A Proteomic Approach to Identify Metalloproteins and Metal-Binding Proteins in Liver from Diabetic Rats

International Journal of Biological Macromolecules 96 (2017) 817–832

Contents lists available at ScienceDirect

International Journal of Biological Macromolecules

j ournal homepage: www.elsevier.com/locate/ijbiomac

A proteomic approach to identify metalloproteins and metal-binding

proteins in liver from diabetic rats

a,∗ a b b

Camila Pereira Braga , José Cavalcante Souza Vieira , Ryan A. Grove , Cory H.T. Boone ,

c c a

Aline de Lima Leite , Marília Afonso Rabelo Buzalaf , Ana Angélica Henrique Fernandes ,

b a

Jiri Adamec , Pedro de Magalhaes Padilha

Department of Chemistry and Biochemistry, Institute of Bioscience of Botucatu, São Paulo State University, Botucatu, SP, Brazil

Department of Biochemistry, University of Nebraska-Lincoln, Lincoln, NE, USA

Bauru Dental School, University of São Paulo- USP, Bauru, SP, Brazil

a r a

t i b s

c l e i n f o t r a c t

Article history: Proteins play crucial roles in biological systems, thus studies comparing the protein pattern present in a

Received 13 September 2016

healthy sample with an affected sample have been widely used for disease biomarker discovery. Although

Received in revised form

proteins containing metal ions constitute only a small proportion of the proteome, they are essential in a

10 December 2016

multitude of structural and functional processes. The correct association between metal ions and proteins

Accepted 21 December 2016

is essential because this binding can signiﬁcantly interfere with normal protein function. Employment of

Available online 3 January 2017

a metalloproteomic study of liver samples from diabetic rats permitted determination of the differential

abundance of copper-, selenium-, zinc- and magnesium-associated proteins between diabetic, diabetic

Keywords:

treatment with insulin and non-diabetic rats. Proteins were detected by ESI–MS/MS. Seventy-ﬁve dif-

Electrospray ionization-tandem mass

spectrometry ferent proteins were found with alterations in the metal ions of interest. The most prominent pathways

Flame atomic absorption spectrometry affected under the diabetic model included: amino-acid metabolism and its derivates, glycogen stor-

Graphite furnace atomic absorption age, metabolism of carbohydrates, redox systems and glucose metabolism. Overall, the current methods

spectrometry employed yielded a greater understanding of metal binding and how type 1 diabetes and insulin treat-

Metalloproteomic

ment can modify some metal bonds in proteins, and therefore affect their mechanism of action and

Type 1 diabetes function.

Two-dimensional electrophoresis

1. Introduction Copper (Cu) is an essential component of many metalloen-

zymes, including cytochrome oxidase, lysyl oxidase, superoxide

Type 1 diabetes mellitus (DM1) is a severe metabolic disorder dismutase, dopamine-ß-hydroxylase and tyrosinase [4]. Superox-

and a public health concern. Clinical complications of DM1 include ide dismutase (SOD) is a metalloprotein that contains Cu and zinc

damage and dysfunction of multiple organs with secondary compli- (Zn) that catalyzes the decomposition of the superoxide anion.

cations, such as atherosclerosis, retinopathy and renal insufﬁciency Therefore, it is a component of the cellular defense system protect-

[1]. ing against oxidative damage. Cu is a vital component of electron

Although metal ions comprise a small proportion of body tis- transfer reactions of SOD, underlying its antioxidant function [5].

sues (4%), they are essential as structural components and in many Experimental data shows that magnesium (Mg) is required

life processes. The main roles of these metal ions can be described as an important cofactor in many enzyme reactions. Importantly

as structural and functional [2]. Metal ions bound to proteins for DM1, Mg participates in phosphorylation reactions of glucose

and metalloproteins represent a large portion of the total protein. metabolism [6]. It is essential in almost all energy transduction sys-

These ions are responsible for many metabolic processes, such as tems in the glycolytic pathway and in oxidative energy metabolism,

energy conversion in photosynthesis and respiration, gene regula- which is required for the synthesis and oxidation of fatty acids,

tion, substrate activation and other catalytic processes, transport protein synthesis, muscle contraction and ATPase activity [7].

and storage [3]. Additionally, Mg participates in the intracellular signaling system,

phosphorylation and dephosphorylation reactions that activate or

inhibit a multitude of enzymes [8].

∗ Selenium (Se) is a nonmetal, essential micronutrient for the

Corresponding author.

E-mail addresses: braga [email protected], braga [email protected] (C.P. Braga). synthesis of selenoproteins, which play an important role in the

http://dx.doi.org/10.1016/j.ijbiomac.2016.12.073

818 C.P. Braga et al. / International Journal of Biological Macromolecules 96 (2017) 817–832

−1

synthesis, metabolism and action of thyroid hormones. Further- a protein pellet with 0.50 mol L NaOH. The total protein concen-

more, Se modiﬁes the expression of selenoproteins, including the tration of the liver samples was determined by the Biuret method

families of peroxidases, selenoenzymes, glutathione and thiore- using bovine serum albumin as the standard. Analytical calibration

−1

doxin [9]. Zn is a component of synthetases and transferases, curves were constructed with concentrations of 10–100 g L from

−1

including DNA and RNA, digestive enzymes, and associates with a stock solution of albumin (100 g L ). The method used 50 mL of

insulin. Zn also participates in metabolic pathways involving pro- sample for the standard and 2.5 mL of Biuret reagent, which was

◦

tein synthesis, carbohydrates, lipids and nucleic acids. In addition, mixed and placed in a water bath at 32 C for 10 min. After 5 min

zinc also plays a role in apoptotically driven programmed cell death at room temperature, absorbance readings were performed in a

[10]. spectrophotometer at a wavelength of 545 nm.

Studies have reported alterations in metal ions by diabetes mel-

litus and suggested that the imbalance of speciﬁc elements may

2.3. Electrophoretic runs (2D-PAGE)

play an important role in normal glucose and insulin metabolism

[11,12]. In the majority of studies, the focus is on analyzing concen-

With the standardization of the gels, the electrophoretic runs

trations and on the status of a single element or combinations of

were performed using liver samples for the different experimen-

elements in blood, plasma or tissues; in our study, the focus is the

tal groups. Six gels were made for each group (pooled). Aliquots of

on the integration of traditional analytical studies with inorganic −1

pooled liver were diluted in a urea solution containing 7 mol L ;

and biochemical studies. For this study, we used a robust method- −1

2 mol L thiourea, 2% (w/v) CHAPS (3-[(3-cholamidopropyl)-

ology to assist in understanding the variability of Cu, Mg, Se and

dimethylammonio]-1-propanesulfonate), 0.5% (v/v) ampholytes at

Zn in diabetes and how treatment with insulin can alter this condi-

a pH ranging from 3 to 10, 0.002% (w/v) bromophenol blue and

tion, providing valuable information about how Cu, Mg, Se and Zn

2.8 mg of dithiothreitol (DTT) were added to this buffer, and the

are distributed with proteins, as well as the individual concentra-

mixture was used in the electrophoretic separations.

tion in speciﬁc proteins, which helps to elucidate the physiological −1

A total of 3 ␮g ␮L protein was added to 13 cm strips contain-

and functional aspects of biomolecules in the liver.

ing polyacrylamide gel with ampholytes immobilized at pH 3–10.

In this context, the present study involves the investigation

These strips were placed onto a ﬁrst dimension focusing for 12 h

Cu, Mg, Se and Zn found in liver samples from diabetic rats

at room temperature to be rehydrated with the protein extract.

using ﬂame atomic absorption spectrometry (FAAS) and graphite

After this period, the strips were placed into an Ettan IPGphor iso-

furnace atomic absorption spectrometry (GFAAS), by protein frac-

electric focusing (IEF) unit (GE Healthcare) for the ﬁrst-dimension

tionation (two-dimensional gel electrophoresis, 2D-PAGE) and

separation, after the strips were reduced for 10 min with a solu-

identiﬁcation by electrospray ionization-tandem mass spectrom- −1

tion containing 6 mol L urea, 2% (w/v) SDS, 30% (v/v) glycerol,

etry (ESI–MS/MS). −1

50 mmol L Tris-HCl, 0.002% (w/v) bromophenol blue and 2% (w/v)

DTT and alkylated for 10 min with a similar solution, but with DTT

2. Material and methods replaced by 2.5% (w/v) iodoacetamide (IAA).

For the second dimension, the strips were placed onto a 10%

␮

2.1. Animals and experimental groups polyacrylamide gel; a piece of ﬁlter paper with 10 L of a molec-

ular weight standard (12–225 kDa range) was placed close to the

A total of 24 Wistar male rats (Rattus norvegicus), 45 days strip in the gel, and they were sealed with a hot solution of 0.5%

old, were used in the experiment; these were kept in individ- (m/v) agarose. The second dimension of the electrophoretic run

ual plastic cages with a controlled temperature and photoperiod, was divided into two stages: 7.5 mA/gel for 30 min and 20 mA/gel

and they received water and a commercial diet (Purina , Labina, for 6 h 40 min.

