Proteotyping of human haptoglobin by MALDI-TOF profiling: Phenotype distribution in a population of...

RESEARCH ARTICLE

Proteotyping of human haptoglobin by MALDI-TOF

profiling: Phenotype distribution in a population of toxic

oil syndrome patients

Carlos Rodriguez1*, Carmen Quero1*, Ana Dominguez1, Miguel Trigo1,Manuel Posada de la Paz 2, Emilio Gelpi1 and Joaquin Abian1

1 CSIC-UAB Proteomics Laboratory, IIBB-CSIC/IDIBAPS, Barcelona, Spain2 CISATER, Instituto de Salud Carlos III, Madrid, Spain

Toxic oil syndrome (TOS) is a disease that appeared in Spain in 1981 as a consequence of theingestion of an aniline-adulterated oil illegally marketed as edible. TOS affected more than20 000 people and produced over 400 deaths in the first 18 months after the outbreak. There isevidence that genetic factors could play a role in the susceptibility of individuals towards thedisease. Recently, we suggested that haptoglobin (Hp) polymorphism could also play a role inTOS. To provide a rapid method for high-throughput Hp phenotyping, we developed a two-stepMALDI-TOF procedure that allows specific identification of the three common Hp a chains.Resolution of the homologous a-1s and a-1f chains, which have a mass difference of only0.043 Da, is obtained after guanidination of the protein with O-methylisourea. We applied thisprocedure to the study of the distribution of the Hp alleles HP1s, HP1f, HP2 in a control versus aTOS-affected population, both originally exposed to the toxic oil. The MALDI-TOF proteotypingmethod was validated by a parallel analysis of the serum samples by 2-DE. Data obtained from 54TOS cases and 48 control individuals indicate significant differences in the distribution of Hpphenotypes in the two populations.

Received: June 12, 2005Revised: October 19, 2005

Accepted: October 21, 2005

Keywords:

Disease markers / Haptoglobin polymorphism / MALDI-TOF proteotyping / Serumproteomics / Toxic oil syndrome

S272 Proteomics 2006, 6, S272–S281

1 Introduction

Haptoglobin (Hp) is a highly abundant plasma glycoprotein(0.38–2.08 g/L) whose major biological function is the bindingof free hemoglobin to prevent kidney damage during hemo-lysis and to allow the recycling of the heme iron [1]. This

binding capacity also protects vascular tissues from oxidativedamage mediated by free hemoglobin [2]. Human Hp is atetramer composed of two beta (or heavy) and two alpha (orlight) chains connected by disulfide bonds that are coded inthe same gene sequence at the 16q22 locus. As far as isknown, the beta subunits always have the same structure [3].On the other hand, three main forms of the alpha subunitcoded by three Hp alleles (HP2, HP1S and HP1F) have beendescribed (a-2, a-1s and a-1f) [4, 5]. The HP2 allele originatesfrom an intragenic duplication of the HP1 alleles that resultsin three different HP2 subtypes (HP2SS, HP2SF and HP2FF), theHP2SF being the dominant subtype in Europe. Taken together,other less common alleles and the HP2SS and HP2FF subtypes,

Correspondence: Dr. Joaquin Abian, Structural and BiologicalMass Spectrometry Unit, Department of Experimental Pathology,IIBB-CSIC/IDIBAPS, Rosselló, 161, 6a i 7a planta, 08036 Barcelona,SpainE-mail: [email protected]: 134-933638301

Abbreviations: Hp, haptoglobin; OR, odd ratio; TOS, toxic oil syn-drome * These authors contributed equally to this work.

DOI 10.1002/pmic.200500434

2006 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.proteomics-journal.com

Proteomics 2006, 6, S272–S281 Clinical Proteomics S273

account only for about 1–5% of the allelic frequency in mostpopulations [6]. The combinations of these main Hp allelesproduce six major expression phenotypes (Hp2-2, Hp2-1s,Hp2-1f, Hp1s-1s, Hp1s-1f, Hp1f-1f).

