Serum Proteomic Signature of Human Chagasic Patients for the Identification of Novel Potential Protein Biomarkers of Disease*

Chagas disease is initiated upon infection by Trypanosoma cruzi. Among the health consequences is a decline in heart function, and the pathophysiological mechanisms underlying this manifestation are not well understood. To explore the possible mechanisms, we employed IgY LC10 affinity chromatography in conjunction with ProteomeLab PF2D and two-dimensional gel electrophoresis to resolve the proteome signature of high and low abundance serum proteins in chagasic patients. MALDI-TOF MS/MS analysis yielded 80 and 14 differentially expressed proteins associated with cardiomyopathy of chagasic and other etiologies, respectively. The extent of oxidative stress-induced carbonyl modifications of the differentially expressed proteins (n = 26) was increased and coupled with a depression of antioxidant proteins. Functional annotation of the top networks developed by ingenuity pathway analysis of proteome database identified dysregulation of inflammation/acute phase response signaling and lipid metabolism relevant to production of prostaglandins and arachidonic acid in chagasic patients. Overlay of the major networks identified prothrombin and plasminogen at a nodal position with connectivity to proteome signature indicative of heart disease (i.e., thrombosis, angiogenesis, vasodilatation of blood vessels or the aorta, and increased permeability of blood vessel and endothelial tubes), and inflammatory responses (e.g., platelet aggregation, complement activation, and phagocyte activation and migration). The detection of cardiac proteins (myosin light chain 2 and myosin heavy chain 11) and increased levels of vinculin and plasminogen provided a comprehensive set of biomarkers of cardiac muscle injury and development of clinical Chagas disease in human patients. These results provide an impetus for biomarker validation in large cohorts of clinically characterized chagasic patients.


Chagas disease is initiated upon infection by Trypanosoma cruzi.
Among the health consequences is a decline in heart function, and the pathophysiological mechanisms underlying this manifestation are not well understood. To explore the possible mechanisms, we employed IgY LC10 affinity chromatography in conjunction with ProteomeLab PF2D and two-dimensional gel electrophoresis to resolve the proteome signature of high and low abundance serum proteins in chagasic patients. MALDI-TOF MS/MS analysis yielded 80 and 14 differentially expressed proteins associated with cardiomyopathy of chagasic and other etiologies, respectively. The extent of oxidative stress-induced carbonyl modifications of the differentially expressed proteins (n ‫؍‬ 26) was increased and coupled with a depression of antioxidant proteins. Functional annotation of the top networks developed by ingenuity pathway analysis of proteome database identified dysregulation of inflammation/acute phase response signaling and lipid metabolism relevant to production of prostaglandins and arachidonic acid in chagasic patients. Overlay of the major networks identified prothrombin and plasminogen at a nodal position with connectivity to proteome signature indicative of heart disease (i.e., thrombosis, angiogenesis, vasodilatation of blood vessels or the aorta, and increased permeability of blood vessel and endothelial tubes), and inflammatory responses (e.g., platelet aggregation, complement activation, and phagocyte activation and migration). The detection of cardiac proteins (myosin light chain 2 and myosin heavy chain 11) and increased levels of vinculin and plasminogen pro- Chagas disease continues to present a serious threat to human health in Latin America and is one of the most important emerging parasitic diseases in developed countries including the United States (1). According to a World Health Organization report released in 2010, an estimated 10 million people are infected, and Ͼ25 million people are at risk of infection in endemic countries (2). It is assessed that Ͼ300,000 infected patients live in the United States (3). Up to 40% of chronically infected individuals develop cardiac alterations, and up to 10% develop digestive, neurological or mixed alterations, resulting in considerable morbidity and mortality (4).
Human plasma and serum are the most easily available, clinically valuable specimens. Besides abundant proteins, e.g., albumin, immunoglobulin, transferrin, haptoglobin, and lipoprotein, plasma and serum contain many other proteins that are secreted or released from cells and tissues throughout the body. In chronic conditions or disease states, as is also noted in chagasic patients (5,6), modification of the proteome manifests as a result of disease-associated changes in specific genes up-or down-regulation, isoform switching, or de novo protein synthesis (7,8). Further, progression of disease severity, presented by an increasing order of cardiac injury and cell death (9,10), may also result in the release of intracellular proteins in the peripheral system, thus altering the proteome profile. It is thus logical to assume that the pathological processes during the development of Chagas disease would cause characteristic changes in the circulating proteins and generate a detectable, disease-specific molecular phenotype.
In addition to changes in proteome profile, post-translational protein modifications in response to disease-associated stress also contribute to protein change resulting from modification of specific amino acids. We have documented in experimental animal models and human chagasic patients that reactive oxygen species (ROS) 1 of inflammatory and mitochondrial origin contribute to oxidative modification of proteins in peripheral blood and myocardium (11)(12)(13)(14). For example, treatment of infected animals with an antioxidant was effective in arresting oxidative cardiac pathology (15) and preventing the loss of cardiac left ventricular function in chronic hearts (16), thus establishing the pathological significance of oxidative overload in Chagas disease. To date, however, proteomic examples of oxidative modification identification in cardiovascular diseases are lacking.
The plasma/serum proteome is the most useful version of the human proteome for clinical investigations and patient diagnostic and treatment purposes. Nevertheless, clinical proteomics, i.e., translation of proteomic techniques to study clinical pathology, has not been widely used. The two-dimensional polyacrylamide gel electrophoresis (2D-GE) method enables identification of major proteins in a tissue or subcellular fraction by mass spectrometric methods and can be used to compare quantities of proteins in related samples (17). However, 2D-GE has many drawbacks, including a narrow dynamic range that requires the multistep removal of abundant proteins before low abundance serum proteins or biomarkers can be detected, problematic solubilization and separation of hydrophobic proteins, and a restricted ability to load limited sample amounts on a gel (ϳ250 g) (18). Poor reproducibility of gel images between experiments, imprecise quantitation, and intensive labor requirements are additional limitations in applying the 2D-GE approach for clinical proteome analysis (19).