Campinas-SP) ad libitum throughout the experimental period. The After the ﬁrst and second dimensions in the electrophoretic

experimental design was approved by the Ethics Committee on runs, the proteins in the gels were ﬁxed in the gels using a staining

the Use of Animals (CEUA) at the Institute of Biosciences/São solution containing 10% (v/v) acetic acid and 40% (v/v) ethanol for

Paulo State University (UNESP), Botucatu, Brazil (protocol: CEUA- 1 h. In the sequence, the proteins were revealed with a colloidal

436/2012). The animals were divided into three groups (n = 8): C Coomassie stain: 8% (m/v) ammonium sulfate, 1.6% (v/v) phospho-

(control group): normal rats, DM1: diabetic rats and DM1 + I: dia- ric acid, 0.08% (m/v) Coomassie blue G-250 and 25% (v/v) methanol

betic rats that received insulin. Diabetes mellitus was induced with for 72 h. Afterwards, the Coomassie stain was removed and the gels

the administration of streptozotocin (STZ; 60 mg/body weight, sin- were washed with ultrapure water. The gels were scanned using an

gle dose, i.p.). Blood glucose was measured 48 h after the STZ ImageScanner III (GE Healthcare), and the images were analyzed by

administration, and animals with glycemic levels greater than ImageMaster 2D Platinum 7.0 (GeneBio, Geneva, Switzerland).

−1

220 mg dL were considered diabetic. The animals received insulin

in the form of Humulin N100UI neutral protamine Hagedorn (NPH;

® 2.4. Copper, magnesium, selenium and zinc mapping by FAAS or

Lilly ) with an initial dose of 3 U/animal; this dose was adjusted

GFAAS

or maintained so that serum glucose levels were kept within the

normal range. At the end of the 30 day experimental period, the ani-

The copper, selenium and zinc in the protein spots identiﬁed

mals were anesthetized (ketamine hydrochloride 10%, 0.1 mL/100 g

were determined by GFAAS, and magnesium was determined by

body weight, i.p.) and sacriﬁced by decapitation, and the liver was

FAAS after mineralizing the samples (spots and feed) as described

collected.

by Moraes et al. [13]. The analyses used two different elec-

trophoretic runs, and gels were obtained in duplicate for each

2.2. Sample preparation run. The copper, magnesium, selenium and zinc determinations

were performed with a Shimadzu AA-6800 atomic absorption spec-

Approximately 1.00 g of pooled sample (liver) was weighed in trometer using wavelengths of 324.7, 285.2, 190.0 and 213.9 nm,

triplicate and ground with 2 mL of ultrapure water, macerated, and respectively. Copper, selenium and zinc operated with a cur-

the protein extracts were separated from the solid portion by cen- rent of 400 mA and magnesium with a current of 10 mA. The

◦

trifugation at 10,000g at 4 C for 10 min in a refrigerated centrifuge. analytical curves were prepared using Merck Titrisol standard

The protein extracts were used to quantify protein resuspension in solutions. The curves for copper and zinc were constructed in

C.P. Braga et al. / International Journal of Biological Macromolecules 96 (2017) 817–832 819

−1

concentration ranges of 5.00–20.00 ␮g L ; for magnesium, the knowledge about reactions, pathways and biological processes.

−1

range was 0.10–0.40 mg L and for selenium the range was Pathways with a false discovery rate (FDR) < 0.05 were considered

−1

10.00–60.00 ␮g L . The region of the gel where no protein spots to be signiﬁcantly enriched.

appeared was used for the analytical blank. The limits of quantiﬁ-

cation (LOQ) for copper, magnesium, selenium and zinc are 0.046,

−1 3. Results and discussion

0.94, 0.083 and 0.023 ␮g L , respectively.

3.1. Protein separation by 2D-PAGE

2.5. Characterization of protein spots by ESI–MS/MS

The total protein concentrations in the pool of pellets in the C,

The protein spots were extracted from the gels using a scalpel, −1

3 DM1 and DM1 + I groups were 131.12, 92.86 and 105.79 g L in

cut into segments of approximately 1 mm and prepared for MS −1

the total extract and 26.09, 20.05 and 21.79 g L precipitated with

according to Shevchenko et al. [14] with some modiﬁcation. The

acetone, respectively. The pellets obtained with the acetone pre-

subsequent procedure with the segments was described by the

cipitation were used to calculate the volume of protein needed to

Waters’ Technical Bulletin, which can be summed up in four steps: −1

obtain 3 ␮g ␮L of protein to apply to the IEF strips. After stan-

a) dye removal (destained with 25 mM ammonium bicarbonate

dardization of the protocol to be followed, the gels obtained from

(Ambic)/acetonitrile (ACN) (50:50 v/v), and after destaining, the

different experimental groups were scanned and analyzed using

fragments were dehydrated with two ACN baths for 10 min and

ImageMaster 2D Platinum 7.0. The gels were compared in pairs

dried at room temperature); b) reduction and alkylation (rehy-

◦ because the imaging is a laborious process. After the identiﬁca-

drated with 20 mM DTT in 50 mM Ambic for 40 min at 56 C; after

tion of equivalent spots through the matching process, we obtained

time, the excess reagent was removed and 55 mM IAA in 50 mM

results of the correlation between the pairs of gels. The correlation

Ambic was added for 30 min at room temperature); c) tryptic diges-

◦ −1 between the gel replicates, encompassing the three electrophoretic

tion of proteins (incubated overnight at 37 C with 10 ng ␮L

runs (six gels from each group studied), were, on average in the C,

trypsin in 25 mM Ambic for 15 min; Trypsin Gold Mass Spectrom-

DM1 and DM1 + I groups, 86%, 79% and 91%, respectively.

etry, Promega, Madison, USA) and d) elution of peptides (extracted

Correlation between the protein spots from gels was deter-

from gel by the addition of extraction buffer) A − 50% ACN with 1%

◦ mined, considering the percentage normalized volume (%V) during

formic acid − to each tube and incubated for 15 min at 40 C under

the matching process. The results obtained were as follows: G1 and

sonication; the supernatant was collected and transferred to a new

G2 (R > 0.75), G1 and G3 (R > 0.90) and G2 and G3 (R > 0.76). Through

tube, and this step was repeated with the extraction of buffer B −

the correlation analysis, we can see that the proteomic proﬁle of the

60% methanol with 1% formic acid − and extraction buffer C − 100%

C group was closer to the proteomic proﬁle of the DM1 + I group

ACN; the extracts were dried in a vacuum centrifuge and peptides

than to the proﬁle of the DM1 group. Most spots were distributed

were dissolved in 10 ␮L 3% ACN with 0.1% formic acid. Aliquots

in the molecular weight (Mw) range of 31–76 kDa with isoelec-

of solutions containing peptides were analyzed by obtaining the ∼

tric points (pI) = 6; in general, the Mw and pI distributions were

mass spectra using the nanoACQUITY UPLC-Xevo QT-MS (Waters,

homogeneous among the different experimental groups.

Manchester, UK) system. Data was acquired over 20 min, and the

scan range was 50–2000 Da. ProteinLynx Global Server (PLGS) ver-

sion 3.0 was used to process and search the continuum LC–MS 3.2. Determination of Cu, Mg, Se and Zn in the spots

data, setting carbamidomethylation of cysteines as the ﬁxed modi-

ﬁcation and oxidation of methionines as the variable modiﬁcation, The concentration of Cu, Mg, Se and Zn does not provide a lot

allowing one missing cleavage and a maximal error tolerance of of information without characterizing the proteins in these protein

10 ppm [15]. The identiﬁcation of proteins was performed using spots. Thus, we converted the estimate of the protein mass in the

the UniProt database (UniProtKB/Swiss-Prot − www.uniprot.org), protein spots and the Cu, Mg, Se and Zn [18,19]. Fig. 1A shows the

and the search was conducted for the species Rattus norvegicus. protein spots containing Cu, Mg, Se and/or Zn; Fig. 1B–D shows the

The UniProt protein IDs were converted to gene symbols in order qualitative analyses that were used to identify the percentage of

to analyze them using Reactome Functional Interaction (FI) [16], a spots with Cu, Se, Mg and Se. In the 75 spots analyzed, the C group

Cytoscape plugin [17]. Reactome uses a comparison with published showed the highest percentage of Cu, the DM1 group showed the

Fig. 1. A. Gel obtained by 2D-PAGE (pH 3–10) for liver tissue. The numbers indicate spots where Cu, Mg, Se and/or Zn as detected and the proteins characterized by ESI–MS/MS.

