Serum Proteome of Leprosy Patients Undergoing ... - ACS Publications

Serum Proteome of Leprosy Patients Undergoing Erythema Nodosum Leprosum Reaction: Regulation of Expression of the Isoforms of Haptoglobin Nishma Gupta,† Nallakandy P. Shankernarayan,‡ and Kuppamuthu Dharmalingam*,† Department of Genetic Engineering, School of Biotechnology, Madurai Kamaraj University, Madurai-625021, Tamil Nadu, India, and Voluntary Health Services, Leprosy Project, Sakthinagar, Erode-638315, Tamil Nadu, India Received April 20, 2007

Validated proteome profile allows better understanding of disease progression, subtype classification, susceptibility patterns, and disease prognosis. Leprosy is a spectral disease, with clinically, histologically, immunologically, and bacteriologically distinguishable subtypes. In addition, a significant fraction of patients undergo immune mediated reactions even after multidrug therapy (MDT). Erythema nodosum leprosum (ENL) is an immune complex mediated reactional condition in leprosy, characterized by a systemic inflammatory condition afflicting borderline lepromatous (BL) and lepromatous leprosy patients (LL). In this study, we have analyzed serum proteome of leprosy patients undergoing ENL reactions and compared it with that of healthy noncontact controls. Depletion of albumin and immunoglobulin G (IgG) was optimized using Aurum serum protein mini kit (Bio-Rad), and then two-dimensional gel electrophoresis (2-DE) of these serum samples was performed. Differentially expressed proteins were identified by MALDI-TOF and MALDI-TOF MS/MS mass spectrometry. Significant increase in one of the isoforms of R2 chain of haptoglobin was observed in ENL condition. In addition, haptoglobin phenotype was determined for healthy controls and leprosy patients. Hp 0-0 phenotype was detected in 21.4% of the ENL patients undergoing treatment, which on follow up examination showed typable phenotype, thus showing a condition of acquired anhaptoglobinemia. Since ENL still remains a threat to leprosy disease management, the above findings may provide new insights in understanding the development and progression of this inflammatory condition. Keywords: aurum kit • ENL • haptoglobin • isoform • leprosy • serum

Introduction Leprosy is a chronic infectious disease caused by Mycobacterium leprae, and the disease mainly affects the skin and peripheral nerves.1 In spite of the effective multidrug therapy (MDT), leprosy affects an estimated 700,000 new individuals each year.2 Among the registered leprosy cases, 83% are concentrated only in six countries: India, Brazil, Burma, Indonesia, Madagascar, and Nepal.1 Continued high incidence level in endemic areas is thus a cause for concern even today. Leprosy presents as a spectral disease manifesting in various clinical forms, ranging from the paucibacillary tuberculoid leprosy (TT) stage to the multibacillary lepromatous leprosy (LL) stage with intermediate borderline forms. The clinical classification of leprosy is complicated by “reactional states”, which represent alterations in pathogen specific host immunity. Two well-recognized reactional states * To whom correspondence should be addressed. Prof. K. Dharmalingam, Department of Genetic Engineering, School of Biotechnology, Madurai Kamaraj University, Madurai-625021. Tamil Nadu, India. E-mail, [email protected]; Fax, +91-452-2459105; Phone, +91-452-2458211. † Madurai Kamaraj University. ‡ Leprosy Project. 10.1021/pr070223p CCC: $37.00

 2007 American Chemical Society

are reversal reaction (RR; Type 1) and erythema nodosum leprosum (ENL; type2). The type 1 reversal reaction is characterized by an increased cell mediated immune response against the antigens of M. leprae. Erythema nodosum leprosum (ENL) is an immune complex mediated reaction occurring in 20% of LL and 10% of BL patients either before or during the course of the antibiotic treatment.1,3 Complexes of M. leprae antigens and their respective antibodies deposit in tissues, followed by neutrophil infiltration and complement activation leading to systemic inflammatory responses. Elevated levels of cytokine tumor necrosis factor-R (TNF-R) may mediate the immunopathologic manifestations of ENL.4 ENL is clinically characterized by eruption of tender, erythematous painful nodules; fever; malaise; and other associated complications such as iritis, neuritis, lymphadenitis, bone pain, arthritis, and proteinuria.1,3 As this reactional state occurs even in patients treated with antibiotics, disease management poses a challenge. Even with sustained exposure to M. leprae, only 0.5% of those exposed individuals develop sub-clinical infection and of these, only 25% succumb to the more serious forms of the disease.5 Comparative sequence analysis of M. leprae genome revealed that clinical isolates of the pathogen from different geographical Journal of Proteome Research 2007, 6, 3669-3679

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research articles regions were highly conserved.6 Therefore the variability in disease progression cannot be entirely ascribed to the variation in the virulence of M. leprae isolates. Further studies have implied genetic predisposition to link the variability in host response to M. leprae infection. TLR2 polymorphism and variations in the regulatory region shared by PARK2 and PACRG genes (associated with Parkinson’s disease) are some of the recent genetic studies, which imply host susceptibility leprosy.2 Also major histocompatability class II antigen, HLA-DR2, has been associated with susceptibility to leprosy in Indian populations.7 However, universal application of these inferences to different populations needs to be validated. The importance of this aspect is seen in studies,8,9 which have focused on IL10 promoter polymorphism in determining the outcome of leprosy and have shown involvement of different haplotypes in susceptibility/resistance to leprosy in Brazilian8 and Indian populations.9 Thus, in addition to genetic predisposition and bacterial virulence, host-pathogen interactions play a very important role in disease progression in leprosy. Nonavailability of animal models to study leprosy makes indirect approaches to study the host pathogen interactions in leprosy as the only option. Studies have mainly focused on cytokine mRNA profiles in lesion biopsies10,11 and peripheral blood immune cells12,13 of leprosy patients. Immunochemical assays and PCR based approaches have also been used to measure host response of selected set of genes. Use of microarray based gene expression profiling has shown that distinction in gene expression correlates with and accurately classifies the clinical forms of the disease.14 But complementing these nucleic acid based studies with protein profiles of patients is lacking. Examination of protein profile in disease condition will give significant insights into protein modifications, proteinprotein interactions, host-pathogen interactions, and disease susceptibility and progression. There are several examples of serum and plasma proteome studies that revealed biomarkers from blood could be a valuable source of information.15 In this study, we used serum samples of leprosy patients undergoing ENL reaction to examine the proteome profile using high-resolution two-dimensional gel electrophoresis (2-DE),16 and the proteins of interest were identified using matrix assisted laser desorption /ionization-time-of-flight (MALDI-TOF) and MALDI-TOF MS/MS mass spectrometry. We observed significant changes in the expression of haptoglobin as well as the changes in the isoforms of polymorphic R chain of haptoglobin in ENL patients when compared to healthy controls. Haptoglobin is a hemoglobin binding acute phase protein, composed of two polypeptide chains R and β linked covalently via disulfide bonds.17 Haptoglobin also shows polymorphism due to R1F, R1S, and R2 forms of R chain which gives rise to atleast six different haptoglobin phenotypes viz 1F-1F, 1S-1S, 1F-1S, 2-1F, 2-1S, and 2-2. We also report here, for the first time, the haptoglobin phenotype pattern in Indian population using 2D analysis. And this study also shows Hp 0-0 condition, as an acquired phenotype, present only in patients undergoing treatment for leprosy. As far as we know, this is the first report of the proteome level analysis of sera from leprosy patients.