The ProteomeLab TM PF2D system offers an alternative approach to protein profiling that addresses issues of complexity and utilization of fractions after analysis. PF2D is designed to allow automated separation of proteins in the first dimension by pI-based chromatofocusing, followed by a second dimension hydrophobicity-based separation of proteins by reverse phase (RP)-HPLC (20). Changing the composition or pH range of elution buffer allows increased resolution of hydrophobic, basic, or acidic proteins, a process that cannot be achieved by 2D-GE (21). Importantly, all protein fractions from PF2D are collected in 96-well plates from which they can be easily lyophilized and submitted for mass spectrometric protein/peptide identification or utilized for immunoblotting, catalytic staining, or other assays. The in-built scanning spectrophotometer allows one to simultaneously monitor the normal and oxidatively modified isoforms of the proteins in second dimension fractions for the automated, efficient detection of disease-associated protein oxidative and other (e.g., phosphorylation) modifications (22).
In this study, we investigated the host physiological and pathological changes at the protein level in chagasic human patients. We employed IgY-12 chemistry to separate the serum-abundant proteins and the PF2D system to develop the protein signature of low abundance serum proteins in Chagas disease patient samples. We also made use of traditional oneand two-dimensional gel electrophoresis and ELISA to identify serum-abundant proteins that were differentially expressed or oxidized in chagasic patients. Our findings of a number of proteins that were differentially expressed in patients provide clues to the pathomechanisms that contribute to Chagas disease.

MATERIALS AND METHODS
Human Subjects-Human sera samples from chagasic and nonchagasic/other cardiomyopathy patients used in this study were obtained from Salta, Argentina and Chiapas, Mexico. Sera samples from seronegative healthy individuals exhibiting no history or clinical symptoms of cardiac disease from the same geographical area were used as controls. All of the procedures were approved by the institutional review boards at the University of Texas Medical Branch, Universidad Nacional de Salta, Argentina, and the Universidad Autó noma de Chiapas, Mexico.
In Argentina, written informed consent was obtained from all individuals (age range: 19 -71 years, 56% females) before their enrollment in the study. Venous blood samples were collected without anticoagulant to obtain serum. Trypanosoma cruzi-specific antibodies in sera samples were monitored by an enzyme-linked immunosorbent assay using a Wiener Chagatest-ELISA recombinant v.4.0 kit comprising six recombinant proteins known to be expressed in mammalian stage of T. cruzi in isolates circulating in Latin America. Briefly, 96-well plates were coated with recombinant proteins and then sequentially incubated with 20-l sera samples (1:20 dilution) and horseradish peroxidase-conjugated human monoclonal anti-IgG and color-developed with chromogenic substrate monitored by spectrometry at 450 nm (cutoff value: average of seronegative samples (Ͻ0.1 optical density) ϩ 0.2 optical density, i.e., Ն0.3). Serological tests were also done following the specifications of the commercial Indirect Heamagglutination test kit (Wiener Chagatest-HAI). Briefly, sera samples (25-l 4-fold dilutions) were mixed with red blood cells sensitized with T. cruzi cytoplasmic and membrane antigens and agglutination-monitored. The titer was defined as the highest serum dilution presenting agglutination (positive Ն 1:16 dilution). Those positive by both tests were identified as seropositive. The clinical data included medical history, physical examination, subjective complaint of frequency and severity of exertional dyspnea, 12-lead electrocardiography at rest to obtain a comprehensive view of cardiac rhythm and conduction abnormalities, 3-lead electrocardiography with exercise to note major cardiac rhythm alterations, transthoracic echocardiogram to obtain objective information regarding the left ventricular contractile function, and chest x-ray to assess cardiomegaly (cardiothoracic ratio Ͼ 0.5). The severity of exertional dyspnea was graded according to the New York Heart Association classification (23). Based on these criteria, seropositive chagasic patients exhibiting no echocardiographic abnormalities, preserved systolic function (ejection fraction Ն 55%), and no left ventricular dilatations, but with negligible to minor electrocardiography alterations were graded as clinically asymptomatic. Seropositive patients were graded clinically symptomatic if they exhibited varying degrees of systolic dysfunction (ejection fraction 40 -54% or less) and/or left ventricular dilatation (end diastolic diameter Ն 57 mm). Seronegative cardiomyopathy patients of other etiologies (CO) were categorized by criteria similar to those of the chagasic patients. Seronegative, healthy controls were recruited from the same geographical area.
In Mexico, human sera samples (age range: 18 -73 years, 55% females) were collected within the framework of a research project on emerging zoonotic diseases conducted jointly by several institutions, including Chiapas State University, Mexican Social Security Institute, Chiapas Health Institute, and University of Texas Medical Branch at Galveston. All of the samples were screened by ELISA, flow cytometry, and Stat-Pak (Chembio Diagnostic Systems, Medford, NY) to distinguish seropositive and seronegative samples before inclusion in the study (24). The seropositive subjects generally represented the indeterminate/asymptomatic form of the disease.
Separation of High Abundance Sera Proteins-To enrich the low abundance proteins for enhanced detection, we employed the IgY-12 high capacity LC10 proteome partitioning system (Beckman Coulter, Brea, CA) in combination with the ProteomeLab PF2D system (Beckman), according to the manufacturer's instructions (details in supplemental File 1). The collected low abundance proteins in the flow through fractions consisted of 4.8 -10% of the proteins present in the original whole serum, and 90 -95.2% of the total proteins were captured as major (abundant) proteins by IgY columns. To obtain sufficient quantities of low abundance proteins for PF2D analysis, we processed five aliquots (total 1250 l) of each sera sample by IgY-12 chromatography. The flow through (enriched low abundance proteins) and eluted (high abundance proteins) serum fractions were neutralized, concentrated by using Amiconா ultracentrifugal filters (3-kDa cutoff; Millipore), and desalted by PD-10 desalting columns (GE Healthcare). Protein content was determined by using the Bradford assay (Bio-Rad).