B. Qualitative analysis of Cu, Mg, Se and Zn in the C group. C. Qualitative analysis of Cu, Mg, Se and Zn in the DM1 group. D. Qualitative analysis of Cu, Mg, Se and Zn in the

DM1 + I group.

820 C.P. Braga et al. / International Journal of Biological Macromolecules 96 (2017) 817–832

highest percentage of Se and the DM1 + I group showed the highest stimulated reactive oxygen species (ROS) [25], which could explain

percentage of Mg. why most protein spots had Se presence, because most Se available

Se exerts a beneﬁcial inﬂuence on health, including the pre- can bind with proteins and interfere in their function and conse-

vention of cancer and neurodegenerative diseases, actuating the quently in their pathways.

immune system and mainly in providing antioxidant capacity The feed was analyzed for Cu, Mg, Se and Zn in order to exclude

through selenoproteins [20]. The literature reports that a number the role of feed in the results of this work. We found the following

−1 −1

of metals (copper, zinc, vanadium and cadmium) have ions that are concentrations: Cu: 2.36 mg kg , Mg: 28.90 mg kg , Se: < LOQ and

−1

capable of eliciting insulin mimetic effects by activating the insulin Zn: 51.40 mg kg . Thus, the feed was observed to be a major source

signaling cascade [21]. Recent epidemiological and intervention of Mg and Zn.

studies related high Se levels to hyperglycemia and dyslipidemia; Table 1 shows the Cu, Mg, Se and Zn concentrations determined

for example, a study conducted using the US National Health and in each protein spot with their respective Mw and pI and the protein

Nutrition Examination Surveys associated a high serum selenium mass measured using ImageMaster 2D Platinum 7.0 software.

concentration with an increased prevalence of diabetes [22,23],

and the French SUVIMAX trial population associated positive cor-

3.3. Protein spot analysis by ESI–MS/MS

relations between plasma Se and fasting plasma glucose [24]. The

effect of Se in carbohydrate metabolism is controversial, and the

This study analyzed 75 spots of protein and found 114 different

adverse effect on the insulin-regulated metabolic pathway could

proteins that were associated with the presence of Cu, Mg, Se and/or

be a fake redox paradox that facilitates insulin action by an insulin-

Zn in the C, DM1 and DM1 + I groups, shown in Table 2. Reactome

Table 1

Values for copper, selenium and zinc concentration, determination by GFAAS and magnesium by FAAS in the protein spots for liver in different experimental groups.

C.P. Braga et al. / International Journal of Biological Macromolecules 96 (2017) 817–832 821

Table 1 (Continued)

FI was used to ﬁnd the pathways of these 114 proteins that were bound enzyme (site–site interactions) and by inducing inactivation

considered signiﬁcantly enriched (FDR < 0.05); the UniProt protein by a slow, tight-binding inhibitor of the free enzyme [27]. The cys-

IDs were converted to gene symbols. Considering FDR < 0.05, 35 teinyl residue at position 273 of the enzyme has been identiﬁed

pathways were considered to be signiﬁcantly enriched (Fig. 2), as a metal ligand [28]. In this study, the presence of Cu was found

and the majority were related to the metabolism of amino acids in the C group and Cu, Mg, Se and Zn were found in the DM1 + I

and derivates (19 genes), glycogen storage diseases (13 genes), group. Cu, Mg and Se have not yet been reported as binding with

metabolism of carbohydrates (13 genes), biological oxidations (12 OTC; it can be suggested that in DM1 + I inactivation of this enzyme

genes) and glucose metabolism (12 genes). by hyperglycemia control is induced by insulin. Arginase-1 (AGR1)

was found in spot ID 79, and sorbitol dehydrogenase (SORD) was

also found in this spot. AGR1 is a binuclear manganese (Mn) metal-

3.3.1. Amino acid metabolism

loenzyme that catalyzes the hydrolysis of arginine to ornithine and

Carbamoyl phosphate synthase [ammonia] (CPS1) is ATP- and

urea [29], and SORD is a Zn metalloenzyme (a tetramer containing

nucleotide-binding, and it is involved in the urea cycle (con-

one Zn atom/subunit) involved in the catalysis of sorbitol/fructose

trols the entry of ammonia into the urea cycle). In the literature,

interconversion [30]. Some alterations in this conversion are associ-

there are no reports that this enzyme has a speciﬁc metal-binding

ated in diabetes with cataract formation, neuropathy, retinopathy

property, but there is a report that it can be inactivated by a

and nephropathy [30]. The results showed in spot 79, the pres-

mixed-function oxidation binding a divalent metal and generally

ence of Zn in C, Se in DM1 and Cu, Mg and Zn in DM1 + I. No

requiring a nucleotide [26]. However, in this work, the most impor-

other previous reports have shown that ARG protein binds with

tant ﬁnd was in the DM1 + I group, Cu and Zn were found; as these

Cu, Mg and Zn, and SORD with Cu and Mg. Beta-ureidopropionase

elements are divalent, it may be inferred that binding with Cu

(UPB1), 4-hydroxyphenylpyruvate dioxygenase (HPD) and fumary-

and Zn can cause the inactivation of this enzyme in the DM1 + I

lacetoacetase (FAH) were identiﬁed in spot 77, where the presence

group associated with the control of hyperglycemia by insulin and

of Mg was found in C, Se in DM1 and Cu, Se, Mg and Zn in DM1 + I.

a decrease in the production of ammonia. Ornithine carbamoyl-

UPB1 is a metalloenzyme that binds two Zn ions per subunit [31],

transferase (OTC) is another enzyme involved in the urea cycle (it

HPD is a metalloenzyme that binds one Fe cation per subunit [32]

catalyzes the formation of L-citrulline from carbamoyl phosphate

2+ 2+

and FAH binds with Ca and Mg [33]. Other proteins associ-

and L-ornithine). A study reported that this enzyme is regulated by

ated with this metabolism such as S-adenosylmethionine synthase

Zn in two different ways: as an allosteric cofactor of the substrate-

822 C.P. Braga et al. / International Journal of Biological Macromolecules 96 (2017) 817–832 Zn Zn

Cu Cu, Cu Cu

= = = =

I I I I Mg Se Se Mg Zn Se Mg

and and

+ + + +

Se;

Zn; Zn; Zn; Zn;

Mg Mg =

and and and and and and and

and DM1 DM1 DM1 DM1

and and and and Cu Cu Cu Cu Cu, Cu Cu Cu, Cu

Zn Se ======DM1

Se; Se I I I Se I Se I Se I Se; Se; I I Se; I

= = + + + = + = + = + = = + + = +

Mg Mg and

Mg,

Mg;

DM1 C DM1 DM1 DM1 DM1 DM1 DM1 DM1 DM1 DM1 DM1 Cu, DM1 DM1 DM1 DM1 and Mg and DM1 DM1 presence

glycine glycine

stress

and and

and

metabolism

Lipid pyrimidine metabolism metabolism metabolism Glycerol metabolism Metabolism associated Amino-acid metabolism Chaperone Choline Choline Creatine metabolism Amino-acid metabolism Valine Amino-acid metabolism Oxidative Mehionine metabolism Transport via

organic

and

pathway

hydrogen

activit

transmembrane

process

to hypoxia,

degradation kinase hormone and

biosynthesis Nucleotide biosynthesis transferase

response regulation compounds

iron

cycle cycle

cyclic peptide S-methylmethionine- homocysteine S-methyltransferase activity Biological Urea Response Stress Protein glycerol Oxireductase Oxireductase Kinase, Urea Oxidoreductase Betaine-homocysteine S-methyltransferase activity, S-adenosylmethionine- homocysteine S-methyltransferase activity ferric Protein redox peroxide, glycerol transporter

(%)

Coverage 10.95 1.82 7.14 10.74 12.21 21.51 24.72 5.51 36.65 35.14 6.06 14.76 15.11 16.76

13 84 74 82 44 79 79 31 33 44 29 65 53 65 Peptides

(Da,

zinc.

