RESEARCH

Proteomic Analysis of Glycated Proteinsfrom Streptozotocin-Induced Diabetic Rat Kidney

Ashok D. Chougale • Shweta P. Bhat • Swapnil V. Bhujbal •

Mandar R. Zambare • Shraddha Puntambekar • Rahul S. Somani •

Ramanamurthy Boppana • Ashok P. Giri • Mahesh J. Kulkarni

Published online: 23 April 2011

� Springer Science+Business Media, LLC 2011

Abstract Glycation of proteins leading to formation of

advanced glycation end products (AGEs) has been con-

sidered as one of the important causes of diabetic

nephropathy. Therefore, in this study, glycated proteins

were detected by anti-AGE antibodies from kidney of

streptozotocin-induced diabetic rat showing nephropathic

symptoms, by using two dimensional electrophoresis and

western blot analysis. These glycated proteins were iden-

tified and characterized by using combination of peptide

mass finger printing and tandem mass spectrometric

approaches. Glycated proteins identified included proteins

from metabolic pathways, oxidative stress, cell signaling,

and transport. Several of the proteins modified by glycation

were involved in glucose metabolism. The extent of gly-

cation was higher in diabetes compared to control, in the

glycated proteins that were common to both control and

diabetic kidney. Two dimensional electrophoresis proteins

profiling of glycated proteins suggest that four of the gly-

cated proteins were significantly up regulated in diabetes.

Keywords Diabetes � Nephropathy � AGE �Glucose toxicity � Post-translation modification

Introduction

Diabetic nephropathy is one of the major complications

caused by hyperglycemic condition. The condition is char-

acterized by glomerular and tubular basement membrane

thickening, microvascular damage, and mesangial extra-

cellular matrix expansion with the accumulation of impaired

proteins modified by advanced glycation end products

(AGEs). Proteins from extracellular matrix, kidney, as well

as proteins from circulation, get AGE modified and trapped

in kidney [1]. In the diabetic kidney, both intracellular and

extracellular AGEs have been observed. Glucose plays main

role in extracellular AGEs formation, which activates the

RAGEs leading to apoptosis and inflammation [2]. Whereas

intracellular AGEs formation are mostly due to various

dicarbonyls. Eventually, both types of the AGEs may be

responsible for the kidney damage [3]. It was reported that

accumulation of AGEs in the kidney was associated with

increased accumulation of AGE peptides in plasma and urine

in diabetic nephropathy [4]. Such AGE accumulation studied

in diffuse and nodular diabetic nephropathy suggests that

carboxy methyl lysine (CML) was the major AGE detected

in diabetic mesangium, glomerular basement membranes,

tubular basement membranes and vessel walls, while pent-

osidine was the major AGE detected in interstitial collagen

[4]. AGE modification in diabetes significantly increased

quantities of methyl glyoxal, an intermediary compound of

AGE reaction, accompanied with a superoxide induced

oxidative damage of proteins [5]. Adaptive response to

oxidative damage was observed in a proteomic analysis of

glomeruli of db/db diabetic mice. The diabetic mice showed

Electronic supplementary material The online version of thisarticle (doi:10.1007/s12033-011-9409-3) contains supplementarymaterial, which is available to authorized users.

A. D. Chougale � S. P. Bhat � S. V. Bhujbal �M. R. Zambare � S. Puntambekar � A. P. Giri �M. J. Kulkarni (&)

Chemical Proteomics Group, Biochemical Sciences Division,

National Chemical Laboratory (CSIR), Pune 411008, India

e-mail: mj.kulkarni@ncl.res.in

R. S. Somani

Sinhgad College of Pharmacy, Vadgaon, Pune, India

R. Boppana

National Centre for Cell Science, Pune, India

123

Mol Biotechnol (2012) 50:28–38

DOI 10.1007/s12033-011-9409-3

up regulation of antioxidative enzymes peroxiredoxin 1 and

3, glutathione peroxidase 1, and super oxide dismutase 1 [6].