Hp belongs to the group of the acute-phase proteins.These plasma proteins, mostly of hepatic origin, contributeto the early and nonspecific innate immune response. Inaddition to its hemoglobin binding function, Hp modulatesthe response to infection and inflammation, and is involvedin immune suppression in cancer and angiogenesis.Changes in Hp plasma levels have been observed in differentpathophysiological conditions, including chronic liver dis-ease, malnutrition, acute myocardial infarction and pro-cesses involving intravascular hemolysis. Hp is up-regulatedin chronic myeloid leukemia cells [7] and hepatocellular car-cinomas in patients with hepatitis C virus [8]. The Hp a-chain shows more than twofold higher levels in ovarian can-cer patients, and has been suggested as a potential bio-marker, especially in combination with CA125 [9].

The various forms of the Hp molecule show differentbiological activities. The Hp1-1 phenotype has a higherhemoglobin binding capacity and antioxidative propertiesthan Hp2-2 [1, 2, 10], and is also a more active inhibitor ofprostaglandin synthesis. The Hp polymorphism has impor-tant clinical consequences for many diseases including car-diovascular, autoimmune and neurological disorders. In thisrespect, the Hp2-2 phenotype is also a major determinant inthe outcome of several infectious diseases like tuberculosis[11] or HIV [12], and the risk of developing cardiovasculardiseases is five times greater among diabetic individuals ofthe Hp2-2 type than in individuals of the Hp1-1 type [13].

In a previous article we suggested that Hp phenotypescould be related to the susceptibility of the individuals to-wards toxic oil syndrome (TOS) [14]. TOS is a disease thatappeared in Spain in 1981 and affected more than20 000 people. Until 1998, about 2500 deaths were related tothe disease [15], and several thousands remain affected withvarious degrees of disability. The disease resulted from theingestion of an illegally commercialized aniline-denaturedrapeseed oil. This oil, imported for industrial purposes, wasrefined and mixed with other oils and sold as edible oil. Al-though several chemical markers of the toxic oil are knownnowadays (anilines, phenylaminopropanediols), the toxicfactor(s) remain obscure.

TOS appeared as a complex symptomatology with aninitial phase characterized by cough, dyspnea, and pulmo-nary infiltrates. If the acute phase was overcome, the mani-festations observed included severe muscle pain, intenseeosinophilia, ascending polyneuropathy, sclerodermiformskin changes and pulmonary hypertension [16]. Epidemio-logical data indicated that the disease often affected closelyrelated people (i.e., families), attributed to the commoningestion of the toxic oil. It was noted, however, that mem-bers of the same family had a different predisposition to thedisease. These findings could be attributable to differences inexposure to the oil and to differing susceptibility to the dis-

ease. In this respect Ladona et al. [17] studied the allelic dis-tribution of CYP1A1, CYP2D6, NAT2, GSTM1, andGSTT1 genes in groups of TOS patients. The control popu-lations were a group of relatives living in the same household(who thus had regular meals with the cases) and a controlgroup of unrelated individuals living in the same neighbor-hood who did not develop the disease. In these studies anincrease in the defective alleles of NAT2 was observed amongcases compared with non-related controls.

In a previous study, we performed a differential proteinexpression analysis in a TOS-affected (case) population rela-tive to a non-affected (control) population with the aim ofidentifying proteins that could be related to organ toxicity[14]. It was observed from the pools of sera that two Hp spotsidentified as the a-1s and a-1f chains showed a significantlydifferent distribution in the two populations studied. Formost of the differentially expressed proteins detected inthese gels it was not possible to establish whether differencesin their levels were related directly to the cause or were aresult of the disease. In the case of the Hp chain spots, how-ever, the phenotypes observed reflected the average genomicbackground of the individuals in a given group, so expres-sion differences could be related to individual susceptibilityto the disease. The results of this study suggested that theHP1S allele was a risk factor for the disease, while the HP1F

was a protection factor. Taking into account the importanceof Hp polymorphism in other disorders where, as in the caseof TOS, inflammation and cardiovascular damage areinvolved, these results supported a possible role of Hp in theetiology of the disease. Due to the low number of control andcase groups screened in these studies and the limitationsderived from the analysis of pools of sera, confirmation ofthese findings required individual phenotyping of a largernumber of control subjects and patients.