Mw theoretical) 164,580 106,037 94,057 106,790 57,477 101,440 101,440 43,045 46,496 57,808 39,929 76,395 80,576 76,395 and/or

(theo-

retical) pI 6.33 7.44 5.12 5.41 5.49 6.15 6.15 6.58 7.63 8.47 8 7.14 6.5 7.14 selenium

653 94 177 2849 1633 1633 7429 1104 1514 6304 2238 717 Score 935 magnesium,

3 2

= =

1 3

1 1 SV SV

= SV

= Aars Ass1

Cps1 Hspa4

= =

1 Rattus copper, 1

= =

SV = = =

PE PE

Pld2) 131

2 norvegicus

1 GN GN GN GN

PE PE synthase = OS = PE

= cytoplasmic

Rattus

1 Rattus 1 Rattus Rattus Rattus

= PE

Rattus Rattus

PE = protein = = = = synthase

(GN

= =

ligase Rattus

Gk Aldh1l1 Aldh1l1 Sardh Aldh6a1 Bhmt Tf Prdx6

SV OS OS OS [acylating] OS =

presence

D2 ======OS OS

ligase

kDa

OS = mitochondrial

70 norvegicus norvegicus norvegicus norvegicus GN GN GN GN GN GN GN GN the

1 1 3 1

dehydrogenase PE

= = = = kinase

with SV SV SV SV

shock Tars

Rattus Rattus Rattus Rattus 3 3 1 1 1 1 2

======

liver

norvegicus norvegicus OS OS OS Protein Carbamoyl-phosphate Phospholipase Heat Alanine–tRNA Glycerol Cytosolic 10-formyltetrahydrofolate dehydrogenase Cytosolic 10-formyltetrahydrofolate dehydrogenase Sarcosine mitochondrial Argininosuccinate Methylmalonate-semialdehyde dehydrogenase mitochondrial Betaine–homocysteine S-methyltransferase Serotransferrin Threonine–tRNA cytoplasmic Peroxiredoxin-6 OS norvegicus PE PE [ammonia] SV SV SV norvegicus norvegicus norvegicus GN norvegicus norvegicus PE PE in

entry

ESI–MS/MS

Protein P07756 P70498 O88600 P50475 Q63060 P28037 P28037 Q64380 P09034 Q02253 O09171 O35244 Q5XHY5 P12346

identiﬁed

Spot 4 27 16 24 20 8 21 10 11 14 15 17 22 Table Proteins

C.P. Braga et al. / International Journal of Biological Macromolecules 96 (2017) 817–832 823 Se

Mg Cu, Cu

= = =

Se I I I Mg Se

and

+ + +

Zn; Zn; Zn;

Mg, Mg and and

DM1 DM1 DM1

and and and Cu Cu Cu, Cu Cu,

Zn = = = = =

Se I Se; I I Se I Se; Se I Se;

= + = + + = + = = + =

Se Zn Mg and

DM1 C DM1 DM1 DM1 DM1 DM1 DM1 DM1 DM1 DM1 DM1 and and Mg and

Chaperone Amino-acid metabolism Chaperone Transport Transport Chaperone Chaperone Other Other Carbohydrate metabolism Carbohydrate metabolism Chaperone Chaperone Chaperone Chaperone Chaperone Chaperone Chaperone Chaperone

of and

process

mRNA cycle

process

response, acid

transcription acid

ion toxic interleukin-4,

activit cellular

transmembrane

to catabolic

regulation

stress

iron amino apoptotic

processing, response response response response response response response response

transport iron

acid

storage

regulation transcription, splicing, ferric Response chaperone, Stress cellular mRNA Stress Iron Transport cellular Tricarboxylic Glycolysis Toxin Stress Stress Stress Stress Stress Stress fatty negative response neuron substance homeostasis transporter

14.49 19 30.66 18.14 15.65 17.03 17.68 27.87 20.07 16.01 10.52 17.94 9.54 26.91 16.54 9.83 6.19 6.32 40.09

55 69 67 91 36 50 57 16 50 60 47 46 49 52 51 48 50 50 50

76,395 73,858 83,281 84,815 51,351 70,871 69,642 20,749 68,731 81,623 71,615 62,200 60,647 72,347 70,549 70,550 70,871 69,642 70,871

7.14 5.97 4.96 4.93 7.58 5.37 5.50 5.98 6.09 7.6 6.75 6.45 6.23 5.07 5.91 5.91 5.37 5.50 5.37

3792 93 2569 8726 897 894 894 3813 2081 633 245 709 98 182 932 220 531 3023 107 2 1

3 B 2

= 2 =

= =

SV SV = Pck1 Hspa9 Hspa8 Hspa5 Hspa1a Hspa8

alpha SV protein protein SV SV

protein 1-like 1A/1

Rattus SV

= subunit 1

Rattus

3 protein

Rattus

1 SV = [GTP]

= 1 =

= 90-beta 90-alpha

norvegicus

= 2

GN GN GN GN GN GN

kDa kDa

kDa 2 =

OS PE PE

OS SV

PE PE

OS subunit PE

= Rattus

PE 1

Rattus

71 71

1 1 70 norvegicus HSP HSP

Rattus Rattus

SV 1 1

= PE

protein protein = =

mitochondrial =

= = = =

Rattus Tf Aco2 Ftl1 Alb Pcca Sdha Pklr

PKLR

1 OS SV

= OS cytosolic SV

= PE

carboxylase

======

OS OS

kDa kDa =

ﬂavoprotein

norvegicus chain

Rattus =

PE protein protein cognate 70 70 cognate norvegicus norvegicus norvegicus norvegicus norvegicus norvegicus norvegicus norvegicus norvegicus GN GN GN GN GN GN GN L protein =

3 1 1 1 2 1

dehydrogenase hydratase protein PE

PE kinase

======

light OS

glucose-regulated albumin

mitochondrial

Rattus SV SV SV SV SV SV

shock shock shock shock shock shock shock-related = Hsp90ab1 Hsp90aa1 Hpx Cct3 Hspa1 Hspa2

Rattus Rattus Rattus Rattus Rattus Rattus Rattus Rattus Rattus

1 1 1 1 2 1

======kDa ======

[ubiquinone] GN GN OS norvegicus PE PE PE PE PE norvegicus norvegicus Serotransferrin Aconitate mitochondrial Heat Heat Hemopexin Stress-70 Heat Ferritin Serum Propionyl-CoA Phosphoenolpyruvate carboxykinase Succinate mitochondrial Pyruvate T-complex 78 Heat Heat Heat Heat OS OS OS OS OS OS norvegicus PE gamma chain 2 norvegicus GN GN GN OS norvegicus norvegicus OS GN

P12346 Q9ER34 P20059 P02793 P02770 P14882 P48721 P07379 P34058 Q920L2 P12928 Q6P502 Q07439 P63018 P14659 P06761 P82995 P63018 P55063

33 36 38–39 31 28 32 35 41 37

824 C.P. Braga et al. / International Journal of Biological Macromolecules 96 (2017) 817–832 Se Se

Zn;

Cu, Cu, Cu, Cu,

= = = =

I I I I

and and

and

+ + + +

Se Mg Mg

= Zn

and DM1 DM1 DM1 DM1

Cu, Cu,

Zn Se Se = =

Zn and Se; I Se; I Se; Se;

DM1

= + = + = =

Se and and

Mg,

Mg Se,

and

= =

C DM1 DM1 DM1 C Cu, DM1 DM1 DM1 Se Mg Mg, Mg presence stress stress

metabolism metabolism metabolism metabolism

Proline Carbohydrate metabolism Metabolism associated Chaperone Chaperone Chaperone Carbohydrate metabolism Proline Chaperone Carbohydrate metabolism Proline Proline Histidine metabolism Other Oxidative Oxidative Lipid Lipid Lipid Lipid

hydrolase hydrolase hydrolase hydrolase

proline

process process, reactive reactive

heat

ester ester ester ester and

process

to to

species species

Transferase response response response response Chaperone transport

biosynthetic Biological Stress Stress Stress Stress Glycolysis/gluconeogenesis Response Oxidoreductase glycolysis/gluconeogenesis pathway Carboxylic Carboxylic Carboxylic Carboxylic Toxin Glycolysis Response Oxidoreductase Glutamate Oxidoreductase Lyase, phosphocreatine biosynthetic oxygen oxygen activity activity activity activity response

