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. 2016 Jan 11;6:587–602. doi: 10.1016/j.dib.2015.12.045

Supporting data for the MS identification of distinct transferrin glycopeptide glycoforms and citrullinated peptides associated with inflammation or autoimmunity

A Rosal-Vela a,1, A Barroso b,1, E Giménez b, S García-Rodríguez b, V Longobardo c, J Postigo d, M Iglesias d, A Lario c, J Merino d, R Merino e, M Zubiaur a, V Sanz-Nebot b, J Sancho a,
PMCID: PMC4731419  PMID: 26909372

Abstract

This data article presents the results of all the statistical analyses applied to the relative intensities of the detected 2D-DiGE protein spots for each of the 3 performed DiGE experiments. The data reveals specific subsets of protein spots with significant differences between WT and CD38-deficient mice with either Collagen-induced arthritis (CIA), or with chronic inflammation induced by CFA, or under steady-state conditions. This article also shows the MS data analyses that allowed the identification of the protein species which serve to discriminate the different experimental groups used in this study. Moreover, the article presents MS data on the citrullinated peptides linked to specific protein species that were generated in CIA+ or CFA-treated mice. Lastly, this data article provides MS data on the efficiency of the analyses of the transferrin (Tf) glycopeptide glycosylation pattern in spleen and serum from CIA+ mice and normal controls. The data supplied in this work is related to the research article entitled “identification of multiple transferrin species in spleen and serum from mice with collagen-induced arthritis which may reflect changes in transferrin glycosylation associated with disease activity: the role of CD38” [1]. All mass spectrometry data have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository with identifiers PRIDE: PXD002644, PRIDE: PXD002643, PRIDE: PXD003183 and PRIDE: PXD003163.

Keywords: Arthritis, Inflammation, Protein species, Transferrin, Glycosylation, CD38, Citrullination


Specifications table

Subject area Biology
More specific subject area Proteomics and glycoproteomics
Type of data Tables, figures and raw data
How data was acquired Scanned 2D-DiGE images were analyzed using the DeCyder7.0 software (GE Healthcare) using the Differential In-gel Analysis (DIA) module to detect and normalize the protein spots. Protein relative abundance across all samples and statistical analyses were performed using the Biological Variation Analysis (BVA) module of the DeCyder software. MS data for protein identification was acquired using a MALDI TOF/TOF UltrafleXtreme (Bruker), or a 4800 MALDI-TOF/TOF Analyzer (AB SCIEX). μLC–TOF–MS data for the analysis of the glycopeptides glycoforms of Tf was acquired with a 1200 series capillary liquid chromatography system (Agilent Technologies) coupled to a 6220 oa-TOF LC/MS mass spectrometer with an orthogonal G1385-44300 interface (Agilent Technologies).
Data format Analyzed (excel files and word tables) and raw data
Experimental factors Mice with Collagen-induced arthritis, or with chronic inflammation, or with no treatment. Protein extraction and/or purification from spleen or serum samples. CyDye labeling. 2-D gel electrophoresis.
Experimental features Protein extracts from mice subjected to different experimental conditions were analyzed by 2D-DiGE, and protein species that differed in abundance were identified by MS/MS. PTMs such as citrullination of the identified proteins, or glycosylation of Tf species were further analyzed by MS.
Data source location UB: Barcelona; UCO: Córdoba; IPBLN: Granada.
Data accessibility Data is within this article. Data also available at the ProteomeXchange Consortium via the PRIDE partner repository, PRIDE: PXD002644, PRIDE: PXD002643, PRIDE: PXD003183 and PRIDE: PXD003163.

Value of the data

  • Application of μLC–TOF–MS for characterization of multiple glycopeptide glycoforms from mouse transferrin.

  • Investigation of altered transferrin glycopeptide glycosylation patterns in inflammatory and/or autoimmune diseases.

  • Mass spectrometry approach to identify new citrullinated peptides in mice with arthritis (CIA model).

  • Properly described approach for 2D-DiGE analysis to identify protein species that differ in abundance due to certain pathologies.

  • Basis for the study of altered protein species associated with inflammatory processes or arthritis in humans.

1. Data

Fig. 1 shows the extracted ion chromatograms (EICs) obtained by µLC–TOF–MS for the most abundant glycopeptide glycoforms of Tf isolated from WT non-immunized serum, Tf standard in a 2D gel, and Tf from a spleen extract in a 2D gel. Table 1, Table 2 in excel format show the list of the protein species identified by MS/MS, displaying the sequence of matched and fragmented peptides of a given protein. Table 3 shows the list of protein species identified by PMF. Table 4, Table 5, Table 6, Table 7, Table 8, Table 9, include the results of all the statistical analyses applied to the relative intensities of the detected 2D-DiGE protein spots for each of the 3 performed DiGE experiments. Table 10a, Table 10b shows the identities of the citrullinated peptides linked to specific protein species in CIA+, or CFA-treated mice. Table 11 shows the peptide coverage of mouse Tf standard digested with trypsin, and Table 12 shows the normalized peak area and %RSD of Tf glycopeptide glycoforms detected by µLC–TOF–MS in the spots of spleen protein extracts subjected to 2D electrophoretic separation and in-gel tryptic digestion.

