cataract is considered as a major cause of visual impairment in diabetic patients , as the incidence and progression of cataract is increasingly elevated in patients with diabetes mellitus . recently , cataract surgery is an effective and the most common surgical ophthalmic procedure worldwide . however , the elucidation of pathomechanisms to delay or prevent the development of cataract in diabetic patients remains a challenge . both diabetes and cataract pose an enormous health and economic burden , particularly in developing countries , where diabetes treatment is insufficient and cataract surgery often inaccessible . the transparency and stability of the eye lens the crystallins comprise 80%-90% of the soluble lens protein mass and are divided into three basic types termed  ,  , and . crystallin proteins located in the central and equatorial regions of the lens are normally stable and do not turnover , since these cells lose their nuclei during the developmental transition from epithelial to fiber cells . it was previously reported that the development of diabetes cataract should be associated with polyol pathway , osmotic stress caused by sorbitol accumulation , and free radical . however , few researches reported differential proteomic analysis of proteins from diabetic cataractous human lenses . in this study , crystallin proteins in the diabetic and age - related cataractous lenses were extracted and compared with those of normal subjects , using two - dimensional electrophoresis ( 2-de ) , maldi - tof - mass spectrometry ( ms ) , and enzyme - linked immunosorbent assay ( elisa ) analysis . this was carried out to gain further insight into the significant observation of differences of age - related and diabetic cataract . the lenses were obtained from 20 age - related cataract patients ( n = 20 , 65.5  6.7 years ) , 20 type i diabetic cataract patients ( n = 20 , 35.5  7.5 years ) and five normal controls ( donated for corneal transplant in accordance with the standardized rules for development and applications of organ transplants and obtained from the eye bank of shanghai , china . ) . the cataractous lenses were carefully examined by ophthalmologists prior to surgical removal and were all detected with nuclear cataract . lenses of age - related and diabetic cataract were detected with grade iii and grade ii of nuclear cataract , respectively . the prestained and unstained molecular weight protein markers were from invitrogen ( carlsbad , ca , usa ) and amersham biosciences ( piscataway , nj , usa ) , respectively . all chemicals for two - dimensional ( 2d ) gel electrophoresis were from either amersham biosciences or bio rad ( hercules , ca , usa ) . louis , mo , usa ) or fisher ( atlanta , ga , usa ) . monoclonal antibodies against beta - crystallin a3 , alpha - crystallin b chain , and beta - crystallin b1 were purchased from sigma . phacoemulsification was used to remove cortical region and was followed by irrigation and aspiration to remove the nuclear region . the recovered lenses should contain 10% of the original cortex and 90% of the nucleus . all samples were collected and stored at -80c before use . each lens sample ( 20 mg ) was mixed with 1 ml phosphate buffered saline ( 20 mm , ph 7.2 ) , disrupted by sonication , and then centrifuged at 10,000 g for 30 min at 4c . to deplete high content of hyaluronic , which could severely interfere the 2-de process , the supernatant was collected and incubated with 25% prechilled trifluoroacetic acid at 4c for 2 h. the result solution was then centrifuged at 10,000 g for 10 min at 4c . the pellet was washed with prechilled acetone for three times and centrifuged at 10,000 g for 10 min at 4c and then dried in vacuum . then the resulting pellets were dissolved in resolubilization buffer ( 5 m urea , 2 m thiourea , 2% 3-[(3-cholamidoproyl)-dimethyl - ammonio-1-propane sulfonate ] ( chaps ) , 2% caprylyl sulfobetaine 3 - 10,2 mm tri - butyl phosphine , 40 mm tris , ph 8.0 ) and incubated with immobiline dry strips ( ph range of 3 - 10 , amersham biosciences ) overnight at 25c . each preparation was subjected to 2d gel electrophoresis ( ief in the first dimension followed by sds - page in the second dimension ) by exactly following the manufacturer 's handbook ( amersham biosciences ) . following the ief separation , the second dimension sds - page was performed with the laemmli method using a 15% polyacrylamide gel of 16  14 cm ( width  height ) . after the first dimensional ief separation , the strips were consecutively treated for 15 min each , first with 100 mm dithiothreitol ( in equilibration buffer : 0.1 m tris , ph 6.8 , containing 6 m urea , 30% glycerol , and 1% sds ) , and next with 300 mm iodoacetamide ( also dissolved in the equilibration buffer ) . 