established , the increase in basp1 levels at the rz is not surprising given its function in regulating the actin cytoskeleton in brain .
changes in protein - protein interaction , that is , between periplakin and vimentin , could alter if attachment to the plasma membrane , serving as a way to modulate the lens cytoskeleton and thus influence membrane shape in the rz .
actin polymerization could also be driving the formation of cellular interdigitations that originate in the rz , as branching f - actin and the clathrin / ap-2 complex were shown to associate with interlocking domains in rat and monkey lenses .
although we do not detect major changes in actin or clathrin abundance in our studies , their involvement in rz morphology can not be ruled out .
it is possible that changes in actin polymerization or interactions with other proteins , in addition to basp1 , are important but remain undetected in our quantitative proteomics studies .
additional work is required to understand how these proteins might be involved in membrane reorganization in the rz . although the switch from vimentin to beaded filaments has been documented in lens , this work is the first to show a rapid if change associated with the human lens rz .
our proteomics results do not definitively prove that the if switch is responsible for the morphologic changes of the rz , but suggest the involvement of beaded filaments in establishing the ball and socket joints that originate in the rz .
this notion is supported by the loss of flap structures and long - range fiber cell stacking in the deep cortex of beaded filament knockout mice .
changes to the if system could allow remodeling of the plasma membrane before the fiber cells return to a more ordered arrangement in the tz .
this phenomenon has only been observed in human and macaque lenses , suggesting that accommodation in long - lived primates could require more extensive preparation of interdigitations between lens fiber cells .
future work should examine human lenses with cataract - causing mutations in beaded filament genes to determine whether the formation of the rz is affected .
using spatially resolved proteomics , we characterized protein changes in the narrow rz and surrounding regions of the human lens outer cortex . membrane - associated ifs switch from vimentin to beaded filaments in this region , with changes in other ifaps , suggesting involvement of the cytoskeleton and associated proteins in the dramatic membrane remodeling in the rz .
consistent quantitative results between three separate lenses suggest the rz region is part of a tightly - regulated differentiation process in human lens .
future work is necessary to characterize the function and necessity of the rz for normal human lens physiology .
 purposeto quantify protein changes in the morphologically distinct remodeling zone ( rz ) and adjacent regions of the human lens outer cortex using spatially directed quantitative proteomics.methodslightly fixed human lens sections were deparaffinized and membranes labeled with fluorescent wheat germ agglutinin ( wga - tritc ) . 
 morphology directed laser capture microdissection ( lcm ) was used to isolate tissue from four distinct regions of human lens outer cortex : differentiating zone ( df ) , rz , transition zone ( tz ) , and inner cortex ( ic ) . 
 liquid chromatography - tandem mass spectrometry ( lc - ms / ms ) of the plasma membrane fraction from three lenses ( 21- , 22- , and 27-year ) revealed changes in major cytoskeletal proteins including vimentin , filensin , and phakinin . 
 peptides from proteins of interest were quantified using multiple reaction monitoring ( mrm ) mass spectrometry and isotopically - labeled internal peptide standards.resultsresults revealed an intermediate filament switch from vimentin to beaded filament proteins filensin and phakinin that occurred at the rz . 
 several other cytoskeletal proteins showed significant changes between regions , while most crystallins remained unchanged . 
 targeted proteomics provided accurate , absolute quantification of these proteins and confirmed vimentin , periplakin , and periaxin decrease from the df to the ic , while filensin , phakinin , and brain acid soluble protein 1 ( basp1 ) increase significantly at the rz.conclusionsmass spectrometry - compatible fixation and morphology directed laser capture enabled proteomic analysis of narrow regions in the human lens outer cortex . 
 results reveal dramatic cytoskeletal protein changes associated with the rz , suggesting that one role of these proteins is in membrane deformation and/or the establishment of ball and socket joints in the human rz . 
Experimental Methods
Materials
Sample Preparation and LCM
Membrane Preparation and Shotgun Proteomics
Quantitative MRM
Results
Lens Fixation for LCM and Liquid Chromatography-Tandem Mass Spectrometry (LC-MS/MS)
Shotgun Proteomics
Quantitative MRM
Discussion
Conclusions
Supplementary Material