Fig. 1.

Fig. 1

Extracted ion chromatograms (EICs) for the most abundant glycopeptide glycoforms of Tf isolated from (a) WT non-immunized serum, (b) Tf standard in a 2D gel (spot 3), and (c) Tf from a spleen extract in a 2D gel (spot 537 equivalent pI to spot 3) by µLC–TOF–MS.

Table 1.

Protein spots from mouse spleen identified by MS/MS using the 4800 MALDI-TOF/TOF (AB Sciex).

Spot numbera Identification Accession Accension numberb Protein score Protein score CI%c Pep.count Total ion scored Total %ion CI MW (theorical)e IEP (theorical)e
533 Serotransferrin TRFE_MOUSE Q921I1 119 100 11 69 100.000 78840.5 6.94
537 Serotransferrin TRFE_MOUSE Q921I1 217 100 18 109 100.000 78840.5 6.94
539 Serotransferrin TRFE_MOUSE Q921I1 461 100 25 266 100.000 78840.5 6.94
614 Stress-induced-phosphoprotein 1 STIP1_MOUSE Q60864 277 100 19 146 100 63,170 6.40
692 Fibrinogen beta chain FIBB_MOUSE Q8K0E8 800 100 25 578 100 55401.9 6.68
697 Catalase CATA_MOUSE P24270 179 100 10 131 100 60012.7 7.72
778 Alpha enolase ENOA_MOUSE P17182 891 100 27 594 100 47453.3 6.37
Beta enolase ENOB_MOUSE P21550 250 100 6 224 100 47337.4 6.73
Gamma enolase ENOG_MOUSE P17183 94 100 5 75 100 47609.1 4.99
906 Voltage-dependent anion selective channel protein 2 VDAC2_MOUSE Q60930 179 100 11 95 100 32339.80 7
981 Proteasome subunit alpha type-1 PSA1_MOUSE Q9R1P4 426 100 10 350 100.000 29812.9 6
982 Carbonic anhydrase 2 CAH2_MOUSE P00920 478 100 17 295 100.000 29128.5 6.49
Carbonic anhydrase 1 CAH1_MOUSE P13634 203 100 7 155 100.000 28360.2 6.44
1001 Proteasome subunit alpha type-6 PSA6_MOUSE Q9QUM9 335 100 10 264 100.000 27,811 6.34
Pyridoxine-5′-phosphate oxidase PNPO_MOUSE Q91XF0 81 99.999 5 57 99.999 30437.1 8.46
1063 Flavin reductase BLVRB_MOUSE Q923D2 777 100 16 583 100.000 22297.4 6.49
1097 ATP synthase subunit d, mitochondrial ATP5H_MOUSE Q9DCX2 243 100 10 156 100.000 18794.6 5.52
1103 Ferritin light chain 1 FRIL1_MOUSE P29391 585 100 14 435 100.000 20846.5 5.66
Ferritin light chain 2 FRIL2_MOUSE P49945 276 100 9 202 100.000 20886.9 6.39
1136 Peptidyl-prolyl cis-trans isomerase A PPIA_MOUSE P17742 340 100 10 249 100 18130.9000 8
1147 Actin-related protein 2/3 complex subunit 5-like protein ARP5L_MOUSE Q9D898 165 100 6 116 100 17026.80 6.32
1171 Nucleoside diphosphate kinase A NDKA_MOUSE P15532 654 100 13 518 100.000 17310.9 6.84
Nucleoside diphosphate kinase B NDKB_MOUSE Q01768 330 100 7 278 100.000 17,466 6.97
1184 Protein S100-A9 S10A9_MOUSE P31725 95 100 1 89 100 13,211 6.64
1313 Protein S100-A8 S10A8_MOUSE P27005 146 100 3 119 100.000 10345.1 5.43

The sequence of matched and fragmented peptides of the identified proteins, plus the ion scores and confidence intervals of the fragmented peptides can be found in the online version of this article (Table 1, .xlsx file) as supplementary material.

a

Spots are named as indicated on the 2-DE gel shown in Fig. 1 in Ref [1].

b

UniProtKB/Swiss-Prot accession number, MASCOT protein score, protein score confidence interval (C.I. %), total ion score, and total ion score confidence interval (C.I. %) are reported for the combined search of MALDI-TOF/TOF MS and MS/MS data (GPS Software, Applied Biosystems).

c

For protein scores, only confidence intervals above 99% were considered as statistically significant.

d

For total ion scores, only confidence intervals above 95% were considered as statistically significant.

e

Theoretical molecular weights and isoelectric points are given for each protein.

Table 2.

Protein spots identified by MS/MS using the MALDI-TOF/TOF UltrafleXtreme (Bruker).