Experimental Section Serum Samples and Collection. Serum samples were collected from healthy controls (n ) 9) in Madurai Kamaraj University, Madurai and from leprosy patients undergoing ENL reaction (n ) 14) at the Voluntary health services center, Sakthi Nagar, Erode, Tamil Nadu, India. The male to female ratio in 3670

Journal of Proteome Research • Vol. 6, No. 9, 2007

Gupta et al. Table 1. Profile of Leprosy Patients Analyzed in This Study patient ID a

age (years)

sex

typeb

bacterial indexc

treatmente

L1 L2 L3 L4 L5 L6 L7 L8 L9 L10 L11 L12a L12b L13a L13b L14a L14b

48 54 44 62 23 31 48 44 33 24 31 49 49 43 43 38 38

F F F M M M M M M M M F F M M F F

LL/ENL LL/ENL LL/ENL LL/ENL LL/ENL LL/ENL LL/ENL LL/ENL LL/ENL LL/ENL LL/ENL LL/ENL BL LL/ENL LL/ENL LL/ENL -

5.0+ 4.0+ 1.0+ 1.5+ 2.2+ 3.0+ 3.0+ 3.0+ 2.0+ 2.0+ 3.0+ 2.5+ N.D.d 3.0 N.D.d 3.5+ -

MDT MDT Thalidomide Thalidomide Thalidomide Thalidomide Thalidomide Thalidomide Thalidomide Thalidomide Thalidomide Untreated MDT + Thalidomide Untreated Thalidomide MDT + Thalidomide Released from treatment

a L12-L14 are three follow up cases where suffix a and b refer to the different stages of treatment. b Patients are immunohistologically classified according to the Ridley and Jopling system of classification (reference 18 in text). Patients were screened for HIV, TB infection, and other routine dermatological complications. c Bacterial index (BI) is a direct measure of the bacillary load of the patient. Slit skin smears of individuals are stained; bacilli count is done and represented in Ridley’s logarithmic scale of 0-6. d Not determined. e Patients were either given MDT, Thalidomide, or a combination of both. MDT treatment includes clofazimine, rifampicin, and dapsone.

both controls and patients was 2:1. The mean age ( SD for controls and ENL patients was 29.8 ( 4.8 and 40.8 ( 11.3 years respectively. Patients were immunohistologically classified according to the Ridley and Jopling system of classification18 and routinely screened for HIV and TB infection and other minor ailments. Clinical profile of leprosy patients is given in Table 1. Blood samples were collected by venipuncture into glass tubes and allowed to clot for 30 min at room temperature. Serum was then separated from the clotted blood by centrifugation at 1300 g for 10 min. Serum was stored in aliquots at -70 °C until further use. Patient sera were collected following the norms prescribed by the Institutional Ethical Committee and Indian Council of Medical research (http://icmr.nic.in). Sample Preparation. For the removal of highly abundant proteins in serum, albumin, and immunoglobulin G (IgG), 60 µL of serum sample was treated with Aurum serum protein mini kit as described by the manufacturer (Bio-Rad) with minor modifications. Briefly, serum was diluted 4-fold with the binding buffer (20 mM Phosphate buffer, pH 7.0) provided in the kit and sonicated (3 cycles of 10 s each) using Vibra cell sonicator. Two hundred microliters of diluted sample was applied to the 2 cm column and vortexed. The column was then incubated for 5 min. Vortexing and incubation was repeated three times and finally the column was centrifuged at 10,000g for 20 s. The unbound protein fraction was collected. The column was washed again with 200 µL of binding buffer, and the supernatant was combined with the earlier fraction. The depleted serum was directly used for one-dimensional SDS-PAGE and 2-DE. Neat serum samples were diluted in SDS buffer (25 mM Tris pH 7.4, 2% SDS, 2.3% DTT) and incubated in boiling water for 3 min before analysis. SDS-PAGE Analysis of Serum Proteins. Whole serum and depleted serum samples were prepared for one-dimensional analysis19 by diluting the samples in 1× loading buffer (0.08 M Tris HCl, pH 6.8, 2.7%SDS, 13.7% glycerol, 0.97 M β-mercap-