25 g trypsin was dissolved in 2.5 ml tosylphenylalanylchloromethane mixed with 250 l of 0.1% redistilled acetonitrile . a 15 l sample of this trypsin solution was activated in 100 l 50 mm nh4hco3 . after washed with dh2o for two times , the gel spots were then washed with 200 l of 25 mm ammonium bicarbonate for three times , followed by dehydrated with acn . after dried down in a vacuum centrifuge , the gel - pieces were incubated in the activated trypsin solution for 16 h at 37c . the resulting peptides were extracted three times by 20 ml aliquots of 5% tfa in 50% acn . maldi - tof - ms analysis was performed using autoflex maldi - tof - ms ( bruker , germany ) . the resulting peptides were dissolved in 0.5% tfa and 5% acn and mixed with matrix ( saturated sinapinic acid in acetoritrile / h2o , containing 0.1% tfa , 1:1 , v / v ) to promote desorption and ionisation . an n2 laser at 337 nm was used to desorb the solute molecules from the sample disc and a voltage of 20 kv were established in the source region . the results were subsequently investigated with the matrix science web site ( http://www.matrixscience.com/ ) using  mascot searchpeptide mass fingerprint  to search for homologous proteins in the ncbinr ( national center for biotechnology information nonredundant ) database with the taxonomy set to to detect the concentrations of beta - crystallin a3 , alpha - crystallin b chain , and beta - crystallin b1 in the soluble protein samples from lenses of normal control , diabetic , and age - related cataract patients , the extracted proteins were applied to elisa analysis according to the method previously reported . monoclonal antibodies were first fixed into the plates and reacted with extracted lens proteins and then hrp - antibodies . finally , the mixtures were reacted with tetramethylbenzidine substrate , after termination with 2 m h2so4 , od450 nm was detected for each well using a microtiter plate reader within 30 min . the lenses were obtained from 20 age - related cataract patients ( n = 20 , 65.5  6.7 years ) , 20 type i diabetic cataract patients ( n = 20 , 35.5  7.5 years ) and five normal controls ( donated for corneal transplant in accordance with the standardized rules for development and applications of organ transplants and obtained from the eye bank of shanghai , china . ) . the cataractous lenses were carefully examined by ophthalmologists prior to surgical removal and were all detected with nuclear cataract . lenses of age - related and diabetic cataract were detected with grade iii and grade ii of nuclear cataract , respectively . the prestained and unstained molecular weight protein markers were from invitrogen ( carlsbad , ca , usa ) and amersham biosciences ( piscataway , nj , usa ) , respectively . all chemicals for two - dimensional ( 2d ) gel electrophoresis were from either amersham biosciences or bio rad ( hercules , ca , usa ) . louis , mo , usa ) or fisher ( atlanta , ga , usa ) . monoclonal antibodies against beta - crystallin a3 , alpha - crystallin b chain , and beta - crystallin b1 were purchased from sigma . phacoemulsification was used to remove cortical region and was followed by irrigation and aspiration to remove the nuclear region . the recovered lenses should contain 10% of the original cortex and 90% of the nucleus . all samples were collected and stored at -80c before use . each lens sample ( 20 mg ) was mixed with 1 ml phosphate buffered saline ( 20 mm , ph 7.2 ) , disrupted by sonication , and then centrifuged at 10,000 g for 30 min at 4c . to deplete high content of hyaluronic , which could severely interfere the 2-de process , the supernatant was collected and incubated with 25% prechilled trifluoroacetic acid at 4c for 2 h. the result solution was then centrifuged at 10,000 g for 10 min at 4c . the pellet was washed with prechilled acetone for three times and centrifuged at 10,000 g for 10 min at 4c and then dried in vacuum . then the resulting pellets were dissolved in resolubilization buffer ( 5 m urea , 2 m thiourea , 2% 3-[(3-cholamidoproyl)-dimethyl - ammonio-1-propane sulfonate ] ( chaps ) , 2% caprylyl sulfobetaine 3 - 10,2 mm tri - butyl phosphine , 40 mm tris , ph 8.0 ) and incubated with immobiline dry strips ( ph range of 3 - 10 , amersham biosciences ) overnight at 25c . each preparation was subjected to 2d gel electrophoresis ( ief in the first dimension followed by sds - page in the second dimension ) by exactly following the manufacturer 's handbook ( amersham biosciences ) . following the ief separation , the second dimension sds - page was performed with the laemmli method using a 15% polyacrylamide gel of 16  14 cm ( width  height ) . after the first dimensional ief separation , the strips were consecutively treated for 15 min each , first with 100 mm dithiothreitol ( in equilibration buffer : 0.1 m tris , ph 6.8 , containing 6 m urea , 30% glycerol , and 1% sds ) , and next with 300 mm iodoacetamide ( also dissolved in the equilibration buffer ) . 