In another proteomic study with diabetes, influence of grape

seed proanthocyanidin extract (GSPE) on proteins involved

in oxidative stress, glycated hemoglobin, and AGE products

was determined. Differentially expressed proteins involved

in oxidative stress, glycosylation damage, and amino acids

metabolism were back regulated to normal levels after GSPE

therapy [7]. This differential expression of proteins could be

due to modification of these proteins by glycation and for-

mation of AGEs. It is important to identify AGE modifica-

tion of proteins and their expression to understand the

molecular mechanism of diabetic nephropathy.

Mass spectrometry has been extensively used to identify

and characterize proteins modified by glycation in both in

vitro and in vivo conditions [8]. In our previous study, we

have used MALDI-TOF-MS to study in vitro glycation of

insulin and albumin [9, 10]. In this study using Anti-AGE

antibodies, we have detected the proteins modified by

AGEs from the kidney of streptozotocin-induced diabetic

rat by using two-dimensional electrophoresis coupled with

western blot analysis. These proteins were characterized

for glycation modification by using peptide mass finger-

printing and tandem mass spectrometric approaches.

Experimental

Chemicals

All chemicals were purchased from Sigma-Aldrich unless

otherwise stated. Bradford protein estimation kit purchased

from the Bio-Rad chemicals. Anti-AGE antibodies obtained

from Millipore, whereas goat anti rabbit IgGs biotin conju-

gate, BCIP (5-Bromo-4-Chloro-30-Indolyphosphate p-Tolui-

dine Salt) and NBT (Nitro-Blue Tetrazolium Chloride)

substrate and streptavidin conjugated alkaline phosphatase

(ALP) were purchased from Bangalore Genei, microalbumin

detection kit was purchased from Chemelex, S.A

Induction of Diabetes and Kidney Sample Collection

All animal experiments were carried out at Sinhgad college

of Pharamacy approved by Institution Animal Ethics

Committee. Diabetes was induced by injecting 55 mg of

STZ/Kg body weight intra-peritoneally to the overnight

fasting Wistar rats. Animals surviving after diabetes

induction were maintained for 100–120 days. The diabetic

status of the rats was monitored by measuring their blood

glucose using glucometer (Entrust, Bayer India) and gly-

cated hemoglobin (HBA1c) using HBA1c analyzer (In2it,

Bio-Rad). Animals with higher HBA1c ([8%) were

selected for kidney collection (Table 1). Kidney tissue was

collected after killing the rats with CO2. The tissue was

washed with cold saline for three to four times and stored at

-80�C.

Microalbuminuria

Microalbumin from rat urine was measured by using the

antibody based kit from Chemelex S. A. according to the

procedure described by the manufacturers.

Histopathology of the Kidney Tissue

After killing the animals by exposing to CO2, kidneys were

removed and immediately placed in 10% formaldehyde.

The kidneys were embedded in paraffin and sectioned

using a microtome to get 5 lm thick sections for histo-

logical studies. Sections were stained with hematoxylin–

eosin stain and observed under microscope at 209

magnification.

Sample Preparation

Kidney was perfused with cold phosphate buffer saline to

remove blood stains prior to homogenization. The tissue

was homogenized to fine powder in liquid nitrogen and

washed two to three times with chilled acetone. Then, the

acetone powder was dissolved in buffer consisting of 7 M

Urea, 2 M Thiourea, 2% CHAPS, 1% DTT, 40 mM Tris,

and centrifuged at 14,0009g for 30 min at 4�C in the

HERMLE centrifuge. The supernatant was collected and

stored in aliquots at -80�C. Protein concentration was

estimated by Bradford method.

Electrophoresis and Western Blotting

Proteins were initially separated by isoelectric focusing, and

second dimension SDS-PAGE of the proteins was done

Table 1 Blood glucose, HbA1C level, and albumin excretion rate of diabetic and control rats

Animal model Body weight (g) Glucose (mg/dl) HbA1 (%) Albumin excretion

rate (lg/24 h)

Control (6) 225 ± 15 60.00 ± 10 4.5 ± 1.8 241.80 ± 71

Diabetic (6) 200 ± 10 371.47 ± 20* 8.5 ± 2.0* 1561.00 ± 37*

Value in parenthesis indicates the total number of rats in the group and (±) indicates the standard deviations. * P \ 0.005

