MALDI-TOF/TOF-MS Reveals Elevated Serum Haptoglobin and Amyloid A in Behcet’s Disease Liming Mao,†,§ Hongtao Dong,†,§ Peizeng Yang,*,‡ Hongyan Zhou,† Xiangkun Huang,† Xiaomin Lin,† and Aize Kijlstra|,⊥ Zhongshan Ophthalmic Center, State Key Laboratory of Ophthalmology of Sun Yat-sen University, Guangzhou, P.R. China, The First Affiliated Hospital, Chongqing Medical University, Chongqing, P.R. China, The Animal Production Division, Animal Sciences Group and Research Center, Wageningen University, Wageningen, The Netherlands, and the Department of Ophthalmology, University of Maastricht, Maastricht, The Netherlands Received April 11, 2008
Behcet’s disease (BD) is a multisystemic autoimmune disease with unclear etiology and pathogenesis. To screen aberrant serum proteins in BD, serum samples were obtained from eight male BD patients with active uveitis and eight male healthy volunteers with informed consent. The serum samples from active BD patients and normal controls were pooled. Highly abundant serum proteins (albumin and IgG) were depleted from these two samples using an affinity capture based kit. The obtained samples were subjected to two-dimensional gel electrophoresis (2-DE). Protein spots were visualized with the “blue silver” staining. Differently expressed proteins were subsequently identified by matrix-assisted laser desorption /ionization tandem time-of-flight mass spectrometry (MALDI-TOF/TOF-MS). Western blot and enzyme-linked immunosorbent assay (ELISA) were performed using the serum samples from 18 patients with active BD, 6 patients with inactive BD, 22 patients with Vogt-Koyanagi-Harada (VKH) syndrome, and 20 healthy volunteers to validate the results of 2-DE and MS. Proteomic profiles of the pooled samples were compared, and approximately 800 protein spots were observed in each of the gels. Expression levels of four of the protein spots in active BD were significantly higher than those in the normal controls. Mass spectrometric protein identification revealed that the four protein spots corresponded to two proteins: haptoglobin (Hp) and serum amyloid A (SAA). Western blot and ELISA showed that Hp was only overexpressed in active BD but not in inactive BD, VKH syndrome, or healthy controls. An obvious band of SAA was detected in 72.2% of the serum samples from BD patients, whereas a vague band of this protein was found in 10.0% of the tested normal samples and 9.1% of VKH samples. Our results revealed a significantly increased expression of Hp and SAA in serum of active BD patients. These two proteins may be involved in the development of BD. Keywords: Behcet’s disease • Serum • Hp • SAA • VKH syndrome
1. Introduction Behcet’s disease (BD) is a multisystem inflammatory disease characterized by recurrent uveitis, oral aphthae, genital ulcers, and erythema nodosum. The prevalence in Asian countries ranges from 13.5 to 20 cases per 100 000.1 Its etiology and pathogenesis are still unknown. Genetic factors are thought to play an important role in the pathogenesis,1,2 while environmental factors as well as viral and bacterial infections may also be involved.1,3 Previous studies have suggested that an autoimmune response is involved in its pathogenesis.1,4,5 The diagnosis of this disease is mainly based on the clinical * To whom correspondence should be addressed. Address: Youyi Road No 1., Chongqing, 400016, P. R. China. E-mail,
[email protected]; phone, 0086-23-89012851; fax, 0086-23-89012851. † State Key Laboratory of Ophthalmology of Sun Yat-sen University. § These authors contributed equally to this work. ‡ Chongqing Medical University. | Wageningen University. ⊥ University of Maastricht.
4500 Journal of Proteome Research 2008, 7, 4500–4507 Published on Web 08/29/2008
manifestations, and to date, no discriminatory laboratory tests are available. Searching for proteins specifically expressed in serum or on/in blood cells from BD patients will greatly contribute to our understanding of its pathogenesis and diagnosis. Serum is a preferred specimen for detecting differently expressed proteins because it can readily be obtained by noninvasive methods. Proteomics has provided a powerful tool for biomarker screening. It has been used for the investigation of serum from patients with cancer6-9 and autoimmune disease.10 However, no proteomic studies have been reported using serum for detecting differently expressed proteins in BD patients. In this study, we investigated the differently expressed proteins using two-dimensional gel electrophoresis (2-DE) and matrix-assisted laser desorption /ionization tandem time-offlight mass spectrometry (MALDI-TOF/TOF-MS). Western blot and enzyme-linked immunosorbent assay (ELISA) were used to validate the proteomic results. Our results demonstrated a 10.1021/pr800279m CCC: $40.75
2008 American Chemical Society
research articles
Elevated Hp and SAA in Behcet’s Disease Table 1. Characteristics of Patients and Healthy Volunteers Involved in This Study 2DE NCa
no. gender (male/female) Age ((SD) Hp (mg/mL),c a
Normal controls.