Spot numbera Identification Accession Accession numberb Protein score Sequence coverage (%) No. of peptides MW (theorical)c IEP (theorical)c
501 Aconitate hydratase, mitochondrial ACON_MOUSE Q99KI0 93.05 3.60 2 85,400 8.08
538 Serotransferrin TRFE_MOUSE Q921I1 96.65 4 3 78,841 6.94
539 Serotransferrin TRFE_MOUSE Q921I1 190.90 5.9 4 78,841 6.94
554 Far upstream element-binding protein 1 FUBP1_MOUSE Q91WJ8 32.75 2.00 1 68,668 7.74
633 Fibrinogen alpha chain FIBA_MOUSE E9PV24 123.51 4.20 3 88,117 5.77
638 Heterogeneous nuclear ribonucleoprotein L HNRPL_MOUSE Q8R081 69.55 4.60 2 64,550 8.33
732 Coronin-1 CORO1A_MOUSE gi|4895037 102.3 2.2 1 51,627 6.05
738 Vimentin VIME_MOUSE P20152 286.83 16.10 5 53,712 5.06
770 Protein disulfide-isomerase A6 precursor PDIA6_MOUSE gi|58037267 117.78 10.6 3 49,058 5.05
898 Actin, cytoplasmic 1 ACTB_MOUSE P60710 177.47 9.10 2 42,052 5.29
Beta-actin-like protein 2 ACTBL_MOUSE Q8BFZ3 129.62 9.00 2 42,319 5.30
F-actin-capping protein subunit alpha-1 CAZA1_MOUSE P47753 92.04 9.80 2 33,090 5.34
972 Tropomyosin alpha-1 chain TPM1_MOUSE P58771 59.03 4.90 1 32,718 4.69
981 Proteasome subunit alpha type-1 PSA1_MOUSE Q9R1P4 133.78 13.7 3 29,813 6.00
1001 Proteasome subunit alpha type-6 PSA6_MOUSE Q9QUM9 177.79 16.3 4 27,811 6.34
1049 Proteasome subunit beta type-10 PSB10_MOUSE O35955 109.4 8.80 3 29,330 6.40
Growth factor receptor-bound protein 2 GRB2_MOUSE Q60631 103.47 11.50 3 25,336 5.89
1103 Ferritin light chain 1 FRIL1_MOUSE P29391 48.36 4.4 1 20,847 5.66
1150 E3 ubiquitin-protein ligase RNF181 RN181_MOUSE Q9CY62 26.01 4.80 1 19,487 5.65
1173 Ubiquitin-conjugating enzyme E2 N UBE2N_MOUSE P61089 104.79 19.70 2 17,184 6.13
1302 N-acyl-aromatic-L-amino acid amidohydrolase (carboxylate-forming) ACY3_MOUSE Q91XE4 23.11 2.50 1 35,720 5.30

The sequence of matched and fragmented peptides plus the ion scores and confidence intervals of the fragmented peptides can be found in the online version of this article (Table 2, .xlsx file) as supplementary material.

a

Spots are named as indicated on the 2-DE gel shown in Fig. 1 in Ref [1].

b

UniProtKB/Swiss-Prot or NCBI accession number.

c

Theoretical molecular weights and isoelectric points are given for each protein.

Table 3.

Protein spots identified by PMF using the MALDI-TOF/TOF UltrafleXtreme (Bruker).

Spot numbera Protein name Accession numberb MW(theorical)c IEP(theorical)c Score Expect Sequence coverage Queries matched Queries searched
572 Prelamin-A/C isoform A precursor (MS) gi|162287370 74,478 6.54 77 0.0034 29 18 73
Prelamin-A/C isoform C (MS) gi|161760667 65,464 6.37 69 0.02 33 17 73
Fibroblast growth factor 22 (MS) gi|12963627 18,972 11.73 72 0.011 61 10 73
983 Mitochondrial peptide methionine sulfoxide reductase Q9D6Y7 26,200 8.6 76 0.00042 34.3 7 37
1104 Low molecular weight phosphotyrosine protein phosphatase Q9D358 18,636 6.30 58.5 2.40E−02 31.6 5 35
a

Spots are named as indicated on the 2-DE gel shown in Fig. 1 in Ref [1].

b

UniProtKB/Swiss-Prot or NCBI accession number.

c

Theoretical molecular weights and isoelectric points are given for each protein.

Table 4.

Spleen protein species that differ in abundance by 2-ANOVA-Mouse in Col II immunized CD38 KO mice versus B6 WT mice.

DeCyder spot no. Protein namea Pvalue (2-ANOVA-Mouse)
B6 WT:
538 Serotransferrin 3.99E−04
633 Fibrinogen alpha chain 6.11E−04
692 Fibrinogen beta chain 1.12E−03
1097 ATP synthase subunit d, mitochondrial 1.30E−03
1302 N-acyl-aromatic-L-amino acid 1.75E−03
amidohydrolase (carboxylate-forming)
539 Serotransferrin 3.53E−03
533 Serotransferrin 8.87E−03
554 Far upstream element-binding protein 1 9.53E−03
537 Serotransferrin 1.61E−02
501 Aconitate hydratase, mitochondrial 1.78E−02
1171 Nucleoside diphosphate kinase A 0.0195
Nucleoside diphosphate kinase B
1103 Ferritin light chain 1 2.06E−02
Ferritin light chain 2
1300 Not identified 0.023
638 Heterogeneous nuclear ribonucleoprotein L 2.89E−02
572 Prelamin-A/C isoform A precursor (PMF) 3.59E−02
Prelamin-A/C isoform C (PMF)
Fibroblast growth factor 22 (PMF)
1313 Protein S100-A8 0.05
CD38 KO:
898 Actin, cytoplasmic 2 6.70E−03
Beta-actin-like protein 2
F-actin-capping protein subunit alpha-1
982 Carbonic anhydrase 2 8.98E−03
Carbonic anhydrase 1
1044 Not identified 0.0202
981 Proteasome subunit alpha type-1 2.39E−02
a

Protein name according to UniProt, or to NCBI.