Serum Proteomics of Leprosy Patients

toethanol, 0.3% bromophenol blue) and kept at 100 °C for 3 min before loading. Two-Dimensional Gel Electrophoresis of Serum Proteins. Protein estimation of samples was done using Bradford’s method.20 For rehydration, serum samples were diluted into a buffer containing 7 M Urea, 2 M Thiourea, 4% CHAPS, 0.5% ampholytes, 50 mM DTT, 2 mM TBP, and 0.004% of bromophenol blue. Wherever applicable, the final concentration of SDS was brought below 0.025% before rehydration. Samples were then applied to an 18 cm, pH 3-10 Nonlinear (NL), or 4-7 linear or 3-5.6 Nonlinear IPG strip (GE Healthcare) by passive rehydration for a minimum of 12 h using the rehydration tray. Rehydrated strips were washed with a stream of water and then focused at a temperature of 20 °C using the IPGphor IEF apparatus (GE Healthcare) as per the following protocol: -500 V for 1 h (step and hold), 1000 V for 1 h (gradient), 8000 V for 3 h (gradient), and 8000 V for 5 h (step and hold). Total volt hours obtained in all experiments is above 54 000. The focused strips were stored at -70 °C until the second dimensional analysis or used immediately. The strips were brought to room temperature and subjected to two-step equilibration (15 min each) to reduce and alkylate the proteins. Equilibration buffers contained 6 M Urea, 2 M Thiourea, 50 mM Tris, 34.5% Glycerol, 2% SDS, and 0.005% bromophenol blue with 2% DTT for the first step and 2.5% iodoacetamide replacing DTT in the second step. Strips were then layered on top of a 12.5% polyacrylamide gel and sealed with 0.5% agarose in electrophoresis buffer. DALT six apparatus (GE Healthcare) was used for second dimension electrophoresis (20 W for 30 min and then 60 W for 5 h). Staining. Gels were stained using colloidal Coomassie Blue, Ammoniacal silver or the MALDI compatible silver staining method. For colloidal Coomassie staining,21 the gels were fixed in 40% methanol and 10% acetic acid in water for an hour. After washing 3 times in water, for 10 min each, gels were stained overnight in Coomassie stain containing 10% phosphoric acid, 10% ammonium sulfate, 0.12% Coomassie Blue G-250, and 20% methanol. Gels were then destained in water. Ammoniacal silver staining22 was used to detect low-abundance proteins and in these gels protein load was 30 µg or less. The gels were washed in water for 5 min. The gels were then fixed in 40% ethanol and 10% acetic acid in water for 1 h and then in 5% ethanol and 5% acetic acid in water for 2 h to overnight. After a single wash with water for 5 min, gels were sensitized in 1% glutraldehyde and 0.5 M sodium acetate. After washing in water for 10 min gels were put in 0.05% 2,7-Naphthalene disulfonic acid for 30 min. This was followed by washing four times in water for 15 min each and staining in ammoniacal silver nitrate solution containing 0.8% w/v silver nitrate, 0.2 N NaOH, and 0.013% Ammonia for 30 min. Staining solution was discarded, and the gels were washed four times in water for 4 min each. Gels were developed in 0.01% citric acid solution containing 0.1% formaldehyde. When the background starts staining, the development was stopped with 5% w/v Tris and 2% v/v acetic acid stop solution. For MALDI compatible silver staining, the following method of Blum was used with minor modifications.23 Gels were fixed in 40% ethanol and 10% acetic acid in water for 1 h after which they were rinsed in 30% ethanol for 20 min. Gels were then given three water washes for 10 min each and then sensitized in 0.02% sodium thiosulfate for 1 min. After water wash for 20 s (3 times), gels were stained in chilled 0.1% silver nitrate solution at 4 °C. After a water wash for 10 s, gels were developed

research articles in 2% sodium carbonate and 0.04% formalin. Subsequently a water wash of 20 s was given. Development was stopped with 5% acetic acid solution, and then the stained gels were washed three times in water for 10 min each. Image Analysis. Image analysis of gels was done using ImageMaster 2D Platinum version 5.0 software (GE healthcare). For comparing the differential expression, all the control and ENL 2D gels were assigned to two different classes. Spots within the region of analysis were detected and then matched between each gel and the reference gel. Visual inspection was used to confirm the data points wherever needed. The quantification value used for comparison was % intensity, which refers to the intensity of the protein spot taken against the total intensity of all proteins in the gel region selected for analysis. To minimize the error between the gels, the ratio of intensity for the isoforms of interest from each sample was calculated. The statistical significance of the differences between the healthy controls and the patients was determined using the Student’s t-test. In-Gel Tryptic Digestion. Selected protein spots stained were excised manually using a 1 mm diameter metal punch from the 2D gel. The colloidal Coomassie stained gel pieces were first washed two times in water for 10 min each. The pieces were destained by incubation in 25 mM ammonium bicarbonate made in 50% acetonitrile, three times for 15 min each. After dehydrating the gel pieces in 100% acetonitrile for 15 min, the gel pieces were dried under vacuum for 30 min. Dried gel pieces were rehydrated for 30 min on ice with 400 ng of trypsin (Promega) made in 5 µL of 100 mM ammonium carbonate in 10% acetonitrile. Excess trypsin was then removed, and the gel pieces were soaked in 20 µL of 40 mM ammonium bicarbonate in 10% acetonitrile and incubated at 37 °C in a water bath for 16 h. The tubes were then briefly centrifuged for 5 s, and the supernatant saved. Peptides were then extracted from gel pieces using 25 µL of 0.1% trifluoroacetic acid (TFA) made in 60% acetonitrile, by sonication for 3 min at a frequency of 2200 MH in Soltech Ultrasonic cleaner (Spinco Biotech limited). The tubes were then centrifuged for 5 s and incubated for 10 min. Supernatant obtained after centrifugation was added to the first supernatant. Twenty microliters of 100% acetonitrile was added to gel pieces for dehydration and vortexed. The tubes were then centrifuged for 5 s and incubated for 10 min. The supernatant was combined with the first two supernatants and these extracted peptides were then vaccum dried for 60-90 min. Dried peptides were dissolved in 5 µL of 0.1% TFA in 5% acetonitrile. Samples were then concentrated and desalted using eppendorhf Perfect pure C18 Tips and eluted in 5 µL of 0.1% TFA in 50% acetonitrile as per the manufacturer’s instructions. MALDI-TOF Spectrometry of Tryptic Digests. R-Cyano-4hydroxy cinnamic acid (CHCA) matrix (10 mg/mL) was made in 70% acetonitrile and 0.03% TFA. Another matrix used was 2 mg/mL of CHCA with 50 mM ammonium monobasic phosphate as additive.24,25 In some cases 2,5-Dihydroxybenzoic acid matrix (50 mg/mL) made in 99% acetone was also used since this matrix gave less nonspecific peaks. Peptide samples were applied on a stainless steel MALDI target plate using the sandwich method. When CHCA matrix was used, 0.5 µL of matrix was first applied on the plate, allowed to dry. Sample (0.5 µL) was applied and then immediately layered on top with 0.5 µL of matrix. Sample was diluted 5 fold in CHCA matrix containing ammonium phosphate, and 1 µL of the sample was applied. Peptide mass spectrum was acquired using Axima CFR Journal of Proteome Research • Vol. 6, No. 9, 2007 3671

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Table 2. Mass Spectrometric Identification of Protein Spots by MALDI-TOF and MALDI-TOF MS/MS Mass Spectrometry spot label

1

identified proteina

database/ accession no.

peptides matchedc

scoreb

Mr/pI 2-Dd

Mr/ pI data base f

SC g (%)

Serum Albumin, Chain A IgH G1 protein Haptoglobin precursor Orosomucoid1 IGKC protein (Ig kappa chain) ApoA1 protein (fragment) Hp 2-alpha Hp 2-alpha

MSDB/1AO6A

94 (64)

15/18

66/4.1

65.6/5.63

28%

NCBI/gi|17939658 Swiss-Prot/HPT_HUMAN

73 (65) 70 (55)

11/35 11/17

59/6.9 45/5.25

51.5/8.45 45.8/6.13

35% 25%

NCBI/gi|5469442 NCBI/gi|47125459

85 (64) 68 (65)

10/50 9/39

45/4.1 28/- e

23.7/4.93 26.1/8.17

44% 35%

MSDB/CAA00975

121 (64)

10/11

28/5.31

28/5.27

44%

NCBI/gi|296653 NCBI/gi|296653

59 (64) 76 (32)