Mol Biotechnol (2012) 50:28–38 29

123

essentially according to Laemmli [11]. Proteins on the gel

were visualized by coomassie brilliant blue (CBB) R-250

staining (Sigma). For protein identification, isoelectric

focusing was carried out by rehydrating 11 cm (pH 3–10) IPG

dry strips (BioRad) with 200 lg of proteins dissolved in

200 ll rehydration buffer (7 M Urea, 2 M thiourea, 40 mM

Tris 1% DTT, and 2% CHAPS) and focused at 8,000 V for

35,000 Vh using Protean IEF Cell (BioRad). Whereas for 2-D

western blot, 7 cm (pH 3–10) IPG dry strips (BioRad) were

rehydrated with 100 lg of protein dissolved in 100 ll rehy-

dration buffer (7 M Urea, 2 M thiourea, 40 mM Tris 1% DTT,

and 2% CHAPS) and focused at 4,000 V for 10,000 Vh using

Protean IEF Cell (BioRad). After isoelectric focusing, the IPG

strips were reduced and alkylated by using equilibration

buffers containing dithiothreitol and iodoacetamide, respec-

tively. Separation in the second dimension was performed on a

12% SDS-PAGE. After electrophoresis, 11 cm gels were used

for CBB staining and 7 cm gels were used for western blot-

ting. Proteins were transferred to PVDF membrane and

incubated overnight at 4�C in blocking buffer containing 2%

milk powder dissolved in 20 mM Tris–Cl (pH 7.5), 0.15 M

NaCl (TBS). The membranes were washed with TBS and

incubated for 1 h at 37�C with diluted (1:5,000) anti-AGE

antibodies (Millipore). After three washes with washing

buffer (TBS containing 0.05% Tween 20), the membranes

were incubated with goat anti rabbit IgGs biotin conjugate

(Bangalore Genei) at a dilution of 1:1,000 for 30 min at room

temperature. Again after three washes with washing buffer,

membranes were incubated with streptavidin-conjugated

alkaline phosphatase at dilution 1:2,000 for 30 min at RT.

Immunodetection was done by adding BCIP/NBT substrate of

ALP in dark.

Image Analysis and In-Gel Digestion

The Coomassie-stained gels (3–10 pH range) were scanned

using a densitometer GS-710 (Bio-Rad Laboratories)

and analyzed using advanced PD-Quest Software version

(Bio-Rad Laboratories). Gel patterns from each independent

analysis were matched and the relative abundance of each

spot was compared between control and diabetic gel. This

analysis was done for three biological replicates. Differen-

tially expressed protein spots were destained with 100 ll of

destaining solution (50 mM ammonium bicarbonate/aceto-

nitrile mixed 1:1 v/v). After thorough rinsing with 100 mM

ammonium bicarbonate, the gel pieces were dehydrated in

100% ACN, which was removed by air drying. The proteins

were in-gel digested overnight at 37�C with 15–20 ll of

20 ng/ll trypsin (Sigma) in 25 mM ammonium bicarbonate

buffer pH 8.4. Peptides were recovered by extraction with

50% ACN5% TFA. Tryptic protein digests were reconsti-

tuted in 50% ACN with 0.1% TFA solvent before subjecting

to mass spectrometric analysis.

Mass Spectrometric Analysis

1 ll of the reconstituted protein digest was premixed with

equal volume of CHCA matrix, vortexed well before

spotting on 96-well MALDI plate. Glu-Fibrinopeptide was

used as an external calibrant (lock mass). Mass spectro-

metric analysis was done on MALDI-Q-TOF (Synapt-

HDMS, M/S Waters). All mass spectra were acquired

200 Hz solid state UV laser in V mode by MassLynx 4.1.

The quadrupole profile was set to 500 m/z for 5% of the

scan time and then ramped to 1,500 m/z for remaining

period of the scan. The instrument mass calibration was

carried with Polyethylene glycol (PEG) MALDI mixture

provided by M/s Waters. Proteins were identified by

MALDI survey method, which uses combination of PMF

and tandem mass spectrometric (MS/MS) approaches for

identification of proteins. In the survey or MS mode, spectra

were recorded in the mass range of 800 to 4,000 m/z for

60 s. MS/MS analysis was done in a data dependent

manner for the top 10 peptides with higher relative inten-

sity for 30 s each. The product ion mass range was set to

100 to 1,500 m/z.