Table 5.

Spleen protein species that differ in abundance by 2-ANOVA-Arthritis test in Col II-immunized CIA+ versus CIA mice.

DeCyder spot no. Protein namea Pvalue (2-ANOVA-Arthritis)
In CIA+:
438 Not identified 0.0162
1150 E3 ubiquitin-protein ligase RNF181 0.0414


 

 


In CIA:
1157 Not identified 0.021
778 Alpha-enolase 0.0365
Beta-enolase
Gamma-enolase
a

Protein Name according to UniProt, or to NCBI.

Table 6.

Spleen protein species that differ in abundance by 2-ANOVA-Interaction in two groups of Col.II-immunized mice (CD38 KO and B6 WT) with two conditions: CIA+ and CIA).

DeCyderspot no. Protein namea Pvalue(2-ANOVA-Mouse) Pvalue(2-ANOVA-Arthritis) Pvalue(2-ANOVA-Interaction)
1302 N-acyl-aromatic-L-amino acid 1.75E−03 0.535 1.27E−04
amidohydrolase (carboxylate-forming)
532 Not identified 0.065 0.585 5.29E−04
538 Serotransferrin 3.99E−04 0.255 8.40E−03
982 Carbonic anhydrase 2 8.98E−03 0.771 8.99E−03
Carbonic anhydrase 1
1001 Proteasome subunit alpha type-6 0.878 0.303 9.65E−03
Pyridoxine-5׳-phosphate oxidase
983 Mitochondrial peptide methionine 0.545 0.849 3.57E−02
sulfoxide reductase (PMF)
1063 Flavin reductase 0.707 0.779 0.0362
537 Serotransferrin 1.61E−02 0.306 4.07E−02
a

Protein name according to UniProt, or to NCBI.

Table 7.

Differences in spleen protein species abundance compared between Col II immunized CD38 KO mice (test group) versus Col II-immunized B6 WT mice (control group).

DeCyder spot no. Protein namea Average ratiob Pvalue (t-test)
Decreased abundance
633 Fibrinogen alpha chain −1.35 1.67E−04
692 Fibrinogen beta chain −1.19 4.53E−04
1097 ATP synthase subunit d, mitochondrial −1.25 4.95E−04
538 Serotransferrin −1.23 1.35E−03
539 Serotransferrin −1.29 2.25E−03
533 Serotransferrin −1.39 4.17E−03
554 Far upstream element-binding protein 1 −1.17 5.26E−03
501 Aconitate hydratase, mitochondrial −1.17 9.81E−03
1103 Ferritin light chain 1 −1.37 0.0124
Ferritin light chain 2
638 Heterogeneous nuclear ribonucleoprotein L −1.23 0.0142
537 Serotransferrin −1.25 0.0208
572 Prelamin-A/C isoform A precursor (MS) −1.18 0.0209
Prelamin-A/C isoform C (MS)
Fibroblast growth factor 22 (MS)
1171 Nucleoside diphosphate kinase A −1.17 0.0258
Nucleoside diphosphate kinase B
697 Catalase −1.16 0.0453
536 Not identified −1.14 0.0475
1300 Not identified −1.33 0.0493


 

 

 


Increased abundance
898 Actin, cytoplasmic 2 1.19 4.37E−03
Beta-actin-like protein 2
F-actin-capping protein subunit alpha-1
1044 Not identified 1.25 0.0163
981 Proteasome subunit alpha type-1 1.27 0.0194
982 Carbonic anhydrase 2 1.16 0.0457
Carbonic anhydrase 1
a

Protein name according to UniProt, or to NCBI.

b

Negative average ratios indicate decreased protein abundance, while positive ratios indicate increased protein abundance in CIA+ CD38 KO relative to that in CIA+ B6 WT mice.

Table 8.

Chronic inflammation model. Differences in spleen protein species abundance compared between CFA/IFA-treated CD38 KO mice (test group) and CFA/IFA-treated B6 WT mice (control group).