b
Western blot BD
BD
VKH
NCa
BD (active)
18 16/2 34.3 ((6.5) /
22 16/6 36.2 ((11.7) /
20 11/9 31.5 ((8.3) 1.09 ( 0.84
15 13/2 33.3 ((6.5) 3.66 ( 2.21
NCa
8 8 20 8/0 8/0 11/9 29.8 ((8.1) 33.3 ((6.9) 32.4 ((9.3) / / /
ELISA BD (inactive)
VKH
6 20 6/0 14/6 38.3 ((7.7) 36.9 ((10.7) 0.95 ( 0.76 0.80 ( 0.73
Hp level in serum determined by ELISA. c Data are represented as means ( SDs.
Table 2. 2-DE and MS Identification of Proteins That Are Pp-Regulated in Serum from BD Patients spot no.a
protein descriptionb
accession no.c
1 2 3 4
Haptoglobin Haptoglobin Haptoglobin Serum amyloid A
gi|3337390 gi|3337390 gi|3337390 gi|337748
protein scored
matched peptides
sequence coverage %e
265 202 129 210
6 9 5 6
18.40% 18.40% 18.40% 68.90%
MW (kD)/pIf
38.2/6.14 38.2/6.14 38.2/6.14 13.5/6.28
fold changeg (BD/NC, n ) 4)
14.9 5.6 9.2 359.4
a Spot numbers correspond to those shown in Figure 1B. b Protein description in NCBI database. c Accession number was recorded as a reference for the identification in NCBI database. d The MASCOT search score of identified proteins. e Coverage of protein sequence by the peptides was used for spot identification. f Measured molecular mass (kDa) and pI. g The ratio of protein expression levels between BD and normal controls (NC) was determined by statistical analysis (Figure 1C, p ) 0.000, n ) 4).
significantly increased level of haptoglobin (Hp) and serum amyloid A (SAA) in serum from active BD patients.
2. Experimental Details 2.1. Clinical Specimen. Eighteen BD patients with active uveitis, six BD patients without active uveitis, 22 Vogt-KoyanagiHarada (VKH) syndrome patients with active uveitis who did not receive any immunosuppressive agent systematically, and 20 healthy individuals were included in the study (Table 1). The diagnosis of BD and VKH syndrome was made, respectively, according to the International Study Group for BD,11and the diagnostic criteria revised for VKH disease by an international committee on nomenclature.12 Serum samples were collected with the informed consent of all patients and the procedures were approved by the Institutional Review Board of the Zhongshan Ophthalmic Center of Sun Yat-sen University. A total amount of 5 mL of blood was collected in 10 mL glass tubes and allowed to clot for 1.5 h at room temperature. The clotted material was removed by centrifugation at 3000g for 10 min. The obtained serum was stored at -80 °C until use. Blood samples from eight male BD patients with active uveitis and eight male healthy individuals were used for the screening study. All the samples from 18 active BD patients, 6 inactive BD patients, 22 VKH syndrome patients, and 20 healthy individuals were used for the validation study. 2.2. Serum Sample Preparation. Serum samples were treated using the ProteoPrep Blue albumin and IgG depletion kit (Sigma-Aldrich) according to the manufacturer’s instructions at room temperature. The total protein concentration of the depleted serum was determined using the Protein Assay Kit (Bio-Rad, Hercules, CA). The depleted serum was stored at -80 °C until use. 2.3. 2-DE and Image Analysis. For 2-DE, the first-dimension separation was carried out with immobiline drystrips (17 cm, pH 4-7; Bio-Rad, Hercules, CA). Each strip was rehydrated for 16 h at room temperature with 340 µL of protein sample, and isoelectric focusing (IEF) was performed in a PROTEAN IEF Cell (Bio-Rad, Hercules, CA). The strips were then equilibrated in equilibration buffer (6 M urea, 2% SDS, 50 mM Tris-HCl, pH 8.8, and 30% glycerol (w/v)) for 30 min. The second-dimension