Protein Identification

The resulting MS, MS/MS data were analyzed by Protein

Lynx Global Survey (PLGS) software (Waters, India). Four

spectra were acquired for each protein. The data were

searched against Rattus norvegicus species database

(downloaded from UNIPROT) using PLGS search engine

with a mass tolerance set to 100 ppm and 0.03 Da for MS

and MS/MS, respectively. Fixed modification was set to

carbamidomethylation and variable modification was set to

methionine oxidation. Only those proteins were considered

identified where a same protein was identified by PLGS

with the top score in all four replications. The protein IDs

were examined for sequence coverage, number of peptides

matched agreement between theoretical and experimental

gel MW, and pI values and matching of major peaks of

PMF with the peptides identified in the protein.

PTM Analysis

After the protein identification by PLGS, the spectra were

again subjected to the PLGS search by including glycation

modifications in the search criteria. Modifications were

defined for Schiff’s base or Amodori product (?162.02),

pentosidine (?58.03) and carboxy methyl lysine (?58.02),

pyralline (?108.02), imidazolone (?142.03), crossline

(?252.11), and 1-Alkyl-2-formyl-3,4-glycosyl-pyrrole

(AFGP) (?270.07) as described [12, 13]. Only those pro-

teins were considered identified where a same protein was

identified by PLGS with the top score in all different

30 Mol Biotechnol (2012) 50:28–38

123

searches with different glycation modifications. Further-

more, each PMF spectrum was manually annotated for the

presence of a peak with increased mass caused by glyca-

tion. And the MS/MS spectrum of glycated peptide

(Amodori product or Schiffs base) was annotated for the

neutral loss or any/all of the following ion (M ? H - 18),

(M ? H - 36), (M ? H - 120), (M ? H - 162) accord-

ing to the procedure described by Brancia et al. [14]. The

same approach was also used to annotate the other glyca-

tion modifications.

Quantification of Glycation

Protein glycation was quantified by comparing the actual

number of protein spots detected by anti-AGE antibody in

the western blot analysis. Wherever a same protein was

detected to be glycated in both control and diabetes, the

extent of glycation was quantified by comparing

(1) number of protein specific glycated peptides in

control and diabetes,

(2) the intensity of protein specific glycated peptide in the

PMF spectrum from control and diabetes,

(3) the densities of glycated spots in control and diabetes

in the western analysis.

Statistical Analysis

Statistical analysis was done for following parameters

(a) Intensity of glycation modified peptides:

Heat map analysis was done to study the extent of gly-

cation modification specific glycated peptides in control and

diabetes. The average ion count of individual modified

peptide from three replication was converted to per cent

relative intensity for generation of heat map analysis. Heat

map analysis was done using MeV Multiexperiment Viewer

Version 4.6 according to Saeed et al. [15]. The relative

intensities were color coded from light green to dark red for

lower intensity to higher intensity, respectively.

(b) For protein expression analysis

The CBB stained 2D gels were analyzed by PDQUEST

image analysis software. The density of each protein was

expressed as mean ± SD for three experiments. The sta-

tistical significance was established by Student’s t-test.

Differences were considered significant if P B 0.05.

Results and Discussion

Identification of Glycated Proteins

Glycation of kidney proteins was studied in STZ-induced

diabetic rats having nephropathic symptoms. The histopa-

thology of glomeruli shows the basement membrane

thickening and mesangial proliferation without nodule

formation in diabetic rat kidney (Fig. 1). The HBA1c was

8.5% and albumin excretion rate (AER) was about

1,561 lg/24 h (Table 1) in diabetic rats, whereas in control

rats the HBA1c was 4.5% and AER was 241 lg/24 h.

These tissues were used to identify and characterize gly-

cated proteins by using combination of approaches

involving 2DE, western analysis with anti-AGE antibodies

and mass spectrometry. The 2DE-western analysis led to

detection of 38 well-resolved glycated protein spots in

diabetes and 17 glycated spots in control sample (Fig. 2).