(%)

Coverage 12.64 19.66 12.64 5.5 37.01 21.14 30.10 17.79 19.07 11.23 11.68 5.35 11.01 14.98 21.49 33.78 20.43 24.57 36.97 5.51

50 49 51 52 44 42 33 44 54 57 36 39 49 46 42 46 42 20 46 31 Peptides

(Da,

Mw 69,642 70,550 70,549 72,347 61,403 59,757 61,869 61,403 62,308 62,495 62,147 61,715 60,647 62,200 59.757 61.849 66,093 61.849 58,914 43,045 theoretical)

(theo-

retical) pI 5.50 5.91 5.91 5.07 6.3 7.07 7.13 6.3 6.29 6.25 6.10 5.64 6.23 6.45 7.07 7.13 7 7.13 5.79 6.58

627 2270 1043 2938 7256 3042 453 12867 4463 590 657 8595 4785 426 372 Score 199 2128 63 345 305 2 1 2 2 2

= = 2 = B = =

SV SV SV SV SV SV Rattus Rattus

Rattus

Gnmt Hspa8 Hspa1a Cbs

SV 2 1 protein = = 1 1 1

= protein

1A/1 1-like

= = 1 Rattus = = =

Rattus

1 1 1

= OS OS = OS

= = = GN GN GN PE PE GN 4 B-1 kDa PE PE PE

kDa

PE OS subunit

= OS

norvegicus norvegicus norvegicus

SV SV SV

70 norvegicus

3 3 3 SV 1

protein protein

1 1 1

SV = = = 1D 1E 2

= = = mitochondrial mitochondrial mitochondrial Pgm1 Pgm1

Ces1d Ces1e Ckm

1 M-type SV =

= = SV SV SV

kDa kDa beta-synthase =

PE PE PE

Rattus Rattus Rattus

norvegicus

PE 1 1 1

Rattus

= = = PE cognate 70 70

norvegicus norvegicus norvegicus norvegicus norvegicus norvegicus norvegicus norvegicus norvegicus norvegicus GN GN GN GN GN L = = = protein =

1 2 3 2

= = = = kinase OS OS OS

PE PE PE

N-methyltransferase

Rattus SV SV SV SV

carboxylesterase carboxylesterase shock shock shock-related shock Aldh4a1 Aldh4a1 Aldh4a1

= Hspa2 Hspa1 Cat Cct3 Cat Cat

Rattus Rattus Rattus Rattus Rattus Rattus Rattus Rattus Rattus Rattus

1 2 1 1

= = =

======

OS GN dehydrogenase OS OS OS norvegicus norvegicus OS PE Heat Heat Heat Heat Phosphoglucomutase-1 Catalase Phosphoglucomutase-1 Cystathionine Liver Liver Carboxylesterase Carboxylesterase T-complex Catalase Delta-1-pyrroline-5-carboxylate dehydrogenase Catalase Delta-1-pyrroline-5-carboxylate dehydrogenase Glycine Creatine norvegicus norvegicus OS 2 gamma OS OS OS GN GN GN GN GN OS GN GN GN norvegicus PE PE PE

entry Protein

9 Delta-1-pyrroline-5-carboxylate 9 9

× × ×

) P38652 P13255 P00564 P63018 Q64573 Q63010 P16303 Q63108 P0C2 P0C2 P32232 P38652 P04762 P04762 P14659 P55063 Q6P502 P0C2 Q07439 P04762 Continued (

ID Protein

Spot 42 43 46 47 45 49 44 Table

C.P. Braga et al. / International Journal of Biological Macromolecules 96 (2017) 817–832 825 Se Se Zn

Cu Cu,

= =

Se I I Mg, Mg

and and

and Se

+ +

= Se;

Se; Zn; Se I Se;

Cu, Mg Mg, Mg

and

= + =

= Zn

I and DM1 DM1

and

Cu Cu, Cu, Cu, +

= = = =

and DM1 DM1 DM1 Mg I Se; I Cu I Se; Se I

= + = + = + = = +

Se Zn Zn Se DM1

Zn; Zn; Zn;

= = =

DM1 C C DM1 DM1 DM1 C DM1 Zn; DM1 DM1 DM1 DM1 and and and Mg, stress stress

metabolism metabolism metabolism

metabolism

Amino-acid metabolism Lipid Energy Amino-acid metabolism Amino-acid metabolism Energy Other Other Other Other Oxidative Energy Oxidative Chaperone Transport Carbohydrate metabolism Carbohydrate metabolism Carbohydrate metabolism

sulfur process process process

catabolic

allosteric

metabolic Hydrogen

Ion carnitine process

metabolic process, -methionine -methionine -methionine

degradation, biosynthesis; biosynthesis; l l l

polyamine homeostasis

Transport homeostasis homeostasis

catabolic

thymine uracil

transporter metabolism;

and metabolism

L-methionine

synthesis, redox catabolic redox redox

transport,

from biosynthesis; S-adenosyl- biosynthesis biosynthesis biosynthesis; and ion microtubule-based microtubule-based Cell sulfur energy beta-alanine cell ATP cell protein gglycolysis/gluconeogenesis gglycolysis/gluconeogenesis gglycolysis/gluconeogenesis amine amino-acid amino-acid amino-acid oxidoreductase, biosynthetic S-adenosyl- S-adenosyl- metabolism regulation process, L-alanine process, transport,

43.37 24.32 26.53 41.39 18.88 12.9 33.47 68.62 14.09 5.77 38.71 19.59 26.96 18.42 17.74 21.16 6.58 19.81

30 30 53 37 39 44 40 36 34 31 31 29 29 40 36 31 32 44

49,801 49,671 56,623 60,806 60,806 56,815 46,301 56,354 48,173 52,532 47,128 47,014 47,141 53,653 55,110 43,698 43,716 61,416

4.79 4.78 5.88 6.41 6.41 6.77 8.06 5.18 5 5.1 6.16 7.8 5.03 6.57 6.08 5.61 5.93 8.05

5873 417 5409 1751 120 188 305 2776 5530 2236 2108 1690 3974 10854 4701 4457 1377 5832 2 1 1 2 2

4 3 2 2 = = =

= =

= = = =

1 SV SV SV

SV SV

SV SV SV SV = A3 A6 Pdia3 Suox Gsr Pdia6 Gpt Rattus

1 1 1

1 SV 1 =

1 Rattus = = = = = 1 1 1 1 1 =

1 = =

= Rattus =

1 PE

= = = = =

= =

PE PE PE OS synthase synthase PE PE GN GN GN GN GN

(Fragment)

PE PE PE PE beta OS

Rattus

OS PE Rattus

Rattus

Rattus Rattus =

Rattus Rattus Rattus

protein = =

Rattus

= = =

chain

OS Tubb4b Tubb5 Ftcd Dpys Atp5b Eno1 Eno3 Eno2 Aldh9a1 Mat1a Mat2a Glud1 =

mitochondrial OS B OS OS chain

======

OS PE OS OS

subunit OS

reductase

norvegicus norvegicus norvegicus norvegicus norvegicus norvegicus GN GN GN GN GN GN GN GN GN GN GN GN dehydrogenase

2 2 2 2 2

= = = = =

type-1 type-2 beta–4 beta-5 aminotransferase disulﬁde-isomerase disulﬁde-isomerase

oxidase

SV SV SV SV SV

Selenbp1

Rattus Rattus Rattus Rattus Rattus Rattus 1 1 synthase 1 1 2 1 1

======

norvegicus norvegicus SV norvegicus Tubulin Tubulin Formimidoyltransferase- cyclodeaminase Protein Sulﬁte Dihydropyrimidinase Glutathione ATP mitochondrial Protein Selenium-binding Alpha-enolase Beta-enolase Gamma-enolase 4-trimethylaminobutyraldehyde dehydrogenase Alanine S-adenosylmethionine S-adenosylmethionine Glutamate mitochondrial norvegicus OS OS norvegicus OS OS norvegicus isoform isoform norvegicus norvegicus PE PE PE PE PE OS OS GN norvegicus SV norvegicus norvegicus norvegicus

Q6P9T8 Q8VIF7 P10860 P25409 P10719 P13444 P69897 Q07116 Q63150 P04764 Q63081 O88618 P07323 Q9JLJ3 P18298 P11598 P70619 P15429

50 70 62 68 60 67 56 65

826 C.P. Braga et al. / International Journal of Biological Macromolecules 96 (2017) 817–832 Zn Se Zn Zn

Mg, Cu, Cu,

= =

Zn Se Se I I and

and

Mg, and and

Cu,

+ +

Zn = Se; =

Se Se, I Se, Se; Se;