21/5.75 21/5.75

42.1/6.25 42.1/6.25

19% 3%

7′ 8

Hp 2-alpha Hp 2-alpha Hp 2-alpha

NCBI/gi|296653 NCBI/gi|296653 NCBI/gi|296653

65 (65) 48 (64) 36 (35)

22/5.8 21/5.5 21/5.5

42.1/6.25 42.1/6.25 42.3/6.23

19% 20% 4%

9

Hp 2-alpha Hp 2-alpha

NCBI/gi|296653 NCBI/gi|296653

76 (65) 144 (51)

21/6.25 21/6.25

42.1/6.25 42.3/6.23

23% 9%

HUMTHYA (transthyretin) Haptoglobin precursor HPT_HUMAN (Haptoglobin alpha) Haptoglobin Hp 2-alpha HUMAPOIV (apolipoprotein a-IV)

MSDB/AAA61181

109 (64)

6/21 TEGDGVYTLNNEK (101-113) 6/21 5/20 LRTEGDGVYTLNNEK (99-113) 7/15 (1) NPANPVQR (136-143) (2) LRTEGDGVYTLNDK (40-53) (3) LRTEGDGVYTLNNEK (99-113) 6/20

17.4/5.66

12.8/5.33

81%

2 3, HPr 4 5 6 7

10 11 12 13 14 Ap

NCBI/gi|306882

66 (65)

5/7

15.4/4.95

45.8/6.2

12%

SwissProt/P00738

45 (60)

3/4

15.4/5.25

38.9/6.13

11%

NCBI/gi|1212947 NCBI/gi|296653 MSDB/AAA51748

41 (64) 68 (64) 152 (64)

4/7 9/40 11/20

15.4/5.8 21/5.2 45/5.2

38.4/6.27 42.1/6.25 43.3/5.22

12% 26% 35%

a Haptoglobin, which exists as a precursor protein, is proteolytically cleaved to form R and β chains. During database search for protein identification, multiple hits in form of various forms of haptoglobin are obtained. Also across the databases, the protein names and accession number are varied. For these cases, we have compared the Mr/pI values of the respective spots from 2D gels with the corresponding spot Mr/pI values in the plasma map in SWISS-2DPAGE database and confirmed the identity. b MOWSE scores greater than the values given in the parenthesis are considered to be significant (p < 0.05). For proteins listed with a score less than the significant score, the identification was done by peptide sequencing of one of the matched peptides by MALDI-TOF-PSD. These identifications done by sequencing are listed in bold in the table. All proteins were also searched across multiple databases to confirm their identity. The hits were simultaneously compared with the respective spots on plasma map given in the SWISS-2DPAGE database (http://www.expasy.org/cgibin/ map2/def?PLASMA_HUMAN) and the identity confirmed. c Represents the peptides matched among the peptide list searched. For proteins identified by sequencing of a matched peptide, the sequence of the peptide is listed along with the peptide coordinates in parenthesis. d Apparent/experimental molecular weight and pI of protein spot on 2-DE gels. e Apparent pI could not be determined as this spot was picked up from a 2D gel, where a nonlinear IPG strip was used in first dimension. f Theoretical molecular weight and pI of the identified protein in database. g Sequence coverage (SC) represents the % sequence covered in the protein by the peptides matched.

plus (KRATOS Shimadzu) MALDI-TOF mass spectrometer in the reflectron mode. The acceleration voltage after pulsed extraction was 20 000. The instrument was calibrated using external standards Bradykinin (757.39 Da), Angiotensin II (1046.54 Da), P14R (1533.85 Da), and ACTH fragment 18-39(2465.19Da). The monoisotopic peak list was generated with Kratos LAUNCHPAD software version 2.4, without using the smoothing function and the peak filter was applied to exclude masses lower than 750 daltons. The signal-to-noise ratio of 20 and above was used for database search. The monoisotopic masses were processed for identification by using the search engine, MASCOT versions 2.1 and 2.2 (www.matrixscience.com). Search was performed in NCBInr, MSDB, and SwissProt databases. The search parameters were as follows: tolerance, 0.05-1 Da; species, Homo sapiens; maximum number of missed cleavages was set to 1 for all samples, except for one sample set at 2 and for two samples set at 3; fixed modification, carbamidomethyl (for cysteine modification by iodoacetamide); variable modifications, oxidation of Methionine and Propianomide (for cysteine modification by acrylamide). For peptide 3672


sequence searching of the MS/MS data obtained by Post source decay (PSD), NCBInr database in MASCOT version 2.1 and 2.2 (www.matrixscience.com) was used. The search parameters for MS/MS data analysis were: precursor-ion and fragment-ion mass tolerance were set to 0.5 and 0.8 Da respectively; fixed modification, carbamidomethyl; variable modifications, oxidation of Methionine and Propianomide; maximum missed cleavages -1; species, Homo sapiens. The criteria for determining protein/ion scores which are significant (p < 0.05) is given in Table 2 legend. The details of the database versions and the entries during the search of peptide mass fingerprint (PMF) and PSD data for each spot is provided in Supplementary Table 1, see Supporting Information.

Results Depletion of Albumin and Immunoglobulin G (IgG). Visualization of low-abundance proteins from human serum requires removal of high-abundance proteins such as albumin


Figure 1. SDS-PAGE analysis of human serum before and after removal of albumin and IgG. Equal amount (10 µg) of protein was loaded in each lane, separated on a 12.5% gel, and stained with colloidal Coomassie Blue as described. Lane 1 is the protein molecular weight marker. Lanes 2 and 4 represent whole serum samples before albumin and IgG removal. Lane 3 and 5 represent albumin and IgG depleted serum samples. Lanes 2 and 3 are serum samples from a healthy control. Lanes 4 and 5 represent serum samples from an ENL patient. A1, A2 represents major protein bands increased in intensity after depletion. B represents band of albumin, which gets depleted after treatment as shown in box 1. D represents band of IgG heavy chain, which gets decreased after treatment as shown in box 2. C1, C2 represents appearance of new bands after depletion (see discussion).