Out of these glycated proteins detected, 12 proteins were

identified in diabetes, and 3 proteins were identified from

control. (Other glycated proteins were not identified as we

did get the same first hit in all the four replications using

PLGS. Henceforth only 12 proteins identified were

Fig. 1 Transverse section of

the kidney glomerulus of

a control rat and b diabetic rat,

indicating basement thickening

Mol Biotechnol (2012) 50:28–38 31

123

considered for this study.) These 12 glycated proteins

identified belong to proteins involved in metabolic path-

ways, oxidative stress, stress proteins, and gene regulation

(Table 2). Glyceraldehyde-3-phosphate dehydrogenase

(GAPDH), malate dehydrogenase (MDH) a-enolase, iso-

citrate dehydogenase, aldehyde dehydrogenase, and pro-

pionyl carboxylase constituted important glycated proteins

involved in metabolic pathways. It was interesting to find

several proteins involved in glucose metabolism were the

targets of glycation. An earlier study has shown that gly-

cation of GAPDH decreases its activity in rabbit muscle

and human erythrocyte [16, 17]. Additionally, it was also

demonstrated that the glycation of GAPDH contributes to

the development of diabetic vascular complications by

activating generation of intracellular advanced glycation

end products [18, 19]. Glycation of isocitrate dehydroge-

nase at its active site lysine residue (K212) reduces the

activity of the enzyme and disturbs the antioxidant system

as it is NADPH dependent enzyme [20]. Similarly, glyca-

tion of mitochondrial isocitrate dehydrogenase was

observed in the kidneys of diabetic rats and in the lenses of

diabetic patients suffering from cataracts, accompanied

with a decrease in the enzyme activity. The glycation-

mediated damage to isocitrate dehydrogenase may result in

the perturbation of cellular antioxidant defense mecha-

nisms and subsequently may contribute to development of

various pathologies associated with long-term complica-

tions of diabetes. The glycation of propionyl CoA car-

boxylase was not known earlier.

Glycation and oxidative stress act synergistically

in developing vascular complications in hyperglycemic

condition [21–23]. In this study, it was observed that the

oxidative enzymes like glutathione S transferase, perrox-

iredoxin and hydroxyacid oxidase were glycated. Glutathi-

one S transferase is an antioxidant protein, whose activity

gets reduced in long-term hyperglycemia. Perhaps, the

decrease in the activity of glutathione S transferase could be

due to glycation [24, 25]. The activity of perroxiredoxin has

been shown to increase in diabetic condition, in our study the

protein was found to be glycated [6]. Glycation of hydroxy

acid oxidase in kidney was also observed in the present study.

Glycation of such proteins largely may affect the oxidative

status of the tissue and leads to the oxidative stress which is

responsible for the damage of tissue.

A B

C D

Fig. 2 Two dimensional

electrophoresis of rat kidney

proteins either stained with

coomassie brilliant blue (a) and

(b), or immunoblotted with

anti-AGE antibodies (c) and

(d) of control and diabetes,

respectively

32 Mol Biotechnol (2012) 50:28–38

123

An elevated level of glucose in diabetes is a stress to the

cells. It has been observed that several stress proteins were

induced in diabetic condition. In this study, stress-induced

proteins such as 78 kDa glucose regulated protein (GRP78)

and a mitochondrial stress 70 protein were found to be

glycated in diabetic condition. These stress proteins act

as molecular chaperones. Crystallin, one of the molecular

chaperones gets glycated in cataract caused by diabetes.

These proteins are known to be involved in folding of

several structural and functional proteins. Glycation of

these proteins may lead to altered or loss of the function

leading to adverse effect on cell metabolism.

Analysis of Glycation Modification

Glycation of proteins leads to formation of different kinds

of AGEs such as carboxy methyl lysine (CML), pentosi-

dine, pyralline, imidazolone B, 1-alky 2 formyl 3-4 glycyl

pyralline (AFGP), and crossline (10). Glucose-derived

Amadori products are thought to be potential precursors of

3-deoxyglucosone (3-DG) in vivo. 3-DG can further react

with proteins to form pyralline or pentosidine. Other AGE,

such as carboxymethyl lysine (CML)-modified adducts of

proteins accumulate readily, and to date, have been iden-

tified as the most prevalent AGE species found in kidney

tissues. High levels of CML and pentosidine, were cor-

related with retinopathy and nephropathy in diabetes

[4, 26]. Formation of the alkyl-2-formyl-3,4-glycosyl-

pyrrole (AFGP) in diabetic condition also reported in the

several studies [27]. Crossline and imidazolone formation

were reported in STZ-induced diabetic rat kidney [26].