Protein Profiling by ProteomeLab PF2D System-Serum protein fractionation using PF2D system was carried out by high performance chromatofocusing in the first dimension and RP-HPLC in the second dimension. Briefly, after equilibration, the samples (2.5 mg of protein in 100 l) were injected onto the chromatofocusing column, and first dimension fractionation was performed using a pH gradient 8.3-3.5, the fractions being collected with the fraction collector/injector module at 0.3-pH intervals. For the second dimension separation, selected first dimension fractions (250 l) were injected into the RP-HPLC column and run for 2 min with solvent A (0.1% trifluoroacetic acid in HPLC water). The column was then eluted with a linear gradient of 0 -100% solvent B (0.08% trifluoroacetic acid in acetonitrile) for 33 min, and second dimension fractions were collected at 30-s intervals in 96-well plates.
ProteoVue software (Eprogen) was utilized to convert chromatographic intensities of fractions collected after a second dimension RP-HPLC fractionation of each of the first dimension fractions collected in pH gradient 8.3-4.0. This analysis produced a highly detailed heat map of each sample, incorporating the dimensions of pI (x axis) and hydrophobicity (y axis). DeltaVue software was employed to compare derived intensity bands from ProteoVue analysis of any two samples, allowing quantitative differences in protein contents measured by peak area (volume). Finally, Karat 32 software was employed to compare significantly different peaks among multiple samples and to identify proteins. Detailed methods for protein profiling by PF2D and comparative proteome analysis are presented in supplemental File 1.
In total, 12 Sypro Ruby-stained two-dimensional gels (n ϭ 18/ group) were digitalized on a ProXPRESS proteomic imaging system (PerkinElmer Life Sciences), and the images were analyzed on Progenesis SameSpotst TM software 2.0 (NonLinear Dynamics). Normalized spot volumes, i.e., the volume of each spot over the volume of all spots in the gel, were used for comparison of the different groups, and candidates were identified as protein spots that changed in expression level by Ͼ2-fold as compared with normal controls. Statistical significance was assessed by the analysis of variance test, and p values of Ͻ0.05 were considered significant for comparison (26).
Detection of Carbonyl Proteins-We employed Western blotting (WB) in conjunction with one-or two-dimensional gel electrophoresis to detect the carbonyl modifications in low abundance and high abundance protein sera fractions, respectively (27, 28) (supplemental File 1).
Mass Spectrometry and Protein Identification-Please see supplemental File 1 for detailed methods for processing of fractionated protein samples collected after second dimension PF2D analysis and protein containing gel spots from two-dimensional gels. Peptide mixtures (1 l) obtained after tryptic digestion were directly spotted onto a MALDI-TOF MS/MS target plate with 1 l of ␣-cyano-4-hydroxycinnamic acid matrix solution (5 mg/ml in 50% acetonitrile). Peptides were analyzed by using a MALDI-TOF/TOF TM ABI 4800 proteomics analyzer (Applied Biosystems peak filtering included the following parameters: minimum S/N filter ϭ 10, maximum missed cleavages ϭ 1, fixed modification of carbamidomethyl, variable modifications caused by oxidation, precursor tolerance ϭ 0.2 Da, MS/MS fragment tolerance ϭ 0.3 Da, mass ϭ monoisotopic, and peptide charges ϭ ϩ1. The significance of a protein match, based on the peptide mass fingerprint in the MS and the MS/MS data from several precursor ions, is presented as expectation values (p Ͻ 0.001). To confirm the identified proteins were of human and not of parasite origin, we also performed a similar search against NCBI nonredundant protein database consisting of T. cruzi sequences.
Functional Analysis-All data sets were assessed by using an online tool (http://www.uniprot.org/uniprot, UniProt release February 8, 2011) to predict the Gene Ontology and recognize the function of the identified proteins. Ingenuity pathways analysis (IPA, Ingenuity Systemsா) was performed to integrate the identified proteins into networks and signaling pathways with biological meaning and significance. IPA was also employed to rapidly gain biological insight and access a comprehensive overview of scientific findings including biological functions, molecular interactions, regulatory events, and pharmacological relevance. An e-value was calculated by estimating the probability of a random set of proteins having a frequency of annotation for that term greater than the frequency obtained in the real set, and a threshold of 10 Ϫ3 was set to retrieve significant molecular functions and biological processes. With these parameters, we were able to highlight the most informative and significantly overrepresented Gene Ontology terms in the data set (29).

Sera Screening for Detection of Disease Biomarkers-
The 96-well microtiter plates were coated overnight at 4°C with 100-l sera samples in 0.1 M carbonate buffer, pH 9.6 (1:50, v/v), and blocked for 2 h at room temperature with 1% nonfat dry milk. The plates were then sequentially incubated with 100 l each of antibodies against MYL2, VCL, and VIM (Santa Cruz, 1:3000) for 2 h, horseradish peroxidase-labeled IgG antibody (Southern Biotech, 1:5000) for 1 h, and Sure blue TMB substrate for 15 min. The reaction was blocked with 2 N sulfuric acid and colorimetric change in absorbance was measured at 450 nm on a SpectraMax 190 microplate reader (Molecular Devices). The ELISA results were normalized to total protein concentration, determined by Bio-Rad protein assay. Plasminogen levels were measured using a sandwich ELISA kit from ICL Inc.

Immunodepletion of High Abundance Proteins in Serum-
Compared with other sample types such as bronchial lavage, cerebrospinal fluid, and tears, plasma/serum consist of a wide dynamic range of protein concentration, and the high abundance proteins interfere with the detection of low abundance potential biomarker proteins (30,31). To enhance our ability to detect low abundance proteins, we employed IgY-12 LC10 immunoaffinity columns and performed HPLC to separate high abundance proteins. Shown in Fig. 1  sentative IgY-12 LC10 affinity chromatographs of sera samples from normal subjects with no disease (NN), seronegative individuals exhibiting cardiomyopathy of other etiologies (CO), seropositive (clinically asymptomatic) subjects from Mexico (CC M ), and chagasic (clinically symptomatic) patients from Argentina (CC A ). The distinct separation of flow through fractions (5-25 min, marked a) enriched in low abundance proteins and column-bound high abundance proteins (eluted during 30 -50 min, marked b) indicate that serum low abundance proteins were effectively depleted of high abundance proteins by IgY-12 LC10 column chromatography. Polyacrylamide gel analysis ( Fig. 1E) confirmed the above results and showed the abundant proteins, e.g., transferrin, albumin, fibrinogen, and haptoglobin, were separated in the bound fraction and not detectable in the low abundance protein fraction for all sera samples.