Reports on procedures for Hp genotyping are scarce. Amethod based on allele-specific PCR amplification capable ofdifferentiating between the six major genotypes [18] and aPCR-based method that differentiates between the HP1 andHP2 alleles have been described [19].

On the other hand, identification of the Hp1-1, Hp2-1 andHp2-2 phenotypes has been usually carried out by permeationchromatography [20], starch gel electrophoresis [4] or PAGE[21] of hemoglobin-supplemented sera. Direct analysis of theHp phenotypes can be also accomplished by PAGE and amethod based on the PhastSystem has been developed toproduce a procedure that could be applied in clinical labora-tories [22]. An ELISA method that can identify the major Hpphenotypes has recently been developed for the same purpose[23]. Hp subtyping was first performed using starch gels inreducing conditions [5] and was later carried out on 1-DE or2-DE followed by CBB or silver staining or by immunoblotting[14]. A procedure using CZE has been reported as an alter-native, faster method for Hp subtyping [24].

To circumvent the technical and economical limitationsof the gel-based approaches, we developed a new MALDI-TOF profiling procedure for Hp subtyping that allows the


S274 C. Rodriguez et al. Proteomics 2006, 6, S272–S281

rapid, automatable analysis of a large number of samples.We validated the method using sera from a small group ofTOS controls (n = 54) and cases (n = 48), which were ana-lyzed in parallel by minigel 2-DE.

2 Material and methods

2.1 Instrumentation and reagents

The equipment for 2-DE consisted of an IPGphor IEF systemfrom Amersham Biosciences (Uppsala, Sweden) and a MiniProtean 2-DE cell from Bio-Rad. Immobiline DryStrips(7 cm, pH 4–7 L) and other reagents for 2-DE (pharmalite 3–10, DryStrip cover fluid, TEMED, ammonium persulfate,Tris-HCl buffer, glycine, bromophenol blue, DeStreak, agar-ose, 87% glycerol and urea) were also from Amersham.Scanning of silver-stained gels was performed with an AgfaDueScan. Duracryl (30%, 0.8% Bis) was provided by Geno-mic Solutions (Ann Arbor, MI, USA) and 2-DE markers byBio-Rad (Hercules, CA, USA). PBS, EDTA, DTT, n-butanol,ammonium carbonate, ammonium hydroxide, sodium car-bonate, sodium acetate, iodoacetamide, thiourea were pro-vided by Sigma (Steinheim, Germany). Silver nitrate, sodiumthiosulfate and all other analytical grade chemicals for elec-trophoresis and staining were obtained from Merck (Darm-stadt, Germany). Water was Milli-Q grade except for therunning SDS buffer.

The MS analyses were accomplished in a Voyager DE-PRO MALDI-TOF mass spectrometer (Applied Biosystems)provided with a 337-nm nitrogen laser. Sinapinic acid(Sigma) and CHCA were used as MALDI matrix for proteo-typing and PMF analyses, respectively. ACN and TFA wereobtained from Merck. O-Methylisourea was from Fluka(Steinheim, Germany). A standard protein mixture (AppliedBiosystems) was used for protein calibration.

2.2 Serum and Hp standards

Gel separation and MALDI-TOF analyses were optimizedusing pools of sera from healthy individuals obtained fromthe Research Center for the Toxic Oil and Rare Diseases(CISATER, Madrid, Spain) and Vall d’Hebron Hospital (Bar-celona). An Hp standard from pooled human plasma con-taining the main Hp phenotypes (2-2, 2-1 and 1-1) wasobtained from Sigma.

2.3 TOS-related serum samples

Control and case serum samples were obtained from theCISATER. A total of 54 patients who were chronically illduring the 1st year of the disease were studied together with48 control individuals corresponding to members of affectedfamilies who at the time of the epidemic lived in the samehousehold as the TOS patients. Control individuals werethus having regular meals with future patients but, despite

this, did not develop TOS. These groups included male andfemale individuals with an age range from 10 to 70 years.The proportions of males were 65% and 75% in control andcase populations, respectively. The samples were stored at2807C until analysis.