Se; I Se Se

and

Mg, Mg, Mg =

and

= + = = =

= +

Zn Zn;

and

and DM1 DM1

Cu, Cu, Cu Cu, Mg Mg Mg, Mg,

Zn Se ======

DM1

DM1 DM1 DM1 DM1 DM1 Zn and DM1 and

DM1 I Se; I Mg I I I I Se; I I

+ = + = + + + + = + +

Zn Zn Zn Se and

Mg,

Mg, Mg Zn; Cu; Cu; Cu; Cu, Cu; Cu;

and

======

DM1 C DM1 Se DM1 C C C DM1 DM1 DM1 DM1 Cu, C C C C and and Mg, Mg DM1 DM1 DM1 DM1 and presence C

metabolism metabolism

Carbohydrate metabolism Amino-acid metabolism metabolism Amino-acid metabolism Other Other Metabolism associated Lipid Transport Amino-acid metabolism Nucleotide Lipid Amino-acid metabolism Carbohydrate metabolism Carbohydrate metabolism Carbohydrate metabolism Carbohydrate metabolism Carbohydrate metabolism Amino-acid metabolism Amino-acid metabolism Other

cycle

catabolic catabolic

catabolism,

acid biosynthesis

biosynthesis biosynthesis;

utilization,

process reductase

dehydrogenase

catabolism biosynthesis Carbohydrate

shunt

tyrosine salvage

cycle cycle

activity Pentose Biological Gluconate purine amino-acid L-phenylalanine Phenylalanine endocytosis Urea Fructose biliverdin Autophagy aspartate Transferase methyltransferase, transferase Urea glycolysis/gluconeogenesis lactate lactate tricarboxylic Glycolysis Amino-acid Tyrosine beta-alanine process proces, activity

(%)

Coverage 3.52 26.59 25.45 15.04 20.47 10.27 41.8 7.84 13.9 51.85 29.21 26.62 24.57 44.63 19.88 2.71 24.26 4.26 25.34 9.01

36 36 33 29 31 18 31 30 28 20 23 20 20 27 29 24 30 19 19 21 Peptides

(Da,

Mw 53,236 40,134 44,042 45,112 45,976 28,405 34,973 38,235 33,566 53,419 31,260 33,906 32,549 39,886 35,828 36,451 35,687 35,684 39,618 46,429 theoretical)

(theo-

retical) pI 6.57 5.72 6.74 6.29 6.67 7.72 6.76 7.14 5.82 6.34 5.52 6.37 7.1 9.12 8.14 8.45 7.56 8.93 8.66 6.73

181 807 8906 1130 2500 110 139 12867 812 180 4190 3136 2812 170 2516 5716 2625 Score 1804 2104 176 3 1

1 1

2 =

= 4

3 3

= =

1 =

= =

= SV SV

= SV

Rattus

SV Rattus SV

Pgd Gnmt Ldha Ldhc Aldob 1

SV Necap2 2

1 =

SV Rattus SV

chain

1 SV

= = = = =

SV =

Rattus =

Rattus

= kinase =

1 1

A C =

= PE

2 = =

OS PE

aldolase PE

GN GN GN GN GN GN PE norvegicus

Rattus OS

PE OS

OS =

PE PE 2 1

PE A

Rattus Rattus PE

= Rattus Rattus Rattus =

= =

OS = = = Rattus

norvegicus

1A1 decarboxylating Adk Upb1 Fah Sord Blvra Aspdh Sult1a1 Mdh2 Got2 SV Gapdh SV Hpd Otc

= protein Rattus

OS OS

======OS OS OS 1

= = OS

norvegicus norvegicus norvegicus norvegicus norvegicus norvegicus GN GN GN GN GN GN GN GN GN GN GN OS GN kinase

1 2 2 1 3 2

Rattus PE reductase

PE carbamoyltransferase aminotransferase

dehydrogenase dehydrogenase ======L-aspartate

ear-binding dehydrogenase

N-methyltransferase

dehydrogenase

SV SV SV SV SV SV OS

Arg1 Ulk3

Rattus Rattus Rattus Rattus Rattus Rattus 1 1 1 1 1 1 1

= =

======

GN OS norvegicus norvegicus OS ULK3 norvegicus OS PE PE PE PE PE Protein 6-phosphogluconate dehydrogenase Adenosine Beta-ureidopropionase 4-hydroxyphenylpyruvate dioxygenase Fumarylacetoacetase Adaptin Arginase-1 Sorbitol Biliverdin Serine/threonine-protein Putative Sulfotransferase Glycine Ornithine mitochondrial Glyceraldehyde-3-phosphate dehydrogenase L-lactate L-lactate Malate mitochondrial Fructose-bisphosphate Aspartate mitochondrial norvegicus norvegicus SV coat-associated dehydrogenase OS norvegicus OS norvegicus GN OS norvegicus norvegicus norvegicus PE norvegicus norvegicus

entry

) Protein P85968 Q64640 Q6P756 P46844 D3ZHP7 Q5I0J9 P13255 P00481 P04642 P00507 P27867 P32755 P17988 P00884 P19629 P04636 P25093 Q03248 P04797 P07824

Continued (

Spot 73 78 86 95 87 91 77 94 92 75 88 79 Table

C.P. Braga et al. / International Journal of Biological Macromolecules 96 (2017) 817–832 827 Zn Zn Zn Zn

Mg Cu, Cu,

= = =

I I I and

and and and and

+ + +

Se; Se;

Se; Se; Cu

Mg Mg Mg Mg

= = =

Zn =

Se; Zn; and and DM1 DM1 DM1 I

Cu, Cu, Cu,

Zn = = =

DM1 DM1 DM1 and Mg Mg I Se; I Se; I Se;

and and

+ = + = + =

Zn Se and

DM1

Cu; Cu Cu, Cu Cu, Cu; Cu Cu; Cu

======

C C C C C C C C C DM1 DM1 Se; DM1 DM1 and Mg Mg, DM1 DM1

stress stress stress stress stress stress stress stress stress stress stress stress

metabolism

Lipid Oxidative Other Other Oxidative Oxidative Oxidative Oxidative Oxidative Oxidative Oxidative Oxidative Other Oxidative Energy Carbohydrate metabolism Carbohydrate metabolism Oxidative Oxidative

steroid process

activity

oxidative oxidative transferase transferase transferase transferase transferase

Protease, Protease, protease protease

to to

regulation transport

apoptotic

regulation regulation

metabolism,

metabolism glutathione glutathione glutathione glutathione glutathione Response Response omega-amidase Antioxidant electron lipid gluconeogenesis gluconeogenesis redox Hydrolase, negative Oxidoreductase, Peroxidase redox Hydrolase, Threonine Threonine activity activity activity activity activity stress stress neuron

21.62 10.11 16.51 30.28 7.11 9.23 8.81 16.55 31.83 6.94 8.46 34.16 5.31 6.84 9.73 18.59 38.81 33.33 6.31

20 21 21 22 15 23 18 21 24 12 23 33 26 17 20 18 15 19 17

25,559 25,607 25,703 25,681 25,914 29,431 28,300 30,701 25,763 34,951 34,835 39,609 36,887 76,395 29,197 28,295 22,305 22,109 22,912

8.89 8.87 6.91 6.84 8.27 6.89 6.86 6.9 9.19 8.62 8.16 5.54 6.76 7.14 6.44 7.14 7.7 8.27 6.96

935 1111 1111 8573 2575 1602 1862 5045 117 107 2858 2650 424 117 1436 322 4320 8422 2729 3 1

= = 1

3 1

= 2 1

1 = =

1 SV SV

Gsta2 Gsta1 Gstm2 Gstm3 Gstm1 Etfa Fbp1 Psmb4 Prdx3 Gpx1 Psmb2

= 1 1 SV

Rattus Rattus Rattus SV SV ======

alpha-2 alpha-1 Mu Yb-3 Mu

= =

1 type-4 type-2

1 = = =

1 1

peroxide

1 =

= cytosolic

= =

GN GN GN GN GN GN GN GN GN GN GN

PE PE

norvegicus OS OS OS norvegicus

Rattus Rattus

1 PE

beta beta PE PE

3 1

= =

1 Rattus

protein

= = ﬂavoprotein

NIT2 OS OS

family SV Sult1b1 Prdx6 Prdx1

Ca3 Ca1 Nit2

Rattus mitochondrial = = = Rattus

2 PE

= =

1 S-transferase S-transferase S-transferase S-transferase S-transferase peroxidase subunit subunit

norvegicus norvegicus norvegicus norvegicus norvegicus norvegicus norvegicus norvegicus norvegicus norvegicus norvegicus GN GN GN GN GN GN 6-bisphosphatase 6-bisphosphatase mitochondrial