PF2D/Mass Spectrometric Analysis of Serum Proteomic Signature of Low Abundance Proteins in Chagasic Patients-We examined the reproducibility of the PF2D system by submitting enriched sera samples from normal healthy subjects to first dimension high performance chromatofocusing on different days (supplemental Fig. S1A) and then submitted triplicates of first dimension fractions for second dimension RP-HPLC analysis (supplemental Fig. S1B). We observed high reproducibility of retention time for both first and second dimension fractions collected for the same and diverse sera samples on different days and obtained precise alignment of peaks among replicate runs. These data showed that it is feasible to resolve complicated protein mixtures with high reproducibility by using the ProteomeLab PF2D system.
For identifying disease-specific serum profiles, we injected 2.5 mg of each enriched sera sample into the first dimension column. In general, for normal subjects as well as patient groups, we recovered 30% of proteins in the 8.3-3.5 pH gradient, 31% before the initiation of the pH gradient (pHϾ8.0), and the remaining 39% eluted when the column was finally washed with high salinity buffer (supplemental Fig. S2). We concentrated on 16 fractions collected in the pH 8.3-3.5 range gradient, which were subjected to a second dimension by using RP-HPLC. Representative virtual two-dimensional gel images of sera proteome of normal, chagasic, and other cardiomyopathy subjects, combining the first and the second dimension and viewing proteins separated by pI and hydrophobicity, are shown in Fig. 2. By using the paired peak function in DeltaVue mapping tools, we aligned and analyzed the second dimension chromatograms for each first dimension fraction of two groups for quantitative and qualitative analysis (supplemental Fig. S3A). A composite MultiVue comparison of peaks during the second dimension fractionation of first dimension fractions (supplemental Fig. S3B) clearly indicated that the shift in retention time for any particular peak among the four groups was less than 10 s and confirmed that we could provide reliable comparative analysis of the second dimension peaks from different samples based upon retention time.
DeltaVue is limited in the simultaneous analysis of more than two samples. We therefore utilized the functions of Karat showed that a majority of peaks were generated from first dimension fractions collected at the pH 4 -8.3 gradient, and there was no statistically significant difference in the number of total peaks (range: 1285-1688) generated for samples from different groups (supplemental Fig. S5, A-D). Of these, the percentage of matching protein peaks among different groups was Ͼ85%, demonstrating an extensive homology in the protein profile of cardiomyopathy patients of chagasic or other etiologies with that of normal subjects. The differentially expressed protein peaks detected in normal controls, seropositive subjects from Mexico, chagasic patients from Argentina, and other cardiomyopathy patients from Argentina, marked with color-coded arrows in Fig. 3 and supplemental Fig. S4, were 27 Ϯ 1.3, 76 Ϯ 6.1, 82 Ϯ 4.5, and 57 Ϯ 5, respectively. These data indicated that (a) some sera proteins, i.e., those that were differentially expressed in normal subjects, were depressed in chagasic patients' sera, and (b) chagasic patients exhibited a higher rate of differentially expressed proteins than noted in patients with cardiomyopathy of other etiologies.
Toward discovery of proteins that are of potential significance in characterizing the disease state and severity, our next task was the identification of differentially expressed protein peaks by mass spectrometry. For this, selected second dimension fractions were either directly subjected to MALDI-TOF MS analysis or further resolved on 8% acrylamide gels to visualize the differences in the polypeptide composition, and gel slices containing differentially expressed protein bands were submitted for MALDI-TOF MS identification. The inset in Fig. 3 shows a typical SDS-PAGE analysis of a second dimension fraction from chagasic patients. The protein peaks for sequencing were chosen based upon their maximal differential expression (Ն2-fold) in seropositive, chagasic groups as compared with normal subjects. The MS and MS/MS spectral data were submitted to the UniProt human proteome database for identification of proteins, and then homology searches were conducted against NCBI and SwissProt databases to validate protein identity. These analyses yielded 108 protein identifications with high probability, some of which were identified more than once. Overall, we identified 16, 13, and 64 low abundance proteins that were overexpressed in normal subjects (i.e., decreased in chagasic subjects), other cardiomyopathy patients, and chagasic patients, respectively (Table I).

2D-GE/Mass Spectrometric Analysis of Serum Proteomic Signature of High Abundance Proteins in Chagasic Patients-
The high abundance serum proteins, eluted from the IgY-12 columns, were resolved by 2D-GE to obtain a disease-specific abundant protein signature. In initial analysis, when using

Proteome profile of low abundance protein sera fractions in human patients with cardiomyopathy of chagasic and other etiologies
Human sera samples were depleted of high abundance proteins using IgY-12 high capacity LC10 proteome partitioning chromatography. The enriched, low abundance proteins were resolved by the ProteomeLab PF-2D approach, and protein fractions eluted after second dimension high performance reverse phase chromatography were collected in 96-well plates. Proteins fractions/protein peaks uniquely detected in sera samples from normal healthy subjects with no disease, cardiomyopathy patients of other etiologies and chagasic patients were subjected to MALDI-TOF MS/MS analysis, and proteins identified by homology search against Swiss-prot database. The putative biological function and cellular location were identified using ingenuity pathway analysis and the UniProt system. The selected, differentially expressed protein fractions were resolved by SDS-PAGE and subjected to Western blotting with anti-DNPH antibody to detect DNPH-derivatized carbonyl post-translational modification.