2.4 Hp phenotyping by 2-DE

Serum samples were diluted in PBS (1:10), and 12 mL ofthese solutions were diluted with 113 mL IPG rehydrationbuffer containing 2% CHAPS, 1% IPG buffer, 8 M urea,60 mM DTT and 0.05% bromophenol blue. The IPG stripswere located in the IPG ceramic holders and rehydrated withthe protein solution for 6 h at 0 V and 6 h at 30 V. Afterrehydration, proteins were separated by IEF using a pro-grammed voltage gradient starting at 30 V and then raised to500 V, 1000 V and 5000 V for 1 h each. The voltage is thenkept at 5000 V until a total of 30 000 Vh.

Proteins were separated in a second dimension by PAGEusing 15% acrylamide gels (9.56760.1 cm). Prior to load-ing the focused IPG strips on the second dimensional gels,IPG strips were incubated for 15 min in 2.5 mL equilibrationbuffer (50 mM Tris, 6 M urea, 30% glycerol, 2% SDS) con-taining 10 mg/mL DTT. This was followed by another 15-min incubation in the same equilibration buffer containing25 mg/mL iodoacetamide. The equilibrated IPG strip wassealed on top of the second dimensional gel using0.5% agarose containing 50 mM Tris-base, 0.1% SDS,192 mM glycine and 0.05% bromophenol blue. Electropho-resis was carried out for 15 min at 30 V and 70 min at 150 V.Protein spots in gels were visualized by silver staining usingconventional protocols [25].

2.5 PMF analysis of gel spots

Hp chain spots were excised and subjected to in-gel digestionwith trypsin as described by Shevchenko et al. [25]. Trypticpeptides were extracted from the gel with ACN/0.5% TFA 1:1and the extracts were evaporated to dryness and redissolvedin ACN/0.1% TFA 1:1. PMF was performed in the VoyagerDE-PRO mass spectrometer. A small aliquot (0.5 mL) of thetryptic extract was mixed with 0.5 mL CHCA (5 mg/mL).External calibration was carried out with a set of syntheticpeptides. When peptides from autolytic degradation of tryp-sin were detected, an internal calibration of the spectra wasperformed.

2.6 Proteotyping by MALDI-TOF profiling

2.6.1 Determination of the main phenotypes 2-2,

2-1 and 1-1

Serum samples (8 mL) were mixed with 2 mL 1 M DTT andwere left for 30 min at room temperature to reduce the di-sulfide bonds between the Hp a and b chains. One microliterof the mixture was diluted 50-fold with water and mixed vig-



orously. An aliquot (1 mL) was loaded onto the MALDI platefollowed by 1 mL of the sinapinic acid matrix (10 mg/mL inACN/TFA 0.1%, 7:3).

The MS analyses were accomplished in the Voyager DE-PRO MALDI-TOF mass spectrometer working in the linearpositive ion mode at 25 kV acceleration voltage. The gridvoltage and guide wire voltage were of 93% and 0.15% of theacceleration voltage, respectively. Delay time was 450 ns andthe low mass gate was set at 850 Da. Laser repetition rate wasset to 20.0 Hz, and data were accumulated between 2000 and25 000 Da. Each data point was the summation of 10 spectra,acquired with 50 laser shots each. Hp a1 and b2 chainsappeared in the spectra at m/z 9192 and 15946, respectively.

2.6.2 Identification of Æ-1s and Æ-1f chains in

individuals having 2-1 or 1-1 phenotypes

Target sera (2 mL) were mixed with 18 mL 2 M O-methyl-isourea. The pH of the mixture was increased to 10.5 withNH4OH and the mixture was incubated for 24 h at 307C.After guanidination, NH4OH was evaporated (30 minuncapped) and 1 mL 1 M DTT was added to 9 mL of the evap-orated solution, which was incubated again for 30 min atroom temperature. After diluting this solution fivefold withwater, the sample was desalted using a C18 micropipetteZipTip. For this purpose, the ZipTip was activated withACN/0.5% TFA 1:1 (10 mL, 56) and washed with 0.1% TFA(10 mL, 26). The sample was loaded, aspirating and dispen-sing the solution ten times, then washed with 0.1% TFA(10 mL, 106) and eluted with ACN/0.5% TFA (4:1) (10 mL,610). The eluted sample was evaporated to a final volume ofabout 3 mL, and then was loaded onto the MALDI-TOF plate.After partial evaporation of the droplets at room temperature(20–257C), 1 mL of the sinapinic acid matrix was added.