25 g trypsin was dissolved in 2.5 ml tosylphenylalanylchloromethane mixed with 250 l of 0.1% redistilled acetonitrile . a 15 l sample of this trypsin solution was activated in 100 l 50 mm nh4hco3 . after washed with dh2o for two times , the gel spots were then washed with 200 l of 25 mm ammonium bicarbonate for three times , followed by dehydrated with acn . after dried down in a vacuum centrifuge , the gel - pieces were incubated in the activated trypsin solution for 16 h at 37c . the resulting peptides were extracted three times by 20 ml aliquots of 5% tfa in 50% acn . maldi - tof - ms analysis was performed using autoflex maldi - tof - ms ( bruker , germany ) . the resulting peptides were dissolved in 0.5% tfa and 5% acn and mixed with matrix ( saturated sinapinic acid in acetoritrile / h2o , containing 0.1% tfa , 1:1 , v / v ) to promote desorption and ionisation . an n2 laser at 337 nm was used to desorb the solute molecules from the sample disc and a voltage of 20 kv were established in the source region . the results were subsequently investigated with the matrix science web site ( http://www.matrixscience.com/ ) using  mascot searchpeptide mass fingerprint  to search for homologous proteins in the ncbinr ( national center for biotechnology information nonredundant ) database with the taxonomy set to to detect the concentrations of beta - crystallin a3 , alpha - crystallin b chain , and beta - crystallin b1 in the soluble protein samples from lenses of normal control , diabetic , and age - related cataract patients , the extracted proteins were applied to elisa analysis according to the method previously reported . monoclonal antibodies were first fixed into the plates and reacted with extracted lens proteins and then hrp - antibodies . finally , the mixtures were reacted with tetramethylbenzidine substrate , after termination with 2 m h2so4 , od450 nm was detected for each well using a microtiter plate reader within 30 min . the soluble proteins extracted from the lenses of age - related cataract patients , type i diabetic cataract patients , and normal controls were subjected to 2-de analysis . the 2-de gels [ figure 1 ] showed that most of the soluble proteins of control , diabetic cataract , and age - related cataract lenses were located in the section of ph 5 - 9 with the relative molecular weight at 14,000 - 97,000 da , while relative molecular weights of more abundant crystallines were localized at 20,000 - 31,000 da . two - dimensional electrophoresis analysis of soluble proteins extracted from normal control , age - related cataractous , and diabetic cataractous lens . five protein spots with significantly differential concentrations were pointed with arrows and then recovered and subjected to mass spectrometry analysis in control , diabetic cataract and age - related cataract groups , 73 , 76 , and 68 protein spots were detected , respectively . a total of five significant differential protein spots were selected by the pdquest software [ figure 1 ] . the 2-de analysis revealed that the type i diabetic cataract lenses contained the highest concentrations of proteins in spot 1 , 2 , 3 , and 4 , while control lenses contained the lowest contents of these proteins . additionally , age - related cataract lenses were detected with the highest concentration of the protein in spot 5 . a total of five selected protein spots were extracted from the gels , hydrolyzed with trypsin , and applied to ms analysis . the resulting ms data were investigated with the matrix science online service to search for homologous proteins . peptides of five crystallin and related proteins were detected : the protein in spot 1 was hydrolyzed into 16 peptides and 7 peptides were identical with segments of beta - crystallin a3 , with the matched sequence coverage at 40% [ figure 2 ] ; the protein in spot 2 was hydrolyzed into 26 peptides , and 6 ones were identical with segments of alpha - crystallin b chain , with the matched sequence coverage at 45% [ figure 3 ] ; the protein in spot 3 was hydrolyzed into 10 peptides , and 7 ones were identical with segments of chain a , crystal structure of truncated human beta - b1-crystallin , with the matched sequence coverage at 49% [ figure 4 ] ; the protein in spot 4 was hydrolyzed into 25 peptides , and 6 ones were identical with