Serum Proteomic Signature in Chagasic Patients
pH 3-10 and pH 4 -9 IPG strips, high abundance sera proteins resolved in the middle portion or on the acidic side of the second dimension 8 -16% gradient gel (supplemental Fig. S6,  A and B). The narrow range IPG strips (pH 4 -7) provided an enhanced resolution of high abundance sera proteins and detection of 5-fold more spots than were detected by using pH 3-10 and pH 4 -9 IPG strips (supplemental Fig. S6C). We therefore used pH 4 -7 IPG strips for further analysis of high abundance protein sera fractions of normal controls, seropositive chagasic subjects, and cardiomyopathy patients of other etiologies. High abundance protein fractions were pooled (three subjects/sample), and at least six samples/group were resolved to obtain 6 gel replicas per group. Representative gel images for each group are shown in Fig. 4 (A-D). Gel images were aligned by using Progenesis SameSpots TM , and densitometric analysis was performed to select differentially expressed protein spots (Fig. 4E). MALDI-TOF MS/MS analysis of 32 protein spots that were differentially expressed in chagasic patients by Ն5-fold as compared with normal controls (p ANOVA Յ 0.01) yielded 71 protein identifications (Table II). Further, we identified 14 low abundance proteins that partitioned with high abundance proteins in a disease-specific manner (Table II). Carbonyl Proteome Profile in Chagas Patients-In prior investigations in our lab, we have shown that the progression of Chagas disease is associated with pathologic oxidative stress. To investigate whether oxidative protein modifications elicit a disease specific serum carbonyl proteome, we incubated the high and low abundance protein sera fractions with DNPH and then performed one-dimensional gel electrophoresis/immunoblotting to detect the DNPH-derivatized carbonyl proteins (supplemental Fig. S7). These data showed that a large number of low abundance (supplemental Fig.  S7A) sera proteins in seropositive/chagasic and other cardiomyopathy subjects were oxidized when compared with those noted in normal controls. The extent of high abundance protein oxidation was more pronounced in chagasic subjects than that noted in other cardiomyopathy patients (supplemental Fig. S7B). To facilitate the identification of oxidized proteins, sera fractions containing high abundance proteins were resolved by 2D-GE, and WB was performed by using anti-DNPH antibody (Fig. 5, A-C). Comparisons of 2D/WB membrane images identified numerous oxidatively modified protein spots in high abundance protein sera fractions from chagasic and other cardiomyopathy subjects (Fig. 5). We also found through comparative analysis of signal densities from SYPRO Ruby-stained gels (Fig. 4) and Western blotting images (Fig. 5) that a majority of oxidized proteins in high abundance protein sera fractions from chagasic subjects were also differentially expressed. In addition to several of the high abundance proteins (albumin, transferring, IgH, IgL), we submitted six protein spots that were oxidized in chagasic samples (marked by arrows, Fig. 5C) to MALDI-TOF MS/MS analysis for protein identification. Oxidized proteins are marked by footnote a in (Table II).

Serum Proteomic Signature in Chagasic Patients
For the identification of oxidized proteins in low abundance protein-enriched sera fractions, second dimension protein fractions from PF2D analysis were resolved on 8% acrylamide gels, and WB was performed with anti-DNPH antibody. Shown in Fig. 5D is a typical SDS-PAGE/WB analysis of ProteomeLab second dimension fractions from a seropositive/chagasic subject. Corresponding Sypro Ruby-stained gels (shown as inset in Fig. 3) were utilized to cut gel slices for MALDI-TOF MS analysis and identification of oxidized low abundance sera proteins. These analyses led to identification of 33 oxidatively modified, low abundance proteins in chagasic subjects as compared with normal controls (listed in Table I).
IPA Network Analysis of the Disease-associated Proteome Signature-The differentially expressed (or oxidized) protein data sets (Tables I and II) were submitted to IPA and UniProt for identifying cellular localization and molecular and biological function. As expected for sera, Ͼ42% of the proteins differentially expressed in chagasic patients were extracellular (supplemental Fig. S8). Detection of the increased release of proteins belonging to cellular compartments, including the cell cytoplasm (13%) and nucleus (10%) in chagasic sera (supplemental Fig. S8) indicated that T. cruzi-induced cellular events and cell injury/cell death, and the resultant release of intracellular proteins, at least partially, contribute to diseasespecific differential sera proteomes.
Following IPA analysis of the differentially expressed proteins in cardiomyopathy patients, we identified four major associated networks (supplemental Table S1). These included: 1) cellular assembly and organization related to cardiovascular diseases (22 proteins, supplemental Fig. S9.1); 2) cardiovascular system development and function (20 proteins, supplemental Fig. S9.2); 3) lipid metabolism, molecular transport and small molecule biochemistry (12 proteins, supplemental Fig. S9.3); and 4) tissue development, gene expression, cardiovascular disease (10 proteins, supplemental Fig.  S9.4). Overlay of the associated networks (supplemental Table S1) identified CLU, KNG1, and PLG as central nodes (supplemental Fig. S9.5). Functional analysis by IPA identified that 29 of the differentially expressed proteins, including 14 of the 22 proteins linked to cellular assembly and organization related to cardiovascular diseases network, were responsible for elicitation of inflammation and an immune response (supplemental Table S2). These included molecules indicative of changes in activation of leukocytes (C4B, F2, GC, IGHG1, IGHG3, KNG1, PLG, PRL, and SERPING1), a quantity of phagocytes (BCL2A1, GCNT1, MYH11, PLG, PRL, and TF), movement, migration, and activation of phagocytes or neu- FIG. 4. 2D-GE analysis of serum fractions containing high abundance proteins. A-D, IgY-12 high capacity LC10 proteome partitioning system was employed to fractionate high abundance sera proteins from normal subjects exhibiting no disease (A), patients exhibiting cardiomyopathy of other etiologies (B), and seropositive chagasic (CC) individuals from Argentina (C) and Mexico (D). High abundance proteins containing sera fractions were run in the first dimension by isoelectric focusing on 11-cm linear pH 4 -7 IPG strips, and second dimension was carried out by SDS-PAGE on an 8 -10% gradient gel. Shown are Sypro Ruby-stained gel images. E, protein spots, marked on C, that were identified to be differentially expressed by Ͼ2-fold in chagasic subjects were submitted to MALDI-TOF MS analysis (listed in Table II).