MALDI-TOFspectra were acquired as indicated above. Thepresence of the a-1s and a-1f chains was determined by thepresence of peaks at approximately m/z 9528 (8 lysine residuesmodified) and 9570 (9 residues modified), respectively.

3 Results and discussion

3.1 Hp typing by MALDI-TOF

The use of MALDI-TOF profiling methods to monitor com-plex protein samples has been reported for different pur-poses, including food control, species classification or, morefrequently, for disease biomarker discovery. In particular,SELDI-TOF instruments have been used successfully in thesearch for protein markers of disease [26]. The high abun-dance of the Hp molecules and the relatively low molecularweights of its a chains made the MALDI-TOF approach agood candidate for their direct monitoring in serum sam-ples. Although the SELDI method has recently enabledcharacterization of the Hp a-chain as a potential serum bio-marker in ovarian cancer using an IMAC chip [9], no simple

MALDI-TOF procedure has been reported for Hp typing. Totest this approach, we analyzed standard Hp samples anduntreated, diluted human sera by linear MALDI-TOF usingsinapinic acid as matrix.

Hp standards produced spectra with several broad sig-nals at about m/z 90 000 which were tentatively explained asthe different tetramer isoforms (not shown). In the case ofuntreated serum samples, the spectrum profiles showedmajor characteristic signals at ca. m/z 15 124, 15 865, 33 204and 66 000, which probably corresponded to the hemoglobinchains and the singly- and doubly-charged molecules ofserum albumin, respectively.

Hp a-1 and a-2 chains were easily detected in theMALDI-TOF spectra obtained from DTT-treated and dilutedserum samples (Fig. 1). Reduction of the sample wasrequired to separate the individual chains of the Hp tetramerthat were covalently linked by interchain disulfide bonds. Inthese spectra, the a-2 chain appeared as a mass signal cen-tered at about m/z 15 945, while the a-1s a-1f chainsappeared unresolved at m/z 9193 (external mass calibration).The expected molecular masses calculated from their aminoacid sequences were 15 946 and 9192, respectively. Identifi-cation of these signals in the spectra was performed by spik-ing serum samples with a Hp standard that contained amixture of the polymorphic forms of the protein. Identifica-tion was also validated with a group of serum samples pre-viously phenotyped by 2-DE (Fig. 2).

The optimal concentration of DTT for Hp reductiondepends on sample composition. Efficient reduction of theHp standard was obtained with 5 mM DTT, while in the caseof serum samples this concentration had to be increased to100–200 mM (Fig. 3).

Direct MALDI analysis of the serum samples did not,however, discriminate between the a-1s and a-1f chains. Theamino acid sequence of these chains differs only at posi-tions 52 and 53, where the pair Asn-Glu replaces Asp-Lys.This produces a mass difference of only 0.043 Da betweenthe two chains. Resolution of our conventional MALDI-TOFsystem falls below 10 000 in the linear mode; thus, in thespectra obtained from serum samples with Hp1s-1f pheno-type the signals from the a-1s and a-1f chains appeared as asingle peak (Fig. 1).

The sequence of the a-1s chain contains 8 Lys residues,while an additional Lys is present in the non-homologousamino acids pair of the a-1f chain. This difference can beexploited through the chemical modification of the Lys resi-dues to produce protein derivatives of different masses thatcould be resolved in the spectra. Conversion of the Lys resi-dues into homoarginine by guanidination at the e-aminogroup of the lysine [27] results in a mass difference of 42 Dabetween the fully modified a-1s and a-1f chains. Thus, afterguanidination, the signal of the unmodified chains atm/z 9192 is shifted by 8 (a-1s) or 9 (a-1f) multiples of 42 Da,resulting in two possible signals appearing at aboutm/z 9528 and 9570, respectively, which allow the specificidentification of the Hp subtypes (Fig. 4).