and IgG. Aurum serum protein mini kit (Bio-Rad), which selectively removes albumin and some isotypes of IgG (IgG1, IgG2, IgG4), was used in this series of experiment. The whole serum samples, before and after depletion using the above kit were analyzed using one-dimensional SDS-PAGE to check the efficiency of the depletion method. As shown in Figure 1, albumin and IgG are efficiently removed after treatment. Removal of these proteins revealed the presence of other comigrating proteins. Within the limits of sensitivity of the staining method, nonspecific loss of proteins was not observed. This method of depletion worked equally well for sera from leprosy patients and healthy controls (Figure 1, compare lane 3 to lane 5). Samples treated using the optimized procedure was then subjected to two-dimensional analysis for comparison of proteome profiles. Both 4-7 and 3-10 pH range 18 cm IPG strips were used in the first dimension to check the reproducibility and efficiency of the depletion method. Though albumin could be removed completely, IgG heavy chains could not be completely removed (Figure 2B). This could be because of comigrating proteins or the increased levels of circulating antibodies in lepromatous leprosy and ENL condition.26-28 Also we used highly sensitive ammoniacal silver staining (detection limit, 1-10 fmoles), unlike the earlier studies,29-31 which used less sensitive staining methods. Comparison of the depletion pattern of IgG heavy chains in controls and leprosy serum samples (Supplementary Figure 1, see Supporting Information) showed that IgG heavy chains were effectively removed in control samples unlike the ENL samples. In Aurum kit treated serum samples, a concomitant increase in the amount of

research articles haptoglobin R chain, haptoglobin precursor, and orosomucoid was observed. In addition, amount of transthyretin increased in Aurum kit treated serum. However, the amount of immunoglobulin light chain and apolipoprotein A1 proteins reduced after the treatment under our experimental conditions (Figure 2). Profile of Haptoglobin Isoforms. Haptoglobin R chain was initially identified by comparing protein profile with the plasma map in SWISS-2DPAGE database (http://www.expasy.org/cgibin/map2/def?PLASMA_HUMAN) based on the calculated pI and molecular weight. The identity of these spots was further confirmed by tryptic in gel digestion and MALDI-TOF mass spectrometry as described under materials and methods. The haptoglobin phenotype for each serum sample was then assigned on the basis of the pattern of haptoglobin R chain observed.17 In order to confirm the presence or absence of weak spots, gels were stained using ammoniacal silver22 and MALDI compatible silver staining.23 Out of the 23 samples analyzed, three phenotypes Hp 2-2, 2-1, 0-0 were distinctly observed. In controls, the incidence of Haptoglobin phenotypes 2-2 and 2-1 is 77.7 and 22.2%, respectively. In leprosy patients, the incidence of Hp 2-2, 2-1, and 0-0 is 57.1, 21.4, and 21.4%, respectively. Hp 1-1 phenotype was not found either in healthy controls or leprosy patients. In control samples, only Hp 2-2 and Hp 2-1 were observed whereas in leprosy serum samples, apart from these 2 phenotypes, Hp 0-0 was also detected. Patients carrying Hp 0-0 phenotype were recruited for a follow up study. 2-DE, MALDI, and MS/MS Analysis of Haptoglobin R Chain Spots. In the Coomassie stained gels, (Figure 3), 3 spots of R2 chains (Experimental mass/pI ) 21 Kda/5.5-6.25, respectively) and 3 spots corresponding to R1 chains (Experimental mass/ pI ) 15.4 Kda/4.95-5.8, respectively) were observed as reported earlier.17 The identity of these spots was also confirmed by MALDI analysis. In the Hp 2-2 phenotype, spots corresponding to R2 chains were observed in all samples and R1 protein spots could not be found (see Figure 3b, c, d, and e). However, in one unique case, (Figure 3c) five spots of R2 chain were observed. In this sample, apart from the usual three spots (spot 7, 8, 9), another spot (no.14) was observed at the acidic extreme and spot number 7 was a doublet (see spot 7′ at the right edge of spot 7 in Figure 3c). The above rare Hp 2-2 phenotype has been reported earlier in Caucasian population.17,32 Database search using MASCOT, identified spots 14, 7, 7′, 8, and 9 as Haptoglobin R2 chains. In the Hp 2-1 phenotype, spots 11, 12, and 13 were identified as haptoglobin precursor, haptoglobin alpha, and haptoglobin, respectively. However, the experimental mass and pI of these isoforms, comparison with the plasma map in SWISS-2DPAGE database and with the haptoglobin R chain pattern in a previous study,17 confirmed their identity as isoforms of R2 and R1 chain. In addition, MALDI-TOF MS/MS analysis was done for some protein spots whose MOWSE scores were less than significant (Refer to Table 2). A representative PSD data (also see Supplementary Figure 2, Supporting Information) is shown in Figure 4A, done for three peptides (m/z values 895.46, 1581.23, 1709.53) obtained in the PMF of spot 9 (Figure 3), which was initially identified as Haptoglobin R-2 protein by peptide mass spectrum (refer to Table 2). Analysis of MS/MS data using the NCBI database reconfirmed the identification. The aminoacid sequence of these peptides is matched (marked in bold in Figure 4B) to position 40-53 (m/z 1581.23), 99-113 (m/z 1709.53), 136-143 (m/z 895.46) of haptoglobin R2 protein. The identification of Journal of Proteome Research • Vol. 6, No. 9, 2007 3673

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Figure 2. 2D protein profile of serum sample of an ENL patient before and after removal of albumin and IgG. (A) Untreated whole serum. (B) Serum treated using Aurum kit. Protein (30 µg) was loaded for IEF (pH 3-10 NL, 18 cm IPG strips were used). Gels (12.5%) were used in the second dimension. Ammoniacal silver staining was performed as described under materials and methods. Circles 1 and 2 in (B) show depletion of albumin and decrease of IgG heavy chains respectively. Circles in B represent proteins depleted or decreased and boxes show protein spots, which increase after Aurum kit treatment (refer to text). Spot details: Spot 1, Albumin; 2, IgG heavy chain; 3, Haptoglobin precursor; 4, Orosomucoid; 5, Ig kappa C region; 6, Apolipoprotein A1; 7, 8, and 9, Haptoglobin R2; 10, Transthyretin (Refer to Table 2 for protein identification details).