Proteome profile of high abundance protein sera fractions in human patients with cardiomyopathy of chagasic and other etiologies
The high abundance proteins in human sera samples from normal healthy subjects and chagasic and other cardiomyopathy patients were separated from low abundance proteins using IgY-12 high capacity LC10 proteome partitioning chromatography and resolved by a 2D-GE approach. The gel images were analyzed on Progenesis SameSpotst TM software, and normalized spot volumes were used for comparison of the two groups. Proteins spots with Ͼ2-fold change in chagasic sera were subjected to MALDI-TOF MS/MS analysis. The putative molecular/biological function and cellular location were identified using ingenuity pathway analysis and UniProt software. NA, no match available in public information databases. Selected, differentially expressed proteins that were recognized by Western blotting with anti-DNPH antibody and consisted DNPH-derivatized carbonyl adducts.  Table S2). The top canonical pathway analysis identified several differentially expressed proteins (ALB, AMBP, C1R, C4B, C4BPA, CFB, CP, F2, FGG, HRG, ITIH2, PLG, SERPINA3, SERPING1, TF, and TTR) that were indicative of the acute phase response signaling pathway (supplemental Fig. S10) and that can be induced in response to infection or triggered by tissue injury and provide protection by using nonspecific defense mechanisms.

Validation of Differentially Expressed Proteins in Chagas
Disease-To validate the T. cruzi-induced differential expression of sera proteins identified by PF2D/2D-GE and mass spectrometry, we chose to perform ELISA with protein-specific antibody to detect the expression levels of VCL, MYL2, VIM, and PLG in chagasic (n ϭ 35) and healthy subjects (n ϭ 20) (Fig. 6). These data exhibited a substantially higher level of VCL, MYL2, and PLG in seropositive chagasic patients compared with normal healthy controls (p Ͻ 0.001). We noticed that ϳ51% of the chagasic patients exhibited VCL levels significantly higher than the mean seropositive values (mean A 450 nm , 0.372 Ϯ 0.114 versus 0.186 Ϯ 0.053; range, 0.240 -0.499 versus 0.101-0.412, seropositive versus seronegative, p Ͻ 0.001; Fig. 6A). Likewise, 56% of the chagasic patients exhibited a significantly higher level of MYL2 than the mean seropositive value (mean A 450 nm , 0.329 Ϯ 0.101 versus 0.173 Ϯ 0.0181; range, 0.121-0.332 versus 0.204 -0.423; seropositive versus seronegative, p Ͻ 0.0001; Fig. 6B). Further, the sera level of PLG was significantly increased in 32% of the chagasic patients (mean value, 216 Ϯ 33 versus 188 Ϯ 40; range, 147-280 versus 100 -220 g/ml; seropositive ver- FIG. 5. Protein carbonyls detected in serum of chagasic subjects. A-C, high abundance proteins containing sera fractions (separated by using the IgY-12 columns) from normal subjects (A), patients exhibiting cardiomyopathy of other etiologies (B), and seropositive chagasic individuals (C) were run in the first dimension by isoelectric focusing on 11-cm linear pH 4 -7 IPG strips. The strips were incubated with DNPH to derivatize carbonyl proteins, and second dimension was carried out by SDS-PAGE on an 8 -10% gradient gel. Shown are the representative images of Western blotting with anti-DNPH antibody. Respective Sypro Ruby-stained gel images are shown in Fig. 4. D, for the detection of protein carbonyls in low abundance sera protein fractions, PF2D second dimension fractions were derivatized with DNPH and then subjected to fourth dimension fractionation on an 8% polyacrylamide gel. Western blotting was performed with anti-DNPH antibody. The Coomassie Blue-stained image is shown as an inset in Fig. 3. Carbonyl proteins were identified by MALDI-TOF MS analysis (listed in Tables I and II). sus seronegative, p Ͻ 0.0001; Fig. 6D). The sera levels of VIM were increased in 10% of the seropositive individuals as compared with those in normal healthy controls (mean A 450 nm , 0.329 Ϯ 0.101 versus 0.173 Ϯ 0.0181; range, 0.204 -0.423 versus 0.121-0.332; seropositive versus seronegative; Fig.  6C); however, these results were not statistically significant. Seronegative patients with cardiac symptoms caused by other etiologies (n ϭ 25) exhibited a marginal, but not statistically significant, increase in VCL, and no increase in MYL2 and VIM levels in sera when compared with these levels in normal controls (data not shown). Together, these data validated the PF2D/mass spectrometry results and led us to suggest that the sera levels of VCL, MYL2, and PLG will be useful indicators of disease and disease severity in human chagasic patients. DISCUSSION In this study, we have developed a comprehensive serum proteome profile of chagasic human patients by utilizing a combination of PF2D ProteomeLab and traditional 2D-GE techniques. Parasite persistence and oxidative damage in the heart are known to be of pathological significance during Chagas disease (32,33). To examine this, we collected sera of clinically diagnosed cardiomyopathy patients with or without Chagas disease and compared with normal healthy controls from the same geographic area to identify the disease-specific serum proteome changes. We identified 14 proteins that were differentially expressed in sera of other cardiomyopathy patients, and 80 and 26 proteins that were differentially expressed or oxidized, respectively, in the sera of chagasic patients. Functional and network analysis assigned a majority of the differentially expressed proteins to molecular pathways associated with inflammatory responses, lipid metabolism, and molecular transport, contributing to cell death and cardiovascular disease, i.e., myocardial infarction, embolism, and hypertension, in chagasic patients. Canonical pathway analysis verified the disease association to dysregulation of the acute phase response signaling pathway. To the best of our knowledge, this is the first study demonstrating the serum oxidative and inflammatory response profile, and serum detection of cardiac proteins parallels the pathologic events contributing to Chagas disease development. Furthermore, we believe the findings have a potential utility in diagnosing disease severity and designing suitable therapy for management of human chagasic patients.