Therefore, we further studied the formation of AGEs in the

proteins detected by anti-AGE antibodies in western

analysis (Fig. 2c, d). All the 12 glycated proteins showed

different AGE-modified peptides in PMF analysis (see

supplementary Table 1). These AGE-modified peptides

were identified as additional peptides after initial identifi-

cation of a protein, which did not involve glycation as

variable modification during the PLGS search. Number of

different glycation-modified peptides for each protein is

depicted by a bar graph (Fig. 3). Representative annotation

of PMF spectra of glycated peptides (schiff’s base) for all

the identified protein was shown in supplementary Fig. 1).

Only six proteins were characterized for AGE modifica-

tions by tandem mass spectrometry (see supplementary

Table 1 and Fig. 2). Among 12 glycated proteins in

diabetes, serum albumin was found to be heavily glycated

as observed with 65 different glycation-modified peptides

(see supplementary Table 1). This was followed by

isocitrate dehydrogenase and malate dehydrogenase.

Majority of the glycated proteins showed imidazolone,

crossline, and AFGP as the major glycation modification,

which was followed by CML, pentosidine, and pyrralineTa

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Mol Biotechnol (2012) 50:28–38 33

123

modifications (Fig. 3). In diabetes, human serum albumin

(HSA) was one of the heavily glycated proteins in the

plasma [28]. The biological impact of glycated albumin on

cell physiology and its role as a biological marker of

diabetes was recently reviewed in great detail [29].

Quantification of Glycation

It was natural to observe more number of glycated proteins

in the kidney of STZ-induced diabetic rat. However, in

addition to those specifically glycated proteins in diabetes,

few glycated proteins were also detected to control by

western blot analysis using anti-AGE antibody. These

glycated proteins were GAPDH, serum albumin, and MDH

(Fig. 4). Therefore, in these glycated proteins a comparison

between control and diabetes was made to establish the

extent of glycation. The extent of glycation was measured

by, (a) density of glycated spot in western blot, (b) number

of glycation modified peptides, and (c) intensity of glyca-

tion modified peptide. In the western blot analysis using

anti-AGE antibodies, it was observed that the density of

glycated forms of serum albumin, GAPDH, and MDH was

higher in diabetes than that of control (Fig. 4a). In all these

three glycated proteins, the number of glycated peptides

were found to be more in diabetes than that of its corre-

sponding control protein (Fig. 4b). These glycated pep-

tides included peptides modified with schiff’s base, CML,

pyralline, pentosidine, imidazolone, crossline, and AFGP.

Furthermore, the intensities of different glycation-modified

peptides were compared between control and diabetes. A

representative mass spectra of PMF analysis of GAPDH

control and diabetes was shown in Fig. 5a. The intensities

of encircled glycated peptide (2,547 m/z) clearly suggest

that extent of glycation was higher in diabetes than control.

Fig. 3 Bar graph representing

number of different glycation

modified peptides in each of the

glycated proteins from kidney

of diabetic rat

34 Mol Biotechnol (2012) 50:28–38

123

Intensities of different glycation-modified peptides of these

three glycated proteins were represented in the form of

Heat map analysis. The intensity of the majority of the

glycation-modified peptides was observed to be higher in

diabetes than control (Fig. 5b) (see supplementary data

sheet 1 and 2 for individual intensity glycated peptide)

suggesting that the extent of glycation was higher in dia-

betes in these three proteins, which were found to be gly-

cated even in control kidney tissue. In proteomic profiling

of the non-enzymatically glycated proteins of human

plasma and erythrocytes, human serum albumin (HSA) and

GAPDH were found to be highly glycated proteins in the

plasma and erythrocytes, respectively [28]. Glycation has

been known to inhibit the activity of the GAPDH leading to

the development of diabetic complications [30–32]. It has

been found that GAPDH activity decreased in patients

susceptible to diabetic kidney disease [33]. Similarly, it has

been observed that the enzyme activity of malate dehy-

drogenase is affected by in vitro glycation at a glucose

concentration as low as 5 mM. Glycation inhibition of

MDH activity is rescued by aspirin and alpha crystal-

line [34]. In yeast, enolase is prime target protein for the

glycation [35, 36].