Figure 1. MALDI-TOF profiles obtained from (A) an Hp standard treated with 5 mM DTT, and (B) a pool of seratreated with 0.2 M DTT. The signals from the a-1 and a-2 chains of the Hp molecule are observed at about m/z 9192and 15946 Da, respectively.

Figure 2. Analysis of four serum samples of different phenotype by 2-DE and MALDI-TOF.



Figure 3. Effect of DTT con-centration on the intensity of thesignals corresponding to the Hpa-1 and a-2 chains.

Figure 4. Resolution of the signals from the a-1 chain after mod-ification of the lysine residues with O-methylisourea. The signalfrom the a-1 chains is shifted from m/z 9192 to m/z 9526 (a-1s)and 9568 (a-1f), resulting in a mass difference of 42 units betweenthese chains.

3.2 Analysis of serum samples from TOS-affected

and control individuals

Our Hp proteotyping method was applied to the analysis ofserum samples from a population of 54 TOS cases and 48 con-trol individuals. For this purpose, the corresponding sera were

first analyzed by direct MALDI-TOF. About 60% of these sam-ples showed the characteristic mass signal from the a-1 chainseither alone or together with the signal derived from the a-2chain. These sera were then treated with O-methylisourea andreanalyzed to discern between the a-1s and a-1f chains.

To validate the method, the samples were analyzed inparallel by 2-DE. The procedure was similar to that reportedpreviously but in this case, 7-cm strips and minigels wereused to speed up the analysis. The percentage of acrylamideand other 2-DE conditions were optimized to obtain clearimages of the Hp a-chain region that would allow rapidevaluation of the Hp-derived spots. In these conditions,removal of major serum proteins such as albumin orimmunoglobulins was not necessary as they did not interferewith the target area of the gel image.

Among the 102 samples analyzed by 2-DE, 4 could not bephenotyped due to the complexity of the spot profiles. Addi-tional interfering spots observed in these samples could beeither genuine components of the proteome or the result ofinadequate storage or handling of the serum sample andconsequent protein degradation. In this respect, several PTMforms of the a-1 and a-2 chains that appear in the same areaof the gel have been described [28, 29] (Fig. 5). Protein spotsderived from deamidation of Asn to Asp at position 5 (N5D)and from the presence of forms with an additional C-termi-nal Arg residue (R84–R143) are generally detected about0.1 pI units to/from the left and right side of the a-2 chain.The corresponding modified forms can also be producedfrom the a-1 chains. Unfortunately, in this case, the deami-dated a1 chain (a-1f, N5D) and the R84 form of the a-2 chaincomigrate with the a-2 and a-1 chains.

The presence of these modified forms was the mainlimitation of the 2-DE approach for Hp phenotyping. Threeof the unassigned samples produced images where the three



Figure 5. 2-DE pattern produced by different post-translationally modified Hp chains in a serum sample that apparently shows the pres-ence of products from the three main alleles of Hp. The location of the different known modified forms is indicated in the zoomed gelimage. Spots 315, 316, 285, 287 and 286 were analyzed by MALDI-TOF PMF (see also Table 1). The signal at m/z 3350.58 in the MALDI-TOFspectrum of the spot initially assigned as the a-1s form, allowed the identification of this spot as the deamidated form of the a-1f chain (A).Spot 315 was identified as the a-1f (B) chain confirming an Hp2-1f phenotype.