The PF2D system represents a promising tool for proteomic mapping because of its superior reproducibility, sensitivity, and automation capacity over the classical 2D gel electrophoresis, as demonstrated by a number of studies in various cell models and tissue types (34 -38). The present work is the first demonstration of using such a protein fractionation technique for a large scale profiling of the serum proteome of chagasic patients. The 12 major proteins accounting for Ͼ90% of the serum protein interfere with the detection of low abundance potential biomarker proteins. The IgY LC10 chromatography removed 90 -95% of the high abundance proteins allowing enrichment of low abundance proteins, yet proteome analysis of high abundance protein fractions by 2D-GE demonstrated that some of the low abundance proteins partitioned with high abundance protein sera fractions (Table II), likely because of binding of low abundance proteins to high abundance proteins. Overall, the PF2D analysis of low abundance protein fractions yielded highly reproducible data, leading to the identification of Ͼ80 disease-associated low abundance proteins (p Ͻ 0.001). Of these, some proteins were identified as differentially expressed in multiple groups. For example, C4BPA was identified to be differentially expressed in normal as well as chagasic patients because of the difference in oxidation; PCID2 was identified to be increased in the sera of chagasic and other cardiomyopathy patients as compared with normal controls, and HRG, SERPINA3, and NUMA1 were identified in different second dimension fractions of chagasic patients (Table I). A similar anomaly has been reported in previous proteomic studies in which the PF2D system was used (39 -41). Several factors including time-based rather than peak-based fractionation during second dimension chromatography and post-translational modifications, e.g., protein carbonylation, phosphorylation resulting in a shift in their pI or molecular weight, may have contributed to variance in partitioning and detection of proteins.
IPA is highly curated and comprehensive software used for the integration of proteins into networks and pathways with biological meaning. Network analysis of serum proteome profiles of chagasic patients identified four major subnetworks linked to the host response to T. cruzi infection and disease development, three of which were directly indicative of alterations in gene expression, cellular assembly, and cardiovascular system development and function leading to cardiovascular disease (supplemental Table S1). Functional annotation associated 29 of the differentially expressed proteins to the inflammatory response category (supplemental Table S2), many of which are known to affect activation of leukocytes, quantity of phagocytes, movement and migration of phagocytes, and aggregation of blood platelets. The presence of a mixed host response, i.e., attempt to decrease (e.g., GCNT1, SERPINA3, SERPIC1, and SERPING1), as well as increase (e.g., F2, GC, IGHA1, IGHA2, IGHG3, IGKC, KNG1, and PRL) immune responses was evident, which may indicate persistence of inflammation, known to be of pathological significance in Chagas disease (42,43). Overall, it appeared that increased complement activation (C4A/C4B, CLU, and KNG1) and activation and proliferation of phagocytes (F2, GC, KNG1, MYH11, PLG, and TF) and leukocytes (C4B, F2, GC, IGHG3, KNG1, PLG, and PRL) were followed by attempts to control cell movement or migration of immune cells (PLG, SERPINA3, SERPINC1, and SERPING1) to prevent tissue damage caused by inflammatory cytotoxic reactions. It is important to note that of the 29 differentially expressed inflammation-associated proteins (supplemental Table S2), seven proteins (ALB, BCL2A1, IGHG3, IGKC, SERPINA3, SERPINC1, and TF) were carbonylated (Tables I and II) in chagasic serum, which indicates that oxidative stress plays an important role in modulating the host immune response against T. cruzi. Other studies have demonstrated that ROS elicit inflammatory cytokines (e.g., TNF-␣, IFN-␥, and IL-1␤) in cardiomyocytes infected by T. cruzi (44,45). Inflammatory pathology was controlled in chronically infected experimental animals and human patients by enhancing the antioxidant status, which was also beneficial in preserving cardiac function during Chagas disease (15,16,46,47). Our recent observations indicate that the mitochondrial release of ROS caused by electron transport chain dysfunction and enhanced release of electrons to molecular oxygen is the primary source of oxidative stress in the heart (13). Further studies will be required to delineate the significance of mitochondrial ROS and classical ROS producers, i.e., NADPH oxidase and myeloperoxidase, known to play an important role in parasite control during the acute phase of infection (24,49,81), in elicitation of inflammatory responses during Chagas disease.
Twenty-three of the differentially expressed proteins in chagasic serum were allotted to the lipid metabolism/molecular transport category (supplemental Table S2). The expression levels of several of the proteins including ALB, APOC3, F2, F2R, KNG1, POMC, PRL, and SERPINC1 that increase quantity, production, release, and transport of lipids, specifically arachidonic acid and prostaglandins, were deregulated in chagasic patients (Tables I and II). Arachidonic acid is the precursor for prostaglandins, which function as autocrine and paracrine mediators that act upon several cell types including platelets and endothelium (50). Heightened platelet reactivity and endothelial cell dysfunction associated with an increased release of 6-keto-prostaglandin F1␣ from endothelium during T. cruzi infection is documented (51) and supported by our observation of the increased expression of several proteins (ALB, F2, GC, KNG1, and SERPINC1) activated to control blood platelet aggregation. Host as well as parasite-derived prostaglandins have been implicated in the pathogenesis of Chagas disease (52), and eicosanoids present during acute infection have been suggested to act as immunomodulators (52) aiding the transition to and maintenance of the chronic phase of the disease (53,54). Treatment with cycloxygenase 2 inhibitors (e.g., aspirin) has been shown to reduce cardiac damage (54) in chagasic experimental models. Our data in this study validate the observations made in experimental models and allow us to propose that dysregulation of lipid mediators and inflammation are interlinked pathological events during human Chagas disease, to be further validated in future studies.