OS 2 3 2 2 2 4 2 2 2 4 1 PE OS

anhydrase anhydrase ======transfer 2

alpha

SV SV SV SV SV SV SV SV SV SV SV

Fam213a Fbp2

Rattus Rattus Rattus Rattus Rattus Rattus Rattus Rattus Rattus Rattus Rattus 2 2 1 1 1 1 1 1 1 1 1 1

= = member

======

OS OS FAM213A OS OS OS OS OS OS isozyme Glutathione Glutathione Glutathione Glutathione Glutathione Carbonic Carbonic Omega-amidase Redox-regulatory Electron Sulfotransferase Fructose-1 Fructose-1 Peroxiredoxin-6 Proteasome Thioredoxin-dependent Glutathione Peroxiredoxin-1 Proteasome norvegicus norvegicus GN GN PE PE PE PE PE PE PE PE PE PE PE OS 1 norvegicus reductase subunit norvegicus norvegicus OS OS norvegicus SV

P04903 P13803 P52847 P04041 O35244 Q9Z0V6 Q63716 B0BNN3 P40307 P00502 P04905 P08010 P14141 Q497B0 P34067 Q6AXX6 P19112 P08009 Q9Z1N1

120 124 98 103 106 107 122 108 113

828 C.P. Braga et al. / International Journal of Biological Macromolecules 96 (2017) 817–832 Mg Mg

and I

Cu, Cu,

Zn Zn; = =

Mg I I

= + +

and Se Zn; and DM1

DM1 DM1 DM1 and Mg Se Se;

and and

= =

Zn Zn Se

Mg, Cu; Cu Cu Cu; Cu; Cu, Cu

======

C DM1 DM1 C C C C C Cu, C and Se and Mg, presence

stress stress stress stress stress stress stress stress stress

metabolism

Energy Oxidative Metabolism associated Oxidative Oxidative Other Other Other Other Oxidative Oxidative Oxidative Oxidative Oxidative Oxidative Other Other Other

levels drug,

protein

Hydrogen

Ion

oxidative nutrient metabolic metabolic metabolic metabolic metabolic

catabolic

Transport process

Sensory Sensory Sensory

to to

response

regulation

synthesis,

transport,

ion stress process, Biological response redox glutathione olfaction, olfaction, Transferase glutathione xenobiotic glutathione glutathione glutathione ATP Transferase Transferase Transferase olfaction, Developmental process process process process process response transduction transduction transduction transport,

(%)

Coverage 13.07 31.56 33.03 18.59 16.29 13.96 3.62 3.6 3.6 3.6 31.46 7.34 21.36 16.61 16.61 25 25.71

18 19 22 18 19 20 19 20 21 20 46 22 29 29 28 30 9 Peptides

(Da,

Mw theoretical) 24,674 22,109 27,439 25,914 25,703 25,319 25,808 25,347 25,510 25,559 25,607 59,754 35,509 35,416 35,365 25,703 12,552

(theo-

retical) pI 8.96 8.27 7.75 8.27 6.91 8.78 5.9 8.42 6.77 8.89 8.87 9.22 5.78 5.57 5.57 6.91 4.86

4035 3785 119 110 87 79 79 79 79 1500 1500 550 3761 1435 62 2872 Score 1811 1

1 2 2 Ste Ste2

SV 1

= =

SV Gstt2 Gstm1 Gstm2 Gsta3 Gsta6 Gsta5 Gsta4 Gsta2 Gsta1 Gstm2

1 =

isoform isoform isoform ======1 theta-2 Mu Mu alpha-3 A6 alpha-5 alpha-4 alpha-2 alpha-1 Mu

GN GN =

PE 1

norvegicus GN GN GN GN GN GN GN GN GN GN PE

[Mn]

alpha PE

Rattus

Hox-A7 Rattus Rattus SV

SV = = 2

OS Sod2 Prdx1 Atp5a1 Rattus

= = = = OS OS

subunit norvegicus norvegicus norvegicus

S-transferase S-transferase S-transferase S-transferase S-transferase S-transferase S-transferase S-transferase S-transferase S-transferase

dismutase PE OS norvegicus norvegicus norvegicus norvegicus norvegicus norvegicus norvegicus norvegicus norvegicus norvegicus protein GN GN GN

3 2 2 3 1 2 2 2 3 1 1 2

sulfotransferase sulfotransferase sulfotransferase ======

Rattus Rattus Rattus SV SV SV SV SV SV SV SV SV SV SV SV

Sult1e1 = = = Hoxa7

Rattus Rattus Rattus Rattus Rattus Rattus Rattus Rattus Rattus Rattus

2 synthase 1 1 1 1 1 1 1 2 1 1 2 1

======

OS OS OS

GN OS OS OS OS OS OS OS Protein Superoxide mitochondrial Peroxiredoxin-1 Glutathione Glutathione Glutathione Glutathione Glutathione Glutathione Glutathione Glutathione Glutathione ATP mitochondrial Estrogen Estrogen Estrogen Glutathione Homeobox (Fragment) norvegicus PE PE PE PE PE PE PE PE PE PE SV PE PE norvegicus norvegicus GN OS OS OS 1 3 2

entry

) Protein P07895 Q63716 P15999 P08010 P09634 Q6AXY0 P46418 P14942 P04903 P00502 P04905 P49889 P52845 P30713 P52844 P08010 P04904

Continued (

Spot 127 140 129 128 142 133 132 Table

C.P. Braga et al. / International Journal of Biological Macromolecules 96 (2017) 817–832 829 Zn Zn

Zn Se

Mg Mg and and

Zn; = = Se;

Se; Se; I Se; I Se Se

and and

= = + = +

Zn; and and

Mg Mg Mg, Mg Mg,

= = = = =

DM1 DM1 DM1 DM1 DM1 Mg Mg I I I I I Se Mg

and

= + + + + + = =

Cu Cu; Cu Cu; Cu; Cu, Cu; Cu;

======

DM1 C C C C C C DM1 DM1 DM1 C C DM1 DM1 DM1 DM1 and

stress stress stress stress

metabolism metabolism

metabolism

Nucleotide Other Other Oxidative Oxidative Oxidative Energy Carbohydrate metabolism Other Lipid Other Energy Other Other Nucleotide Oxidative

redox

process

response response response

cellular

synthesis

transferase transferase

cell

response catabolic transport transport, biosynthesis;

response biosynthesis

immune immune immune

regulation

acid

differentiation nucleotides glycolysis/gluconeogenesis Oxidoreductase bile epithelial innate electron innate cellular cofactor innate immune redox electron glutathione glutathione homeostasis activity activity state NAD(+) transport

18.62 28.34 26.38 10.44 20.08 5.11 5.26 22.06 27.62 21.69 38.41 21.11 26.27 33.49 36.24 14.98

28 21 101 20 25 24 17 44 100 25 21 18 17 22 22 20

37,453 40,675 37,378 33,407 28,577 34,951 26,176 23,407 32,582 28,577 32,302 22,109 27,687 25,703 25,914 25,438

6.16 8.35 6.18 7.77 5.77 8.62 5.5 5.1 5.57 5.77 6.46 8.27 7.61 6.91 8.27 8.89

2741 1131 182 106 5621 2130 10826 3089 782 2026 1544 733 245 2132 278 7067 1

= 3

2 1

AK3 = 2 1 =

SV 1

SV Tst Etfa Apcs Pnp Gstm2 Gstm1

1 SV = SV

======1 Mu Mu

= PE

1 norvegicus =

GN GN GN GN GN GN PE 1 1 = Rattus reductase

norvegicus

inhibitor

PE =

Rattus complex complex

norvegicus norvegicus

Rattus

= =

Rattus

SV phosphorylase

SV SV OS

= 1

Rattus

ﬂavoprotein ﬂavoprotein Rattus OS SV 1

2 2 Akr1d1 Prdx1 Etfb Ak3 =

Haao

[NAD(+)] P-component = = Rattus 1

= = mitochondrial

= = = = OS

Rattus Rattus

= =

OS = =

aldehyde PE

S-transferase S-transferase activator activator

PE PE

sulfurtransferase

norvegicus norvegicus norvegicus norvegicus norvegicus norvegicus norvegicus GN GN GN GN GN