a chains seemed to be present, and thus suggested thepresence of modified forms of the a-1s or a-1f chain thatwere interfering with the identification (Fig. 5). To identifythe Hp composition, the gel spots were subjected to a PMFanalysis that allowed the assignment of each isoform on thebasis of specific tryptic peptide signals observed in theMALDI spectra [28, 30, 31], as shown for one of these sam-ples (a TOS control) in Fig. 5 and Table 1. The presence ofthe Lys53 residue in the a-1f chain produces a specific tryp-tic fragment that is observed at m/z 1580.8 in the MALDIspectrum. This ion is absent in the spectra from the a-1schain that holds an Asn residue at position 53; hence, thisdifference can be used to identify the presence of thedeamidated form of the a-1f chain at the location of the a-1sspot. In addition, amidated forms of the Hp chains producean N-terminal fragment at m/z 3350.5, while non-amidatedmolecules produce a characteristic ion at 3349.5. Moreover,both a-1s and a-1f chains produce a tryptic C-terminalfragment that appears at m/z 2578. In the Arg-extendedforms of the Hp a-1 chain this ion is absent and the corre-sponding C-terminal peptide ion appears at m/z 2734. Inthis way, the PMF analysis of the spots in the unassignedsera allowed their classification as Hp2-1s or Hp2-1 f. Addi-

Table 1. MALDI-TOF identification of gel spots in Fig. 6 on thebasis of characteristic tryptic peptide ions expected forthe different protein chains. The phenotype was identi-fied as Hp 2-1f

Spot no. Observed ions Identified chain Expected ions

315 3349.611580.82

a-1f 3349.661580.79

316 3350.58 a-1f,N5D 3350.52285 3349.61

2962.34a-2 3349.54

2962.35287 3350.53

2962.34a-2,N5D 3350.64

2962.35286 3349.63

2962.36a-2,R143 3349.54

2962.35

tional spots at the location of the a-1f (sample Hp2-1f) and a-1s (sample Hp2-1s) chains were identified as the a-1s,R anda-1f,N5D forms, respectively.

These samples were readily identified by MALDI-TOF,thus confirming their previous assignment by 2-DE andPMF. The presence of modified forms did not interfere with



Figure 6. Serum sample from a TOS patient with an uncharacteristic 2-DE profile. Characterization of the phenotype as an Hp2-1s pheno-type was performed by MALDI-TOF despite the complexity of the sample and the apparent low levels of the Hp molecules reflected in the 2-DE images. The direct MALDI-TOF analysis indicated the presence of the Hp a-1 (signal at m/z 9192) (A) and a-2 (signal at m/z 15943)(B) chains. After guanidination, a signal at m/z 9532 reveals the presence of an a-1s chain (C).

the mass analysis as the different species produced deriva-tives with different masses, thus allowing an unambiguousidentification. The confidence of the MALDI-TOF data wasalso shown in the case of two mismatched samples that weredifferently assigned by 2-DE and MALDI. A reanalysis by 2-DE demonstrated that these mismatches were the result ofthe lack of clearly visible Hp spots at the correspondinglocation and to the presence of faint spots that producedfalse-positive identifications, respectively.

Specific physiological or pathological processes could beresponsible for important changes in the proteome imagesthat can interfere with Hp pattern recognition. Furthermore,sample handling can produce protein degradation productsor fragments of proteins migrating at the location of the Hpchains that may also be responsible for interferences in the 2-DE analysis. This may be applicable to one sample that couldnot be phenotyped by 2-DE (Fig. 6). This sample produced alarge number of spots with an unusual distribution in theHp area that was difficult to interpret. Despite the problemsencountered in the 2-DE analysis, this sample was readilyidentified as Hp2-1s by MALDI-TOF prototyping, indicatingthe higher sensitivity and specificity of the latter approach.

3.3 Hp phenotype distribution

The distribution of Hp alleles varies greatly between humanpopulations, and there are important differences betweenethnic groups. The analysis of Hp distribution is thus a use-

ful tool for genetic population studies [1]. Numerous Hpsubtype distribution studies in different populations includ-ing small, ethnically well-defined communities have beenreported [1, 6, 18, 20]. In a study on four populations fromSouth America, Nigeria and India, the relative distributionsranged from 54% to 79% for HP2, 15% to 28% for HP1S and5% to 19% for HP1F [32]. Surveys in Spain showed a dis-tribution of about 63% and 60% (HP2), 24% and 23% (HP1S)and 14% and 17% (HP1F) for Barcelona (Mediterraneancoast) and Madrid (central area), respectively [33, 34]. In thecase of our population, which corresponds to individuals ofthe central area of Spain, these values were of 57% (HP2),31% (HP1S) and 12% (HP1F) (Table 2). The observed devia-tion from the reported values is mainly accounted for by thecontribution of the case population where the HP1S and HP1F