When categorized in relation to disease severity, it became apparent that chagasic patients exhibited proteome signa-  (ALB, AMBP, TTR, CFB,  HRG, TF, ITIH2, C4BP, C4, FGG, CP, SERPINA2, SERPING1, and SERPINF), were linked to PLG and/or F2 nodes. PLG, when activated to function as serine protease (plasmin), directly attacks fibrin blood clots (55). Prothrombin (coaglulation factor II) is proteolytically activated to form thrombin that acts as a trypsin-like serine protease. F2 complements PLG function, contributing platelet activation and aggregation via activation of protease-activated receptors on the cell membranes and catalyzing polymerization of soluble fibrinogen into insoluble fibrin (56). A deficiency of, as well as up-regulation of, PLG and F2 beyond physiological levels can lead to multiple pathological outcomes, including thrombosis caused by partial or inadequate degradation of blood clots, defective wound healing, and excessive bleeding (55)(56)(57). Plasmin is postulated to influence the progression of cardiovascular diseases through activation of collagenases and matrix metalloproteinases and resultant degradation of matrix proteins (58) and has also been implicated in metabolic syndrome (59). PLG, F2, and their receptors are also closely linked to regulation of cardiovascular inflammatory responses and have been suggested by others to influence cell migration through regulation of growth factor and chemokine pathways and acute phase response signaling through activation of C3 component of complement system (60). Plasmin-mediated fibrin degradation products have vascular permeability-inducing effects and are considered a cardiovascular disease risk factor in aging (61). An increase in thrombolytic risk factors is documented in chagasic patients (62). Our network analysis provides the first indication of the central role of PLG and F2 in Chagas disease. Dissection of the mechanisms altered by PLG and F2 in an effort to understand their roles in vivo represents a future challenge in unraveling the pathogenesis of Chagas disease.
In prior studies, we and others have documented oxidative stress pathogenesis in experimental models of Chagas disease (32,33) and shown that enhancing antioxidant status was beneficial in controlling cardiac tissue damage and subsequent loss of contractile function (16). In this study, five proteins associated with free radical scavenging, i.e., ALB, CLU, CP, F2, and PRODH were identified, all of which were either depressed or oxidized in chagasic patients. CLU (Clustrin) is suggested to prevent cell death and the associated cytotoxicity of superoxide (63), and CP (ceruloplasmin) is related to reduction of superoxide through its antioxidant properties independently of its function in iron metabolism (64). A positive correlation between ceruloplasmin and the incidence of atrial fibrillation has been documented in a population-based cohort study (65). PRODH (proline dehydrogenase) modulates the intracellular redox environment and protects mammalian cells against oxidative stress (66). Albumin is present in large amounts in plasma and sera, and 70 -80% of Cys 34 in albumin contains a free sulfhydryl group. Through the reduced Cys 34 , ALB is able to scavenge hydroxyl radicals (67). Cys 34 and Met residues (six) are also oxidation-sensitive amino acids in ALB. Increased glycation of ALB occurs in diabetes mellitus, which is one of the pathological conditions associated with an early occurrence of vascular complications (68). Our observation of the decreased expression or increased oxidation of these proteins in chagasic patients pro-vides the proteomic signature of compromised antioxidant status.
Four proteins (VIM, MYL2, MYH11, and VCL) directly linked to cardiac muscle functions deserve to be further discussed. VIM is a member of the intermediate filament network, and along with microtubules and actin microfilaments, it plays an important role in maintaining cell shape and integrity of cytoplasm and stabilizing cytoskeletal interactions (69). Vimentin is also shown to be localized in common carotid artery and heart valves and serves as a target antigen of peripheral and heart-infiltrating T cells during valvular disease (70). The increased detection of VIM in the heart is indicative of the occurrence of a fibrotic process, because infiltrating fibroblasts replace damaged cardiomyocytes in disease conditions (70) and has been identified by proteomic inventory of myocardial proteins in patients with Chagas disease (71). The increased plasma detection of VIM in 10% of the seropositive chagasic patients may be indicative of its significance in relation to disease severity and requires further investigation in large cohort studies. MYL2 is a cardiac-specific protein.
MYL2 dimerizes with cardiac myosin ␤ (or slow) heavy chain, and its phosphorylation by Ca 2ϩ triggers cardiac contractions. Mutations in MYL2 or abnormalities in MYL2 expression are associated with cardiomyopathy (72), heart failure (73), left ventricular hypertrophy (74), and familial hypertrophy (75). MYH11 is a smooth muscle myosin belonging to the myosin heavy chain family. It functions as a major contractile protein, utilizing the energy of ATP hydrolysis to move actin filaments and produce muscle force. MYH11 mutations are documented to result in a distinct aortic and occlusive vascular pathology (76). Deletion, splicing, and mis-sense mutations in MYH11 have been identified in patients with familial thoracic aortic aneurysm and dissections with patent ductus arteriosus type 4, which is one of the most severe cardiovascular conditions in adults (77). Studies have shown that MYH11 heterozygous mutations lead to early severe decrease in the elasticity of the aortic wall, a finding consistent with the role of smooth muscle cells in maintaining the mechanical properties of the thoracic aorta (78). Vinculin plays a pivotal role in cell adhesion and migration by providing the link between the actin cytoskeleton and the transmembrane receptors integrin and cadherin (79). VCL is a cytoskeletal protein associated with cell to cell and cell-matrix junctions, where it is thought to function as one of several interacting proteins involved in anchoring F-actin to the membrane. Defects in VCL are the cause of dilated cardiomyopathy (80). The absence of VCL demonstrates a decrease in the spreading of cells that was accompanied by reduced stress fiber formation and restored by its overexpression (48). The sera release of MYL2 and MYH11 and up-regulation of VCL provide a comprehensive set of biomarkers of cardiac muscle injury and development of clinical Chagas disease in human patients.
In summary, we were successful in employing PF2D and 2D-GE to resolve low abundance and high abundance sera proteins for the development of a proteomic signature of Chagas disease. The functional analysis of differentially expressed/oxidized sera proteins suggested that dysregulated inflammation/acute phase response signaling and lipid metabolism relevant to production of prostaglandins were associated with cell death and cardiovascular disease in chagasic patients. Further studies are required to investigate the causal and mechanistic relationship of altered lipid metabolism in Chagas disease. Our ability to detect significantly higher levels of MYL2, VCL, and PLG in the sera of chagasic subjects indicates that these protein biomarkers are independent of the parasite lineage and the genetic or ethnic background of the host and thus have potential utility for diagnosis of cardiac muscle injury and development of clinical Chagas disease in human patients. These results provide an impetus for biomarker validation in large cohorts of clinically characterized chagasic patients.