segments of beta - crystallin b1 , with the matched sequence coverage at 34% [ figure 5 ] ; and the protein in spot 5 was hydrolyzed into 11 peptides , and 5 ones were identical with segments of an interesting unnamed protein product , which is highly similar to alpha - crystallin b chain , containing an alpha - crystallin a chain region at n terminal , and an alpha - crystallin - hsps_p23-like domain , the matched sequence coverage was 49% [ figure 6 ] . mass spectrometry analysis of spot 1 the protein extracted from spot 1 was digested with trypsin and 16 peptides were detected . the protein was then identified as gi|12056461 , beta - crystallin a3 with seven matched peptides mass spectrometry analysis of spot 2 the protein extracted from spot 2 was digested with trypsin and 26 peptides were detected . the protein was then identified as gi|4503057 , alpha - crystallin b chain with six matched peptides mass spectrometry analysis of spot 3 the protein extracted from spot 3 was digested with trypsin and 10 peptides were detected . the protein was then identified as gi|42543230 , crystal structure of truncated human beta - b1-crystallin with seven matched peptides mass spectrometry analysis of spot 4 the protein extracted from spot 4 was digested with trypsin and 25 peptides were detected . the protein was then identified as gi|4503061 beta - crystallin b1 with six matched peptides mass spectrometry analysis of spot 5 the protein extracted from spot 5 was digested with trypsin and 11 peptides were detected . the protein was then identified as gi|194380530 , unnamed protein product with five matched peptides in elisa analysis , the concentrations of beta - crystallin a3 , alpha - crystallin b chain , and alpha - crystallin chain a in the three protein samples from lenses of control , diabetic , and age - related cataract patients were determined . the results revealed that type i diabetic cataract lenses contained much more beta - crystallin a3 , alpha - crystallin b chain , and beta - crystallin b1 than the other two groups ; additionally , the concentrations of the three proteins in age - related cataract lenses were detected as a little higher than those of control subjects [ figure 7 ] . enzyme - linked immunosorbent assay analysis the protein concentrations of beta - crystallin a3 , alpha - crystallin b chain , and beta - crystallin b1 were determined in lenses of normal control , type i diabetic cataract and age - related cataract patients the soluble proteins extracted from the lenses of age - related cataract patients , type i diabetic cataract patients , and normal controls were subjected to 2-de analysis . the 2-de gels [ figure 1 ] showed that most of the soluble proteins of control , diabetic cataract , and age - related cataract lenses were located in the section of ph 5 - 9 with the relative molecular weight at 14,000 - 97,000 da , while relative molecular weights of more abundant crystallines were localized at 20,000 - 31,000 da . two - dimensional electrophoresis analysis of soluble proteins extracted from normal control , age - related cataractous , and diabetic cataractous lens . five protein spots with significantly differential concentrations were pointed with arrows and then recovered and subjected to mass spectrometry analysis in control , diabetic cataract and age - related cataract groups , 73 , 76 , and 68 protein spots were detected , respectively . a total of five significant differential protein spots were selected by the pdquest software [ figure 1 ] . the 2-de analysis revealed that the type i diabetic cataract lenses contained the highest concentrations of proteins in spot 1 , 2 , 3 , and 4 , while control lenses contained the lowest contents of these proteins . additionally , age - related cataract lenses were detected with the highest concentration of the protein in spot 5 . a total of five selected protein spots were extracted from the gels , hydrolyzed with trypsin , and applied to ms analysis . the resulting ms data were investigated with the matrix science online service to search for homologous proteins . peptides of five crystallin and related proteins were detected : the protein in spot 1 was hydrolyzed into 16 peptides and 7 peptides were identical with segments of beta - crystallin a3 , with the matched sequence coverage at 40% [ figure 2 ] ; the protein in spot 2 was hydrolyzed into 26 peptides , and 6 ones were identical with segments of alpha - crystallin b chain , with the matched sequence coverage at 45% [ figure 3 ] ; the protein in spot 3 was hydrolyzed into 10 peptides , and 7 ones were identical with segments of chain a , crystal structure of truncated human beta - b1-crystallin , with the matched sequence coverage at 49% [ figure 4 ] ; the protein in spot 4 was hydrolyzed into 25 peptides , and 6 ones were identical with segments of beta - crystallin b1 , with the matched sequence coverage at 34% [ figure 5 ] ; and the protein in spot 5 was hydrolyzed into 11 peptides , and 5 ones were identical with segments of an interesting unnamed protein product , which is highly similar to alpha - crystallin b chain , containing an alpha - crystallin a chain region at n terminal , and an alpha - crystallin - hsps_p23-like domain , the matched sequence coverage was 49% [ figure 6 ] . mass spectrometry analysis of spot 1 the protein extracted from spot 1 was digested with trypsin and 16 peptides were detected . the protein was then identified as gi|12056461 , beta - crystallin a3 with seven matched peptides mass spectrometry analysis of spot 2 the protein extracted from spot 2 was digested with trypsin and 26 peptides were detected . the protein was then identified as gi|4503057 , alpha - crystallin b chain with six matched peptides mass spectrometry analysis of spot 3 the protein extracted from spot 3 was digested with trypsin and 10 peptides were detected . the protein was then identified as gi|42543230 , crystal structure of truncated human beta - b1-crystallin with seven matched peptides mass spectrometry analysis of spot 4 the protein extracted from spot 4 was digested with trypsin and 25 peptides were detected . the protein was then identified as gi|4503061 beta - crystallin b1 with six matched peptides mass spectrometry analysis of spot 5 the protein extracted from spot 5 was digested with trypsin and 11 peptides were detected . in elisa analysis , the concentrations of beta - crystallin a3 , alpha - crystallin b chain , and alpha - crystallin chain a in the three protein samples from lenses of control , diabetic , and age - related cataract patients were determined . the results revealed that type i diabetic cataract lenses contained much more beta - crystallin a3 , alpha - crystallin b chain , and beta - crystallin b1 than the other two groups ; additionally , the concentrations of the three proteins in age - related cataract lenses were detected as a little higher than those of control subjects [ figure 7 ] . enzyme - linked immunosorbent assay analysis the protein concentrations of beta - crystallin a3 , alpha - crystallin b chain , and beta - crystallin b1 were determined in lenses of normal control , type i diabetic cataract and age - related cataract patients diabetes is a known risk factor for cataract formation . in view of the prevailing and predicted outbreak of diabetes in developing countries like india , diabetic cataract may become a leading cause of blindness , along with age - related cataract . in the present study , we comparatively analyzed the separated protein spots on 2-de gels of normal control , age - related , and diabetic cataract lenses . no distinction was made in this study regarding changes between the cortical and nuclear regions of these lenses . in 2-de and ms analysis , five significantly differential protein spots were detected , including beta - crystallin a3 , alpha - crystallin b chain , chain a of crystal structure of truncated human beta - b1-crystallin , beta - crystallin b1 , and an unnamed protein product . the gels revealed that type i diabetic cataract lenses should contain much more beta - crystallin a3 , alpha - crystallin b chain , chain a , crystal structure of truncated human beta - b1-crystallin and beta - crystallin b1 than the other two groups . and in elisa analysis , type i diabetic cataract lenses were also proved to contain more contents of beta - crystallin a3 , alpha - crystallin b chain , and beta - crystallin b1 than age - related cataract and control lenses [ figure 7 ] . it was interesting that a significant protein spot was detected in age - related cataract lenses ; it was not entitled and could only be identified from a cdna sequence cloned from human hippocampus . it was highly similar to alpha - crystallin b chain , containing an alpha crystallin a chain region at n terminal , and an alpha - crystallin - hsps_p23-like domain , seldom mentioned in cataract , and its role and principle in cataract were still unclear . however , this protein might be helpful in the further research on the mechanism of age - related cataract . as previously reported , beta - crystallin oligomers were suggested to play a critical role in maintenance of lens transparency ; and the truncation of beta - b1-crystallin was associated with its structure and stability . and diabetic may enhance nh2 - and cooh - terminal extensions of beta - b1-crystalline , which could accelerate