Figure 3. Coomassie stained 2D gels (Partial view) showing haptoglobin R chain phenotype. (a) and (b) are control samples. (c), (d), (e), and (f) are serum samples from ENL patients. Spot 10, transthyretin. Spot numbers 7, 7′, 8, 9, and 14 indicate haptoglobin R2 chain isoforms. Spot numbers 11, 12, and 13 indicate haptoglobin R1 chain isoforms.

the protein using Mascot search in NCBInr database is given in Figure 4B. This search returned three different matches of identical scores that were significant (p < 0.05) and all of these belonged to the various forms of haptoglobin. We have confirmed that the spot is haptoglobin R2 protein by comparing pI and molecular weight values from the 2D data with corresponding spot values from plasma map in SWISS-2D PAGE database. Differential Expression of Haptoglobin Alpha Isoform. In 7 out of the 11 (typable haptoglobin phenotype) ENL cases (63.6%) analyzed, spot number 9 of the R2 chain was upregulated when compared to control samples, which could have clinical significance (see discussion). Interestingly, all the 7 ENL cases, in which this upregulation was observed, were on thalidomide treatment only. To minimize the error in calculating the intensity difference of spot 9 between the gels, the upregulation of spot 9 was analyzed by calculating the ratio of 3674


% intensity spot 7 to 9 across all gels. As shown in Figure 5A, the ratio of % intensity of spot 7 to 9 is higher in controls (1.432 ( 0.262) compared to the ENL patients (1.104 ( 0.088), and the difference is statistically significant (p ) 0.0078). In comparison, there is no significant difference (Figure 5B) in the ratio of % intensity of spot 7 to 8 in controls (2.79 ( 0.514) and ENL patients (2.51 ( 1.000). The spot 9 represents the most basic protein species among the isoforms of Haptoglobin R2 chains. The modification in this isoform is attributed to the presence of C-terminal arginine at the 143rd position.17 PSD analysis of the peptide peak (m/z value 895. 46) which covers this region (Figure 4) confirmed the peptide sequence as NPANPVQR (amino acid position 136-143). Acquired Anhaptoglobinemia. Among all the control (n ) 9) and ENL (n ) 14) samples examined, three ENL patients (L12b, L13b, and L14a in Table 1) showed Hp 0-0 phenotype. Rare form of primary anhaptoglobinemia, due to the deletion


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Figure 4. Identification of Haptoglobin R2 protein by MS/MS. (A) Post source decay (PSD) spectrum of peptide with ion signal at m/z 895.46, 1581.23, and 1709.53 was done to obtain the amino acid sequence. These peptide peaks were part of the peptide mass fingerprint (PMF) data of spot 9 of Figure 3. (B) Mascot search results of the PSD data is shown. Using NCBInr database the sequence was identified (p < 0.05) corresponding to a stretch of amino acids 40-53, 99-113, and 136-143 of haptoglobin R2 protein. The identification as haptoglobin protein was because the alpha-2 chain is also contained in the precursor protein. The same sequence was also matched to Hp 2-alpha protein with identical score (see circled text in figure).

of the haptoglobin gene, was reported from Asian population.33 However, secondary anhaptoglobinemia is more prevalent under pathological conditions.33,34 To identify the nature of Hp 0-0 in our samples, we examined the serum of the Hp 0-0 patient after the patient was released from treatment. Serum of this patient showed reversion of Hp 0-0 to Hp 2-1 (Figure 6) phenotype. As shown in Figure 6A, during treatment, absence of haptoglobin R1 and R2 chains (Box 2) is accompanied by the absence of haptoglobin precursor chain (Box 1) as well. The absence of Haptoglobin chains in the serum profile in Hp 0-0 cases was further confirmed by the presence of transthyretin (T) in the expected haptoglobin R chain region (Figure 6A). The identity of this protein was confirmed by MALDI-TOF analysis. Around 45 kDa region, where the haptoglobin precursor chains proteins are present, APO-AIV protein (Ap) could

be detected. This data also confirms that the absence of haptoglobin precursor spots was not due to experimental conditions. In addition, the spots corresponding to haptoglobin isoforms could not be detected even after ammoniacal silver staining (Supplementary Figure 3, see Supporting Information). In the follow-up study, the serum sample of the above Hp 0-0 phenotype patient collected after the completion of the multi drug and thalidomide treatment (Figure 6B) showed the presence of haptoglobin R2 and R1 (Box 4 and 5) proteins as well as the haptoglobin precursor protein (Box 3). In case of the other two Hp 0-0 patients, serum samples collected before the treatment showed Hp 2-2 phenotype. Aurum kit treated serum of one of these anhaptoglobinemic patients was analyzed using 3-5.6 pH range IPG strip (Figure 7), to resolve the isoforms clearly. This analysis also confirmed Journal of Proteome Research • Vol. 6, No. 9, 2007 3675

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methods. Further, Aurum kit has been validated for biomarker studies in clinical settings in a recent study, where, Haptoglobin-1 precursor was identified as a biomarker in serum of ovarian cancer patients.30 One-dimensional SDS-PAGE analysis was initially used for optimizing the method under our experimental conditions. With the removal of albumin and IgG, increased abundance of existing proteins, new proteins, and comigrating proteins could be detected. Enhanced visualization of various protein spots was reported in depleted serum by 2D analysis, but the identity of these spots was not determined.31 In our experiments, increased abundance of haptoglobin R chain, haptoglobin precursor, orosomucoid, and transthyretin proteins was observed in depleted serum. On the other hand, we consistently observed the nonspecific decrease of ApoA1 protein. An earlier report using Aurum kit showed the nonspecific depletion of proteins transferrin and complement C3,29 but loss of ApoA1 protein was not reported. In some samples in our study, decreased levels of transferrin protein was seen. Therefore, the proteins, which showed nonspecific loss, were not used for comparative studies in depleted serum samples, and care was taken to make sure our data was not obscured by this technical drawback.

Figure 5. Relative levels of haptoglobin R2 isoforms. (A) Ratio of % intensity of spot 7 to spot 9 in control (n ) 8) and ENL (n ) 7) patients and (B) the ratio of % intensity of spot 7 to spot 8 in control (n ) 8) and ENL (n ) 6) patients. Bars represent Mean ( SD values. Statistically, the difference is significant (p ) 0.0078) in (A) and is not significant (p ) 0.51) in (B).

the above results with respect to the absence of haptoglobin precursor and alpha chain spots in the patient during treatment. This clearly shows that haptoglobin expression is affected during drug treatment. The isoform migrating at the extreme basic end (represented by spot 9 in Figure 3) is missing in this narrow range IPG separation covering pH 3-5.6, because the pI of this isoform is 6.25 (see Table 2). One of these patients is still under treatment, but the second one has been released from treatment and now shows Hp 2-2 phenotype (Supplementary Figure 4, see Supporting Information). These results show that the Hp 0-0 phenotypes in our study group are acquired anhaptoglobinemia and arose due to the treatment.