DeCyder spot no. Protein namea Average ratiob P value (t-test)
Decreased abundance
1049 Proteasome subunit beta type-10 −1.18 4.06E−03
Growth factor receptor-bound protein 2
1330 Not identified −1.31 0.0106
972 Tropomyosin alpha-1 chain −1.11 0.0163
1173 Ubiquitin-conjugating enzyme E2 N −1.13 0.0217
1103 Ferritin light chain 1 −1.62 0.0343
Ferritin light chain 2
738 Vimentin −1.22 0.0351
1104 Low molecular weight phosphotyrosine
protein phosphatase (PMF) −1.28 0.0384
732 Coronin-1 −1.1 0.0434
Increased abundance
1313 Protein S100-A8 1.37 0.0236
a

Protein name according to UniProt, or to NCBI.

b

Negative average ratios indicate decreased protein abundance, while positive ratios indicate increased protein abundance in CFA/IFA-treated CD38 KO relative to that in CFA/IFA-treated B6 WT mice.

Table 9.

Non-immunized control mice. Differences in spleen protein species abundance compared between non-immunized CD38 KO mice (test group) and non-immunized B6 WT mice (control group).

DeCyder spot no. Protein namea Average ratiob Pvalue (t-test)
Decreased abundance
538 Serotransferrin −1.26 4.04E−03
539 Serotransferrin −1.23 0.0185
537 Serotransferrin −1.22 0.0221
638 Heterogeneous nuclear ribonucleoprotein L −1.13 0.0266
770 Protein disulfide-isomerase A6 −1.11 0.0437
532 Not identified −1.2 0.0477
Increased abundance
1295 Not identified 1.33 0.0162
a

Protein name according to UniProt, or to NCBI.

b

Negative average ratios indicate decreased protein abundance, while positive ratios indicate increased protein abundance in CFA/IFA-treated CD38 KO relative to that in CFA/IFA-treated B6 WT mice.

Table 10a.

Citrullinated protein species and peptidesa detected in spleen from collagen-induced arthritis, or CFA-treated mice. TOF/TOF 4800.

Spot number Identification Accesion Accension number Protein score Protein score CI% Pep. count Total ion score Total % ion CI MW (theoretical) IEP (theoretical)

537 Serotransferrin TRFE_MOUSE Q921I1 172 100 20 107 100.00 78840.50 6.94
614 Stress-induced-phosphoprotein 1 STIP1_MOUSE Q60864 238 100 27 145 100.00 63169.60 6.40
697 Catalase CATA_MOUSE Q8C6E3 174 100 15 131 100.00 60082.80 7.73
778 Alpha enolase ENOA_MOUSE P17182 940 100 33 675 100.00 47453.30 6.37
Beta enolase ENOB_MOUSE P21550 260 100 12 224 100.00 47337.40 6.73
Gamma enolase ENOG_MOUSE P17183 102 100 10 75 100.00 47609.10 4.99
981 Proteasome subunit alpha type-1 PSA1_MOUSE Q9R1P4 415 100 14 350 100.00 29812.90 6.00
Proteasome subunit alpha type PSMA1_MOUSE Q8BTU5 399 100 12 349 100.00 29732.80 5.78
1001 Proteasome subunit alpha type-6 PSA6_MOUSE Q9QUM9 325 100 13 264 100.00 27811.00 6.34
Pyridoxine-5׳-phosphate oxidase PNPO_MOUSE Q91XF0 81 99.999 5 57 100.00 30437.10 8.46
1103 Ferritin light chain 2 FRIL2_MOUSE P49945 251 100 10 202 100.00 20886.90 6.39
1136 Peptidyl-prolyl cis–trans isomerase A PPIA_MOUSE P17742 320 100 11 249 100.00 18130.90 7.74
1171 Nucleoside diphosphate kinase A NDKA_MOUSE P15532 726 100 17 591 100.00 17310.90 6.84
Nucleoside diphosphate kinase B NDKB_MOUSE Q01768 321 100 9 278 100.00 17466.00 6.97
a

The sequence of matched citrullinated peptides of a given protein, and the positions of the deiminated arginines are shown in online version of this article as Supplementary material (Table 10, xlsx. File).

Table 10b.

Citrullinated protein species and peptidesa detected in spleen from collagen-induced arthritis, or CFA-treated mice. TOF/TOF UltrafleXtreme.

Spot number Protein name Accession Accesion number MW (theoretical) IEP (theoretical) Score Sequence coverage (%) Queries matched Queries searched

633 Fibrinogen alpha chain FIBA_MOUSE E9PV24 87.40 5.78 101.00 17.00 15 26
732 Coronin-1A COR1A_MOUSE O89053 51.00 6.04 83.40 36.00 16 75
898 F-actin-capping protein subunit alpha-1 CAZA1_MOUSE P47753 32.90 5.34 77.90 43.00 12 65
972 Tropomyosin alpha-3 chain TPM3_MOUSE P21107 33.00 4.68 60.50 38.60 10 40
a

The sequence of matched citrullinated peptides of a given protein, and the positions of the deiminated arginines are shown in online version of this article as Supplementary material (Table 10, .xlsx file).

Table 11.

Detected peptides in a tryptic digest of standard mTf analyzed by µLC–MS–TOF.