separation was performed on homemade 12% polyacrylamide gels on the Bio-Rad system at a constant current of 25 mA/gel at 20 °C for 10 h. Each pooled sample was run four times to minimize run-to-run variation. The gels were visualized with the “blue silver” protocol.13 The stained gels were scanned in an Imagescanner (GE healthcare, Piscataway, NJ) operated by Labscan 3.0 software (GE healthcare, Piscataway, NJ). Image analysis including spot detection, quantification, and normalization was performed using the ImageMaster 2D Elite software 5.0 (GE healthcare, Piscataway, NJ). The relative intensities of spots were used for comparison between the two groups and only the significantly different spots were selected for further analysis by MALDITOF/TOF-MS. 2.4. Protein Identification by MALDI-TOF/TOF-MS. Differentially expressed protein spots were excised from the gels. The gel pieces were extensively washed with ammonium bicarbonate. The protein in the dried gel plug was digested overnight at 37 °C with sequencing-grade modified trypsin (Promega, Madison, WI). The tryptic digests were recovered by incubation with 50% acetonitrile (CAN) /0.1% trifluoroacetic acid (TFA), and the isolated peptides were concentrated under nitrogen gas and then subjected to mass spectrometry. A database search against Swiss-Prot was performed with MASCOT software (Matrix science, Boston, MA). 2.5. Hp and SAA Detection by Western Blot. For Western blot analysis on serum samples, there is no stably expressed protein to be used as internal control. Total serum protein concentration was precisely determined by the DC Protein assay kit using gamma globulin as a standard (500-0111, BioRad, Richmond, CA) and an equal protein amount was loaded onto 12% SDS-PAGE gels. The protein samples were separated and transferred onto PVDF membrane for 1.5 h at 110 V. After blocking the membrane with 5% milk solution in TBS for 1 h at room temperature, the membrane was incubated with mouse monoclonal antibodies against human Hp (1:4 000, ab13429, Abcam, Cambridge, U.K.), and serum amyloid A (1: 2000, ab687, Abcam, Cambridge, U.K.), respectively. The immunoreactive bands were visualized using Phototope-HRP Western Blot Detection System (Cell Signaling, Danvers, MA). Journal of Proteome Research • Vol. 7, No. 10, 2008 4501
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Mao et al.
Figure 1. Representative 2-DE images of serum proteome before (A) and after (B and C) the depletion of albumin and IgG are shown. Each pooled serum sample was run four times. The images were analyzed using ImageMaster 2D Elite software 5.0 and relative intensity of protein spots were obtained. Quantitative comparison revealed that the expression levels of four protein spots (spots 1-4) were significantly up-regulated in active BD as compared with those in normal controls (NC) (p ) 0.000, n ) 4, D). Detailed patterns of these four spots were shown in four pairs of gels (E).
2.6. Hp Detection by ELISA. The serum Hp level was measured by ELISA. A 96-well microplate was coated with the diluted capture antibody (1:2000, ab13429, Abcam, Cambridge, U.K.) and incubated overnight at room temperature. After a wash with TPBS, the microplate was blocked with 1% BSA at room temperature for 2 h. After a wash step, samples and standards (4890-0504, AbD Serotec, Oxford, U.K.) were added to the wells and incubated at room temperature for 2 h. HRPconjugated sheep polyclonal antibody (1:1000, ab 35312, Abcam, Cambridge, U.K.) was used for detection of Hp and it was incubated at room temperature for 2 h. Substrate solution was added to each well after washing with TPBS and the solution was incubated at room temperature for 20 min. Stop solution 4502
Journal of Proteome Research • Vol. 7, No. 10, 2008
was added and the optical density at 450 nm was determined immediately. 2.7. Statistical Analyses. All the data are expressed as the mean ( SD. Differences between groups were analyzed for statistical significance by analysis of variance and the t-test using SPSS software (version 13.0). A p-value of