Discussion In addition to the ease of sample acquisition and processing, serum represents an important clinical sample reflecting the physiological or disease status of the host. In this report, sera from clinically well documented leprosy patients were used to examine the proteome profile. In leprosy patients, there is a close correlation between the systemic changes in circulating blood and peripheral tissue lesions,13 and hence, this approach will be of significance in understanding the disease. Albumin together with immunoglobulin G constitutes 60-80% of the total serum protein.29 Depletion of these two abundant proteins allows increased sample loading capacity, and consequently, low-abundance proteins can be detected. Comparative studies of various commercially available albumin and IgG depletion kits showed that antibody-based LC columns were superior in depletion efficiency and reproducibility29 when compared to affinity based depletion methods. We chose Aurum serum protein minikit (Bio-Rad) for the ease of use and the high sample capacity of the column when compared to other 3676


In this study, differential expression of haptoglobin and its isoforms was observed in serum of leprosy patients undergoing ENL reaction when compared to healthy controls. Therefore, we examined these changes in detail. Major biological functions of haptoglobin, including its antioxidant activity, bacteriostatic effect, and anti-inflammatory activity are mediated through capturing of hemoglobin during hemolysis.35,36 In humans, haptoglobin is coded by three alleles- Hp1F, Hp1S, and Hp2.17 The β chain coded by all three alleles is identical and polymorphism is the result of variant R chains. Hp1F and Hp1S alleles code for R chain polypeptides R1F and R1S, respectively, which are of equal length but differ in two amino acid residues. The amino acids aspartic acid and lysine at position 52 and 53 in R1F chain are replaced by asparagine and glutamic acid in R1S chain. Hp2 allele codes for R chain polypeptide R2 and is a partial gene duplication product arising from nonhomologous crossing over between Hp1F and Hp1S alleles. These 3 alleles give rise to six haptoglobin phenotypes: 1F-1F, 1S-1S, 1F-1S, 2-1F, 2-1S, and 2-2. These phenotypes have been recently identified by analyzing haptoglobin R chain tryptic peptides by MALDI-TOF MS and MALDI-quadrapole ion trap-TOFMS.17 However, conventional haptoglobin phenotyping methods36 can only discriminate 3 major phenotypes: Hp 2-2, Hp 2-1, and Hp 1-1. Data from this study shows, among the samples examined, in controls 77.7% and in leprosy patients 57.1% are Hp 2-2 phenotypes. It has been shown earlier also that Hp 2-2 phenotype is predominant in Indian populations.36-38 But these studies measured the hemoglobin binding capacity of haptoglobin using conventional methods such as onedimensional polyacrylamide disc gel electrophoresis37,38 or gel filtration technique,39 which do not resolve the haptoglobin R and β isoforms. 2-DE gives information about the levels of each isoform, which is an important indicator of maturation, and degradation process of haptoglobin in plasma. The reported Hp 2-2 frequency in the previous reports with respect to controls is similar to our data, but leprosy samples in our study showed lower Hp 2-2 frequency. The variance between our data and the earlier reports could be due to the high frequency of Hp 0-0 found in our studies. The geographical distribution of the populations examined could also be a likely explanation. Hp 1-1 phenotype was shown to be very rare in Indian


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Figure 6. Coomassie stained 2D gels of serum sample of an anhaptoglobinemic ENL patient. (A) Serum protein profile of an ENL patient undergoing MDT and thalidomide treatment. (B) Protein profile of the same patient after the completion of the treatment. Two hundred and fifty micrograms of depleted serum protein was loaded for IEF (pH 4-7, 18 cm IPG strips were used). Box 1 and 3 depict areas showing absence and presence of haptoglobin precursor chains respectively. Box 2 depicts absence of Haptoglobin R chains in the sample during treatment. Box 4 and 5 depict appearance of Haptoglobin R chains (circled spots in the box) after the completion of treatment. Spot details: T-transthyretin, Ap-apolipoprotein A-IV, HPr- Haptoglobin precursor chain, HR2-haptoglobin R2 chains, HR1haptoglobin R1 chains.

Figure 7. Silver stained 2D gels of serum sample of an anhaptoglobinemic leprosy patient undergoing ENL reaction. (A) Serum protein profile of an ENL patient before treatment. (B) Protein profile of the same patient during thalidomide treatment. Thirty micrograms of depleted serum protein was loaded for IEF (pH 3-5.6, 18 cm IPG strips were used). Box 1 and 3 depict areas showing presence (circled in box) and absence of haptoglobin precursor chains respectively. Box 2 depicts presence of Haptoglobin R chains (circled spots in the box) in the sample before treatment. Box 4 depicts disappearance of Haptoglobin R chains during treatment. Spot details: T-transthyretin, HPr- Haptoglobin precursor chain, HR2-haptoglobin R2 chains.

populations.36 Data in this report clearly shows that Hp 1-1 phenotype is absent in both control and leprosy cases examined. Hp 1-1 protein complex (R-β)2 is 86 KDa in size, and these are shown to transmigrate across vascular epithelium freely unlike Hp 2-1 and Hp 2-2 complexes, which form linear and circular multimers respectively. Population studies in Israel, Belgium, and the United States has shown that when compared to Hp 1-1 phenotype, diabetic patients with Hp 2-2 phenotype have increased risk of developing cardiovascular diseases.40 High rate of death and heart failure has been predicted in diabetic patients with acute myocardial infarction if they are Hp 2-2 and Hp 2-1 phenotype.41 Hp 2-2 phenotype is overrepresented in tuberculosis patients with advanced destruction and dissemination and in autoimmune diseases.36 In contrast, Hp 1-1 phenotype population is shown to be more prone to breast cancer and cervical carcinoma.36 Lower effectiveness of multimers in their ability to bind hemoglobin, poor antioxidant capacity, and inefficient inhibition of pros-

taglandin synthesis36 indicate perhaps the predisposition of Indian population, which is predominantly Hp 2-2 phenotype, to chronic diseases under certain conditions. Hp 0-0 phenotype was detected in 21.4% of the leprosy samples analyzed. The frequency observed is 3-fold higher when compared to earlier reports.37,38 Compared to the conventional haptoglobin phenotyping methods used in these studies,37-39 2D analysis and sensitive staining methods used in this study are more accurate, reliable, and give information about haptoglobin isoforms that the earlier studies missed completely. This anhaptoglobinemic condition is acquired, because the follow up study of patients released from drug therapy showed that the patients regained haptoglobin levels. In all these anhaptoglobinemic sera, the haptoglobin precursor chains were also not detected. Haptoglobin precursor is a single polypeptide chain synthesized in liver, containing a signal peptide and R and β chains.17 As proteolytic processing of the precursor leads to the formation of separate haptoglobin R and Journal of Proteome Research • Vol. 6, No. 9, 2007 3677