Detected peptides in mTf standard
VPDK
TVK
WCAVSEHENTK
CISFR
DHMK
TVLPPDGPR
LACVK
K
TSYPDCIK
AISASEADAMTLDGGWVYDA GLTPNNLKPVAAEFYGSVEH PQTYYYAVAVVK X
K
GTDFQLNQLEGK
K
SCHTGLGR
SAGWVIPIGLLFCK
LSEPR
SPLEK
AVSSFFSGSCVPCADPVAFP K
LCQLCPGCGCSSTQPFFGYV GAFK
CLK
DGGGDVAFVK
HTTIFEVLPEK
ADR
DQYELLCLDNTR
KPVDQYEDCYLAR
IPSHAVVAR
K
NNGK X
EDLIWEILK
VAQEHFGK
GK
SK
DFQLFSSPLGK
DLLFK
DSAFGLLR
VPPR
MDYR
LYLGHNYVTAIR
NQQEGVCPEGSIDNSPVK
WCALSHLER
TK
CDEWSIISEGK
IECESAETTEDCIEK
IVNGEADAMTLDGGHAYIAGQCGLVPVMAEYYESSNCAIPSQQGIFPK
GYYAVAVVK
ASDTSITWNNLK
GK
K
SCHTGVDR
TAGWNIPMGMLYNR
INHCK
FDEFFSQGCAPGYEK
CAPNNK
EEYNGYTGAFR
CLVEK
GDVAFVK
HQTVLDNTEGK
NPAEWAK
NLK
QEDFELLCPDGTR
KPVK
DFASCHLAQAPNHVVVSR
K
EK
AAR
VK
AVLTSQETLFGGSDCTGNFC LFK
STTK
DLLFR
DDTK X
CFVK
LPEGTTPEK
YLGAEYMQSVGNMR
K
CSTSR
LLEACTFHK
H
Total number of amino acids 678
Number of amino acids detected 618
Coverage (%) 91

Table 12.

Normalized peak area and %RSD of Tf glycopeptide glycoforms detected in the spots of spleen protein extracts subjected to 2D electrophoretic separation and in-gel tryptic digestion.

Glycoforms Spot 532
Spot 533
Spot 536
Spot 537
Spot 539
Anorm %RSD Anorm %RSD Anorm %RSD Anorm %RSD Anorm %RSD
2Ant/1NeuGc 7.7 6.1 17.0 18.1 15.4 6.4
2Ant/1NeuGc1Fuc 5.5 1.2
2Ant/2NeuGc 54.7 3.7 56.9 7.1 54.9 7.9 76.5 5.5 137.8 0.8
2Ant/2NeuGc1Fuc 29.8 6.7 25.9 7.5 16.2 20.7 26.1 17.1
2Ant/3NeuGc 17.7 5.5 29.8 15.6 8.7 4.9
2Ant/3NeuGc1Fuc 2.6 3.3

Anorm: normalized peak areas were calculated as: (Glycoform peak area/peptide 354–364 (CDEWSIISEGK) peak area)×100.

2. Experimental design, materials and methods

2.1. Mice

WT mice were purchased from Harlan Ibérica (Barcelona, Spain). Mice deficient in CD38 (CD38-KO) were backcrossed onto the B6 background for more than 12 generations, as described previously [3]. All studies with live animals were approved by the IPBLN and Universidad de Cantabria Institutional Laboratory Animal Care and Use Committees.

2.2. Induction and assessment of arthritis

For the induction of CIA, 8–12 weeks-old male mice were immunized as previously described [4], [5].

2.3. Protein extraction from spleen preparations

Proteins were extracted from spleen by using the MicroRotofor Lysis Kit (for mammalian tissues and cells) (Bio-Rad, Ref #163-2141), following the manufacturer׳s instructions, which includes the use of mini-grinders for effective disruption of cells and tissues. The excess of salts and other contaminants were removed using the Bio-Rad׳s ReadyPrep 2-D cleanup kit. Samples were then resuspended in a DIGE-compatible buffer (7 M urea, 2 M thiourea, 4% CHAPS, 20 mM Tris, pH 8.5), quantified using the RC DC assay, and kept at −20 °C until further use.

2.4. Design of DiGE experiments

Unless otherwise indicated in each DiGE experiment conducted, four biological replicates of each condition were compared, comprising protein samples derived from four CD38-KO mice and four WT mice as previously described [1], [6].

2.5. DiGE labeling and two-dimensional gel electrophoresis

Samples were aliquoted at 45 μg, and the pooled internal standard was made with 23 μg of each of the sixteen test samples combined. The proteins were labeled with 400 pmol (in 1 μL of anhydrous DMF) of CyDye per 50 μg of protein as per the manufacturer׳s instructions (GE Healthcare). After labeling, the appropriate samples were combined for each gel. Each combined sample (~50 μL) was made up to 200 μL with Readyprep Rehydration/Sample buffer (8 M urea, 2% CHAPS, 50 mM dithiothreitol (DTT), 0.2% (w/v) Bio-Lyte® 3/10 ampholytes, and Bromophenol Blue (trace)).