lens opacity and oligomer formation . in our study , higher contents of beta - b1-crystalline and truncated human beta - b1-crystallin were determined in diabetic cataract lenses , and it was consistent with early reports . alpha - crystallin was reported to constitute the major portion of eye lens cytoplasm and its concentration in the lens could reach up to 50% of the total proteins ; it displayed chaperone - like activity in suppressing the aggregation of various proteins and in preventing inactivation of enzymes due to heat and other stress conditions . alpha - crystallin , especially alpha - crystallin b , was also known to undergo extensive posttranslational modifications including oxidation , mixed disulfide formation , truncation , and glycation during aging , and was believed to be the key structural and functional element for maintaining the transparency of the lens . furthermore , it was known that the chaperone activity of -crystallin is compromised in various types of cataract , including diabetic cataract . it was reported that alpha - crystallin from diabetic rat and human lenses had shown a substantial loss in their chaperone function , in addition , -crystallin chaperone activity was also found to be impaired in galactosemic rat lenses . owing to the loss of its chaperone function , more alpha - crystallin should be expressed by the surround cells and tissues ; therefore , in this research , significantly higher alpha - crystallin b chain concentration could be detected in type i diabetic lenses . additionally , the contents of beta - crystallin a3 and was also studied in age - related cataracts lenses . in this study , proteins extracted from control , type i diabetic , and age - related cataracts lenses were applied to 2-de , ms , and elisa analysis . type i diabetic cataract lenses should contain the highest contents of beta - crystallin a3 , alpha - crystallin b chain , beta - crystallin b1 , and chain a of crystal structure of truncated human beta - b1-crystallin . compared with control subjects , age - related cataracts lenses revealed slightly higher concentrations of the four proteins above ; additionally , gi|194380530 , an unnamed crystalline - like protein was also detected in this group with significantly higher content . furthermore , the identification of these differential proteins could help to diagnose the certain types and grades of nuclear cataract in clinical research and treatment , and could also bring benefits in the further research on the principle of cataract . the lens samples could only be extracted and studied through surgery after cataract was formed and detected ; therefore , to reveal the pathogenesis in the early stage of cataract , more attention could be devoted to gene research . to monitor the expression levels of the genes for these differential proteins
backgound : to investigate the differential lens proteomics between diabetic cataract , age - related cataract , and natural subjects.materials and methods : two - dimensional electrophoresis ( 2-de ) , mass spectrometry ( ms ) , and enzyme - linked immunosorbent assay ( elisa ) were employed . 
 total soluble proteins in lenses of type i diabetic cataract , age - related cataract ( nondiabetic ) patients , and normal control were extracted and subjected to 2-de . 
 the differential protein spots were recovered , digested with trypsin , and further applied to maldi - tof - ms . 
 elisa analysis was used to determine the levels of differential proteins in lenses of three groups.results:2-de analysis reflected that lens proteins of normal control , diabetic , and age - related cataract subjects were in the section of ph 5 - 9 and the relative molecular weights were 14 - 97 kda , while relative molecular weight of more abundant crystallines was localized at 20 - 31 kda . 
 five differential protein spots were detected and identified using maldi - tof - ms , including beta - crystallin a3 , alpha - crystallin b chain , chain a of crystal structure of truncated human beta - b1-crystallin , beta - crystallin b1 , and an interesting unnamed protein product highly similar to alpha - crystallin b chain , respectively . 
 elisa analysis revealed that lenses of diabetic cataract patients should contain significantly more concentrations of beta - crystallin a3 , alpha - crystallin b chain , and beta - crystallin b1 than those of age - related cataract patients and normal control.conclusion:this study clearly reflected the differential proteins of diabetic cataract , age - related cataract lenses compared with natural subjects , and it is helpful for the further research on the principles and mechanisms of different types of cataract .