are over- and underrepresented, respectively. The odds ratio(OR) for the presence of the allele HP1F in control versus casesis 3.36 (1.24–9.08; 95% confidence interval, CI), while forallele HP1S the OR is 0.45 (0.20–1.00; 95% CI). These resultsconfirm our previous findings from the analysis of pools ofsera that suggested that the allele HP1S could be a risk factorfor the disease, while the allele HP1F could be a protectionfactor. In comparison, the OR for the HP2 allele was 1.18(0.48–2.91; 95% CI).

The comparison of phenotype distributions (Table 3)showed significant differences between control and cases ofthe Hp2-1s and 2-1f phenotypes (p = 0.03 and p = 1.461027,respectively). Nearly 70% of the Hp2-1s individuals were



Table 2. Allele distributions (in%) of Hp in the studied population

Control Cases Total

HP1S 24.0 38.0 31.4HP1F 17.7 6.5 11.8HP2 58.3 55.6 56.9

Table 3. Phenotype distributions (in%) of Hp in the studied pop-ulation

Control Cases Total Control Cases Total

Hp 1-1 11 14 25 1S-1S 4 9 131S-1F 6 5 111F-1F 1 0 1

Hp 2-1 18 20 37 2-1S 9 18 262-1F 9 2 11

Hp 2-2 19 20 38 2-2 19 20 38

cases, while only 20% with the Hp2-1f phenotype were inthis group. The OR for Hp2-1s in control versus cases was0.46 (p = 0.09, 95% CI = 0.184–1.158), while for Hp2-1f was6.0 (p = 0.01, 95% CI = 1.223–29.348). The Hp2-2 phenotypeshowed a similar proportion in case and controls, suggestingthat the HP2 allele is not related to the susceptibility to thedisease. The same trend is observed for the Hp1s-1f pheno-type, although in this case this result would be consistentwith an antagonistic effect between the Hp1s and 1f chains.These results suggest that the Hp1f-1f phenotype could con-fer resistance to the disease. This could not be proved, due tothe presence of only one individual with this phenotype inour population (a control individual). The contrary trendcould be expected for Hp1s-1s individuals with a proportionof 70% of cases. As before, the low number of individualsresulted in a low confidence for the OR value. This value was,in any case, lower than those calculated for the other pheno-types, and similar to the OR of the Hp2-1s (0.45 versus 0.46,respectively). It should be noted that, in comparison to acontrolled toxicological experiment, the theoretical case/control ratio for a given phenotype could be higher than thatobserved in our population because a number of individualsin the control population were probably not exposed to thetoxic. These differences would be relatively more importantin cases when a phenotype confers a high risk for the diseaseas could be the case of the Hp1s-1s.

4 Concluding remarks

We have developed a MALDI-TOF profiling method for thesubtyping of Hp that outperforms the speed and efficiency ofgel-based procedures and makes no use of immunodetectionprocedures. The analysis is carried out in two steps: deter-

mination of the presence of the a-1 and a-2 chains, followedby subtyping of the a-1 chain in the corresponding samplesas a-1s and/or a-1f. The technique is not affected by thepresence of some known modified Hp chains that can inter-fere with the detection in 2-D gels and can be applied for thelarge scale typing of Hp.

We applied this method to the analysis of a group ofserum samples from TOS patients confirming our previousresults obtained from pools of sera that indicated that thepresence of the HP1s allele of Hp was a risk factor for theTOS disease, while the HP1F was a protective factor. Hp,which modulates many aspects of the acute-phase response,is known to have important physiological functions, many ofwhich are related to its antioxidant properties.

This work was supported by WHO projects EU/03/016989and EU 05/025039.

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