2-DE was carried out using the Protean IEF cell and Criterion electrophoresis cell systems (Bio-Rad, Hercules, CA, USA) as previously described [7], with the following modifications: (1) First-dimension IPG strips (Bio-Rad: 11 cm, linear pH 3-10 gradient); (2) Active in-gel rehydration at 50 V, 12 h at 20 °C; (3) The IPG strips were focused in a one-step procedure, at 8000 V for a total of 35,000  Vh at 20 °C with a current limit of 50 μA/strip.

After electrophoresis, one of the gels was pre-scanned using the Typhoon 9400 variable mode imager at each of the appropriate CyDye excitation wavelengths (Cy3 (532 nm), Cy5 (633 nm), Cy2 (488 nm)), in order to determine the appropriate laser intensity for each CyDye. Thereafter, each of the analytical gels was scanned at this optimum laser intensity at a 2resolution of 100 μm. Gels were then fixed and stained with SYPRO Ruby (Bio-Rad) and re-scanned using the 488 nm laser. Scanned images were analyzed using the DeCyder7.0 software (GE Healthcare) using the Differential In-gel Analysis (DIA) module to detect and normalize the protein spots. Standard was used to normalize gels by calculating the standardized abundance of each spot, i.e., the ratio of either Cy3 or Cy5 signal to that of Cy2.

2.6. Protein identification by MALDI-TOF/TOF MS/MS

In-gel digestion of proteins has been described previously [8]. A set of protein spots were identified by MS/MS using a 4800 MALDI-TOF/TOF Analyzer (AB SCIEX) in automatic mode with the settings described previously [6]. Protein identification was assigned by peptide mass fingerprinting and confirmed by MS/MS analysis of at least three peptides in each sample. Mascot 2.0 search engine (Matrixscience) was used for protein identification running on GPS software (Applied Biosystems) against the SwissProt Mus musculus database (uniprot_sprot_26042011.fasta). The search setting allowed one missed cleavage with the selected trypsin enzyme, a MS/MS fragment tolerance of 0.2 Da and a precursor mass tolerance of 100 ppm.

Other spots were identified by MS/MS using a MALDI TOF/TOF UltrafleXtreme (Bruker) in manual mode as previously described [6]. Fragment selection criteria were a minimum S/N ratio of 15, a maximum number of peaks set at 200. For each precursor selected for MS/MS analysis, fragment mass values in the range from 13 Da to 4 Da below precursor mass were used to peptide identification.

Protein identification was assigned by peptide mass fingerprinting and confirmed by MS/MS analysis of 5 peptides. Mascot Server 2.4 (Matrixscience) and ProteinScape 3.1 (Bruker) were used for protein identification against the SwissProt Mus musculus database (SwissProt_2015_06.fasta and NCBInr_20150409.fasta). The search setting allowed two missed cleavage with the selected trypsin enzyme, fixed modification was cysteine carbamidomethylation and variable modification was methionine oxidation, a MS/MS fragment tolerance of 0.5 Da and a precursor mass tolerance of 50 ppm, unless otherwise indicated.

The MS spectra of the identified proteins were further examined in order to detect the presence of citrullinated proteins. Protein citrullination (o deimination) is the enzymatic conversion of peptidyl-arginine residues to peptidyl-citruline, mediated by the family of calcium-dependent peptidylarginine deiminases (PADs) [9]. The search setting for this PTM with MASCOT was performed as in the previous paragraph, including as variable modification the deamination of arginine, with the following considerations [10]: (a) for one citrullinated arginine, the peptide theoretical mass increase is 0.98 Da and the modified peptide, losing one amino group, becomes more acidic; (b) citrullinated arginine residues are not likely to be cleaved by trypsin, so that a minimum number of one missed cleavage must be specified; (c) a peptide that includes a C-terminal citrullinated arginine must be rejected; (d) citrullinated peptides generate an unusual isotopic mass cluster as compared with that of unmodified peptides.

2.7. μLC–TOF–MS

The µLC–TOF–MS experiments were performed in a 1200 series capillary liquid chromatography system coupled to a 6220 oa-TOF mass spectrometer with an orthogonal G1385–44300 interface (Agilent Technologies). LC and MS control, separation, data acquisition and processing were performed using MassHunter workstation software (Agilent Technologies). The oa-TOF mass spectrometer was tuned and calibrated following the manufacturer׳s instructions. Once a day, or even twice a day when required, a “Quick Tune” of the instrument was carried out in positive mode followed by a mass-axis calibration to ensure accurate mass assignments. In order to enhance detection sensitivity of glycopeptides, no internal recalibration was used [11]. MS measurement parameters were as described in a previous work [12]: capillary voltage 4000 V, drying gas (N2) temperature 200 °C,drying gasflow rate 4 L min−1,nebulizer gas (N2) 15 psig, fragmentor voltage 215 V, skimmer voltage 60 V, OCT 1 RF Vpp voltage 300 V. Data were collected in profile (continuum) at 1 spectrum s−1 (approx. 10,000 transients/spectrum) between m/z 100 and 3200, working in the highest resolution mode (4 GHz). For separation, a Zorbax 300SB-C18 column (3.5 m particle diameter, 300 A° pore diameter, 150 mM×0.3 mm LT×id, Agilent Technologies) was used. Experiments were performed at room temperature with gradient elution at a flow rate of 4 µL min−1. Eluting solvents were A: water with 0.1% (v/v) formic acid, and B: acetonitrile with 0.1% (v/v) formic acid. Solvents were degassed for 10 min by sonication before use. The optimum elution program was: solvent B from 10% to 60% (v/v) within 45 min as linear gradient, followed by cleaning and re-equilibration steps of B: 60% to 100% (v/v) (5 min), 100% (v/v) (10 min), 100% to 10% (v/v) (5 min) and 10% (v/v) (10 min). Before analysis, samples were filtered using a 0.22 µm polyvinylidene difluoride centrifugal filter (Ultrafree-MC, Millipore, Bedford, MA, USA) at 12,000 rpm for 4 min. Sample injection was performed with an autosampler refrigerated at 4 °C and the injection volume was 1 μL when analyzing Tf isolated from serum samples and digested with trypsin, and 5 μL when analyzing Tf in-gel digests.