Expression of Glycated Proteins

Image analysis of glycated proteins was carried out to

study their expression levels. Figure 6 (see also

supplementary Fig. 3) and Table 2 depict the differentially

expressed glycated proteins. Four of the glycated proteins

were up significantly regulated (more than 2-folds) in

diabetic conditions. While we did not observe a significant

down regulation among the identified glycated proteins.

These up-regulated glycated proteins (P \ 0.05) include

GAPDH, glutathione S transferase-alpha 1, peroxiredoxin

1, and stress 70 protein. Expression of malate dehydroge-

nase, glutathione s transferase-P, alpha enolase, GRP78,

and propionyl caboxylase did not change much. The pro-

teins that were up regulated in our study were also found to

be up regulated in different studies carried out elsewhere.

GAPDH has been reported to be up regulated in diabetic

rat liver [37]. An increased expression of glutathione

S-transferase was observed in renal proximal tubules in the

early stages of diabetes [38]. In a proteomic analysis of

glomeruli of db/db diabetic mice, peroxiredoxin 1 and 3,

were up regulated in an adaptive response to oxidative

stress [6]. It was interesting to observe the increased levels

of these four glycated proteins in the diabetic conditions.

AGE modification of proteins results in irreversible change

in conformation leading to reduced biological activity of

many important proteins like insulin [39], crystalline [40],

glucose-6-phosphate dehydrogenase [41], aldehyde reduc-

tase [42], glutathione reductase [43], Cu–Zn superoxide

dismutase [44], sorbitol dehydrogenase [45], isotrate

dehydrogenase [20] which were affected by glycation. On

account of AGE modification, cells may up regulate

A B

Fig. 4 a Western blot indicating differential glycation of the glycated proteins found in both control and diabetes and b a bar graph representing

total number of different glycation modified peptides in these proteins

Mol Biotechnol (2012) 50:28–38 35

123

Fig. 5 a Representative peptide

mass finger printing analysis of

glyceraldehyde 3 phosphate

dehydrogenase from control

(i) and diabetes (ii). The

encircled peaks correspond to

mass of glycated peptides.

b Heat map analysis of

intensities of different

glycation-modified peptides of

glycated proteins from control

and diabetes

36 Mol Biotechnol (2012) 50:28–38

123

expression of those glycated proteins to compensate for the

loss of biological activity caused by glycation. In fact,

glycation-induced inactivation of sorbitol dehydrogenase

was accompanied with increased levels of mRNA and

protein in liver of STZ-induced diabetic rats [45]. Alter-

natively, the increased level of such proteins could be due

to formation of protease resistant protein aggregates caused

by AGE modification as well as by decreased proteolytic

activity. It has been observed that the glomerular protein-

ase activity was decreased in the streptozotocin-induced

diabetic rats [46]. Additionally, methyl glyoxal covalently

modifies the 20S proteasome, decreasing its activity in the

diabetic kidney [47]. All these factors may contribute

toward elevated levels of glycated proteins.

In conclusion, we have comprehensively analyzed the

glycation of kidney proteins from STZ-induced diabetic

and control rats. The number of glycated proteins detected

in the diabetic kidney were more than that of control.

Additionally, the extent of glycation was higher in few

glycated proteins that were present in both control and

diabetic kidney. Moreover, some of these glycated proteins

were found to be up regulated in diabetes. Functional

characterization of such glycated proteins may help better

in understanding their role in development of nephropathy.

Acknowledgments This work was carried out as a part of network

project NWP-4 supported by Council of Scientific and Industrial

Research, India. Authors thank Dr. Vidya Gupta, Chair, Biochemical

Sciences, Dr. S. Sivaram and Dr. Sourav Pal, Director, NCL for their

support and encouragement. We also thank Dr. Chetan Gadgil and

Mr. Avinash Ghanate for their inputs in Heatmap analysis. ADC also

thanks Department of Biotechnology, India for the post-doctoral

fellowship.

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