OS 3 4 2 1 2 2

phosphotransferase OS OS B1

======2 transfer transfer

1 alpha 1 beta

nucleoside amyloid

SV SV SV SV SV SV

Gpd1 Akr7a2 Psme1 Arhgdia Psme1

Rattus Rattus Rattus Rattus Rattus Rattus Rattus 1 GDP-dissociation 1 1 2 1 1 1

= = = = =

======

4-dioxygenase

OS member OS subunit subunit GN OS OS Glycerol-3-phosphate dehydrogenase cytoplasmic Aﬂatoxin 3-oxo-5-beta-steroid 4-dehydrogenase Thiosulfate Proteasome Electron Serum Rho 3-hydroxyanthranilate 3 Proteasome Purine Peroxiredoxin-1 Electron Glutathione Glutathione GTP:AMP mitochondrial OS GN GN OS norvegicus norvegicus norvegicus GN PE PE PE PE PE PE norvegicus OS GN norvegicus subunit subunit SV

O35077 P31210 P24329 Q63797 P85973 Q63716 Q5XI73 P29411 P04905 P13803 Q68FU3 P08010 Q63797 P23680 Q8CG45 P46953

143 149 145 148 161 156 160 147 150 146

830 C.P. Braga et al. / International Journal of Biological Macromolecules 96 (2017) 817–832

Fig. 2. Pathways with FDR < 0.05 using the Reactome FI.

Genes related in each pathway. Sulﬁde oxidation to sulfate: Tst,Suox; Phenylalanine and tyrosine catabolism: Fah,Hpd; Uptake and function of diphtheria

toxin: Hsp90ab1,Hsp90aa1; Purine catabolism: Pnp,Gpx1,Cat; Attenuation phase: Hsp90ab1,Hsp90aa1,Hspa8; Citric acid cycle (TCA cycle): Mdh2,Aco2,Sdha; Urea

cycle: Ass1,Arg1,Cps1,Otc; HSF1-dependent transactivation: Hspa1l,Hsp90ab1,Hsp90aa1,Hspa8; Sulfur amino acid metabolism: Mat1a,Tst,Suox,Cbs; Purine metabolism:

PNP,GPX1,CAT,ADK; Cellular response to heat stress: Pnp,Gpx1,Cat,Adk; Pyruvate metabolism and Citric Acid (TCA) cycle: Ldha,Mdh2,Aco2,Sdha; Glycolysis:

Aldob,Pklr,Eno2,Eno3,Gapdh,Eno1; Metabolism of nucleotides: Pnp,Gpx1,Gsr,Cat,Adk,Dpys; Detoxiﬁcation of Reactive Oxygen Species: Prdx3,Prdx1,Gpx1,Gsr,Cat,Prdx6,Sod2; Glu-

tathione conjugation: Gstm1,Gstm2,Gstm3,Gsta1, Gsta2,Gsta3,Gsta4; The citric acid (TCA) cycle and respiratory electron transport: Ldha,Mdh2,Atp5b,Aco2,Etfb,Etfa,Sdha,Atp5a1;

Gluconeogenesis: Got2, Fbp1, Fbp2, Mdh2, Aldob, Eno2, Eno3, Gapdh, Eno1, Pck1; Defective TPMT causes Thiopurine S-methyltransferase deﬁciency (TPMT deﬁ-

ciency), Defective SLC35D1 causes Schneckenbecken dysplasia (SCHBCKD), Defective GSS causes Glutathione synthetase deﬁciency (GSS deﬁciency), Defective

MAT1A causes Methionine adenosyltransferase deﬁciency (MATD), Defective AHCY causes Hypermethioninemia with S-adenosylhomocysteine hydrolase deﬁciency

(HMAHCHD), Defective GGT1 causes Glutathionuria (GLUTH), Defective OPLAH causes 5-oxoprolinase deﬁciency (OPLAHD), Defective UGT1A4 causes hyperbiliru-

binemia, Phase II conjugation, Defective GCLC causes Hemolytic anemia due to gamma-glutamylcysteine synthetase deﬁciency (HAGGSD) and Defective UGT1A1

causes hyperbilirubinemia: Gstm1,Gstm2,Gstm3,Mat1a,Sult1a1,Sult1e1,Gsta1,Gsta2,Gsta3,Gsta4,Mat2a; Cellular responses to stress: Hspa1l, Hsp90ab1, Prdx3, Prdx1, Gpx1,

Gsr, Cat, Hsp90aa1, Prdx6, Hspa8, Sod2; Glucose metabolism: Got2, Fbp1, Fbp2, Pgm1, Mdh2, Aldob, Pklr, Eno2, Eno3, Gapdh, Eno1, Pck1; Biologicaloxidations:

Gstm1,Gstm2,Gstm3,Mat1a,Sult1a1,Akr7a2,Sult1e1,Gsta1,Gsta2,Gsta3,Gsta4,Mat2a; Glycogen storage diseases and Metabolism of carbohydrates: Pgd, Got2, Fbp1, Fbp2, Pgm1,

Mdh2, Aldob, Pklr, Eno2, Eno3, Gapdh, Eno1, Pck1; Metabolism of amino acids and derivatives: Fah, Got2, Mat1a, Ass1, Glud1, Arg1, Psmb4, Psmb2, Aldh4a1, Cps1, Tst, Hpd, Suox,

Otc, Ckm, Psme1, Haao, Ftcd, Cbs.

isoform type 1 and 2 (MAT1A and MAT2A) are reported in the lit- the presence of Se in DM1 and Cu and Mg in DM1 + I. In spot

erature as metal ion-binding proteins; this protein has Mg-ﬁnger 42, HSPA8, HSPA1A, HSPA2 and HSPA1L showed the presence of

metal-binding domains [34]. MAT1A and MAT2A catalyze the for- Se in DM1 and Cu, Mg and Se in DM1 + I. The literature reported

mation of S-adenosylmethionine from methionine and ATP [34]. metal binding just in the small heat shock protein ␣-crystallin that

Most signiﬁcant was the protein bound in DM1 and DM1 + I with binds with Cu in the core domain, inducing increased dynamics

Zn, as Zn and Mg have the same properties that can interfere with at the dimer interface and modulating anti-aggregation [37]. The

some modiﬁcations in these proteins. Propionyl-CoA carboxylase literature does not have any other report of metal binding or asso-

alpha chain (PCCA) was detected with Cu in C. Cu is expected to ciation with metals in heat shock proteins; our study could suggest

bind by amino acids containing side chains with soft or border- some alterations to treatment with insulin in that the ions can bind

line ligands such as histidine, cysteine and methionine [35], which with these proteins to protect them from oxidative stress gener-

could explain this presence because PCCA is composed of 737 amino ated in the diabetic condictions. Peroxiredoxins are responsible

acids where 2.4% are histidine, 1.5% cysteine and 2.8% methionine for the degradation of peroxides and thiol-dependent enzymes;

(UniProtKB/Swiss-Prot − www.uniprot.org). peroxiredoxin-1 (PRX1) was found in three spots (124, 128 and

156) with the presence of Cu detected in C and Se in DM1. Another

isoform of PRX (peroxiredoxin-6, PRX6) was found in two spots:

3.3.2. Chaperone and oxidative stress metabolism

spot 27 with Cu and Mg in DM1, and spot 120 with Cu and Zn in C,

Heat shock proteins protect other proteins from stress, promote

Se in DM1 and Cu, Mg, Se and Zn in DM1 + I. The literature does not

protein folding and prevent protein aggregation [36]. In spot 10,

report any binding or association of these enzymes with Cu, Mg, Se

heat shock 70 kDa protein 4 (HSPA4) showed the presence of Cu

and Zn; in DM1 the PRX-1 and 6 had a presence of Se that could

and Se in DM1 + I. In spot 33, stress-70 protein (HSPA9) and heat

change the function of these proteins in the degradation of perox-

shock cognate 71 kDa (HSPA8) showed the presence of Cu, Mg,

ides. Different isoforms of glutathione S-transferase were found in

Se and Zn. In spot 41, 78 kDa glucose-regulated protein (HSPA5),

this study. This enzyme plays a key role in enzymatic detoxiﬁcation

heat shock 70 kDa protein 1-like and 1A/1 B (HSPA1L and HSPA1A),

[38]. The results showed association with Cu in C, Se in DM1 and

HSPA8 and heat shock-related 70 kDa protein 2 (HSPA2) showed

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