2.8. μLC–TOF–MS data analysis

Prior to data analysis, a database with the exact monoisotopic mass of the different glycopeptide glycoforms of mouse Tf was created using Excel. To calculate the monoisotopic mass of each glycopeptide glycoform, it was necessary to calculate the elemental composition of all the glycopeptides taking into account the peptide and glycan contribution. First, the peptide sequence of mouse Tf was obtained from UniProt Knowledgebase (Q921l1), which also includes information about which cysteines and asparagines are involved in disulfide bonds and in N-glycosylation points, respectively. Afterwards, the theoretical sequence of each peptide and glycopeptide that would be obtained after tryptic digestion is obtained using the proteomic tool PeptideMass from the Expasy bioinformatics resource program. Subsequently, using the ProtParam tool from Expasy the elemental composition of the peptide sequence of the glycopeptide is obtained. Furthermore, the elemental composition of each glycan is calculated as the sum of the elemental composition of each monosaccharide that forms the glycan. Ion source webpage was used to obtain the elemental composition of each monosaccharide. Finally, the elemental composition of the peptide is added to obtain the molecular formula of each possible glycopeptides glycoform and thus, the monoisotopic mass with four decimals. Afterwards, the mass-to-charge values (m/z) for each glycopeptide glycoform are calculated up to a z value of 5 considering proton adducts (i.e. [M+H]+, [M+2H]2+, [M+3H]3+, [M+4H]4+ and [M+5H]5+)

Finally, the data analysis is carried out using the software MassHunter Qualitative (Agilent Technologies). All the previously calculated m/z values for each glycopeptide glycoform are extracted together to obtain an extracted ion chromatogram (EIC) of that glycopeptide specie, as can be observed in Fig. 1, which shows the EIC for some glycopeptide glycoforms in three different samples. If more than one of the extracted masses is detected in one chromatographic peak of the EIC, the presence of the corresponding glycopeptide glycoforms can be confirmed.

Table 1, Table 2 can be found in the online version of this article (.xlsx files). They show the list of protein species identified by MS/MS, displaying the sequence of matched and fragmented peptides of a given protein. Ion scores and confidence intervals of the fragmented peptides are also shown.

Acknowledgments

We thank to Dr. Frances E. Lund (Department of Microbiology, University of Alabama at Birmingham, Birmingham, Alabama, USA) for the gift of the CD38-/- mice and helpful discussions; to Pilar Navarro-Cuesta for her technical assistance.

The proteomic analyses were performed in the proteomics facilities of IPBLN-CSIC (Granada) and UCO-SCAI (Córdoba). Both facilities belong to ProteoRed, PRB2-ISCIII, supported by grant PT13/0001.

This work was supported in part by the European Commission in collaboration with the following Funding agencies:

Ministerio de Economía y Competitividad (MINECO) del Gobierno de España Grant number SAF2011-27261 (awarded to J.S.), Grant number CTQ2011-27130 (awarded to V. S-N), Grant number SAF2011-22463 (to R.M.; cofunded by the European Regional Development Fund), and Grant number SAF2012-34059 (to J.M.; cofunded by the European Regional Development Fund); Consejería de Innovación, Ciencia y Empresa de la Junta de Andalucí;a, Grant number P08-CTS-04046 (awarded to J.S.). From the MINECO del Gobierno de España, Grant number IPT2011-1527-010000, associated to Fibrostatin SL (awarded to J.M). A.R.V. was supported by a felllowship-contract from Consejería de Innovación, Ciencia y Empresa de la Junta de Andalucía. S.G.R. was supported by a JAEDoc contract from CSIC and a MINECO contract. A.B. was supported by a FPU fellowship from the Ministry of Education, Culture and Sport.

Footnotes

Appendix A

Supplementary data associated with this article can be found in the online version at doi:10.1016/j.dib.2015.12.045.

Appendix A. Supplementary material

Supplementary material

mmc1.pdf (1.2MB, pdf)

Supplementary material

mmc2.zip (309.5KB, zip)

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Supplementary material

mmc1.pdf (1.2MB, pdf)

Supplementary material

mmc2.zip (309.5KB, zip)

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