research articles β chains, the absence of precursor confirms the absence of expression of this gene in these patients. Even though anhaptoglobinemic condition was reported in other diseases such as in malaria and sickle cell anaemia,34 and in leprosy,37,38 the status of haptoglobin precursor was not studied in these cases. The mechanism of selective loss of translational competence is not clear. Additional possibility for the loss of haptoglobin could be selective proteolytic degradation or loss from the blood circulation following hemolysis. Although dapsone induced hemolysis leading to hypohaptoglobinemia in leprosy has been shown in a previous study,39 our study showed anhaptoglobinemic condition not only in MDT (includes dapsone) treated, but also in one rare case of only thalidomide treatment. Thalidomide, an immunomodulatory drug, is used in treating ENL cases as it ameliorates the inflammatory condition rapidly and avoids the side effects of steroids.42 Thus, it will be interesting to further study leprosy cases under different drug regimen to understand the clinical significance behind the development of this anhaptoglobinemic condition. ENL represents an inflammatory condition in the lepromatous end of leprosy characterized by the systemic increase in acute phase markers such as TNF-R,4 C-reactive protein (CRP),43,44 Serum Amyloid A (SAA),44 and Erythrocyte sedimentation rate (ESR).45 The ENL patients in this study show elevated ESR values in the range of 30-145 mm/hr (66.53 ( 43.46, Mean ( SD), compared to normal ESR values of 0-5 mm/hr for males and 0-7 mm/hr for females. In 63.6% of these ENL patients, one of the isoforms of the haptoglobin R2 chain (spot 9 in Figure 3) showed increased expression when compared to controls. The uniqueness of this haptoglobin R2 isoform is that it has an arginine containing C-terminus unlike the other two isoforms (represented by spot 7 and 8 in Figure 3). This arginine residue is the single amino acid separating the R and β chains of Haptoglobin and is the site of proteolysis leading to the conversion of the precursor to separate R and β chains.17 In this study, the MS/MS data of one of the peaks in the PMF of spot 9 also confirms the presence of C-terminal arginine. Canine blood analysis from an animal undergoing acute inflammation reaction has shown that 30% of the haptoglobin R chains have C-terminal arginine residue.17 Hepatic synthesis of haptoglobin is known to be induced by TNF-R,36 a cytokine with increased systemic concentrations in ENL patients.4 Thus, in ENL condition, increased in haptoglobin synthesis would result in presence of more of newly synthesized haptoglobin R chains with the C-terminal arginine residue. Deamidation of asparagine residue, a post translational modification of haptoglobin, which results in Hp R isoforms with negatively charged chains (spot 8 in Figure 3), has been proposed to affect the molecular clock of protein such as protein turnover, development and aging in peptide model studies.17 Thus, relative amounts of haptoglobin R chain isoforms in a physiological or diseased state would give information about haptoglobin turnover. Though the variations in haptoglobin levels and its potential as a biomarker has been described in various diseases such as Schizophrenia,46 Ovarian cancer,47,48 Hypercholesterolemia,49 and various other cancerous conditions,15 the specific, most basic alpha isoform increase shown in this study in a diseased condition has not been reported before. Increase in serum haptoglobin alpha levels has been shown in Ovarian cancer,47 but here total alpha levels were measured by ELISA and western blot. In our study we specifically show the differential expression among alpha isoforms itself. Preliminary follow up studies 3678


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(data not shown) of a patient has shown that, post-treatment, the isoform levels are comparable to that of healthy controls. The results are being validated for all the ENL samples in this study. The nature and amount of the isoforms in ENL cases will be important to understand the clinical status of this reactional condition.

Concluding Remarks Our study showing the demonstrable variation that is statistically significant (p ) 0.0078), with respect to haptoglobin isoforms levels could be studied further for its use as a putative diagnostic marker in ENL cases. Further, the drug induced anhaptoglobinemia demonstrated in this report is reversible after the termination of treatment. The clinical significance of this finding needs further studies. Also, these acute phase changes could either be used as independent biomarkers before the clinical signs develop or could be used to confirm the diagnostic criteria. In a similar acute phase response condition, rising levels of haptoglobin in ovarian cancer patients were shown to respond to cisplatin therapy.50 Thus, in a reactional condition like ENL, validating clinical biomarkers responding to drug therapy will help in better disease management and treatment.

Acknowledgment. K.D. thanks the Department of Biotechnology, New Delhi, India for grants CGSEM/ BT/03/002/ 87-Vol. IV and BT/PR6937/MED/14/905/2005 and the Indian Council of Medical Research for grant 63/21/002-BMS. The financial support from University Grants Commission, Govt. Of India under the programme-Centre for potential in the subject of Genomic Sciences, School of Biotechnology, F.1-41/ 2001 (CPP2) is acknowledged. N.G. was a recipient of Senior Research Fellowship from the Council of Scientific and Industrial Research, New Delhi, India. We thank Rachel Martin, Matt Openshaw, and H. R. Prashanth for their help in mass spectrometry. Supporting Information Available: Supplementary Figures 1, 2, 3, and 4 show IgG heavy chain regions after Aurum kit treatment, MS/MS data analysis, silver stained 2D gel of an anhaptoglobinemic patient, and follow up 2D gels (partial view) of an anhaptoglobinemic patient, respectively. Supplementary Table 1 contains the details of database versions and protein entries at the time of PMF search for protein identification. This material is available free of charge via the Internet at http://pubs.acs.org References (1) Britton, W. J.; Lockwood, D. N. J. Lancet 2004, 363, 1209-1219. (2) Alcaı¨s, A.; Mira, M.; Casanova, J. L.; Schurr, E.; Abel, L. Curr. Opin. Immunol. 2005, 17, 44-48. (3) Hastings, R. C.; Gillis, T. P.; Krahenbuhl, J. L.; Franzblau, S. G. Clin. Microbiol. Rev. 1988, 1, 330-348. (4) Barnes, P. F.; Chatterjee, D.; Brennan, P. J.; Rea, T. H.; Modlin, R. L. Infect. Immun. 1992, 60, 1441-1446. (5) Steger, J. W.; Barrett, T. L. In Military Dermatology, Part III: Disease and The Environment; James, W. D., Ed.; Office of the Surgeon General at TMM publications, Borden Institute: Washington, DC 1994; pp 319-354. (6) Monot, M.; Honore´, N.; Garnier, T.; Araoz, R. Coppe´e, J. Y.; Lacroix, C.; Sow, S.; Spencer, J. S.; Truman, R. W.; Williams, D. L.; Gelber, R.; Virmond, M.; Flageul, B.; Cho, S. N.; Ji, B.; PanizMondolfi, A.; Convit, J.; Young, S.; Fine, P. E.; Rasolofo, V.; Brennan, P. J.; Cole, S. T. Science 2005, 308, 1040-1042. (7) Hill, A. V. S. Ann. Rev. Immunol. 1998, 16, 593-617. (8) Moraes, M. O.; Pacheco, A. G.; Schonkeren, J. J.; Vanderborght, P. R.; Nery, J. A.; Santos, A. R.; Moraes, M. E.; Moraes, J. R.; Ottenhoff, T. H.; Sampaio, E. P.; Huizinga, T. W.; Sarno, E. N. Genes Immun. 2004, 5, 592-595.

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