Protein expression patterns associated with progression of chronic obstructive pulmonary disease in bronchoalveolar lavage of smokers

BACKGROUND: We modeled the expression of proteins in baseline bronchoalveolar lavage (BAL) samples from asymptomatic 60-year-old lifelong current smokers or healthy never-smokers, who were reevaluated after 6 to 7 years to record clinical outcome. METHODS: Applying a technology toolbox consisting of replicate 2-dimensional gel separations, image annotation, and mass spectrometry identification, we catalogued a global set of proteins that were differentially expressed in individuals by presence, absence, and intensity scores. RESULTS: By use of multivariate analysis, we selected a subset of proteins that accurately separated smokers from never-smokers based on composite scoring. Follow-up after 6 to 7 years identified a group of individuals who had progressed to chronic obstructive pulmonary disease (COPD), Global Initiative for Chronic Obstructive Lung Disease stage 2. The baseline BAL samples of these eventual COPD patients shared a distinct protein expression profile that could be identified using partial least-squares discriminant analysis. This pattern was not observed in BAL samples of asymptomatic smokers free of COPD at 6- to 7-year follow-up. CONCLUSIONS: Our model suggests that certain patterns of protein expression occurring in the airways of long-term smokers may be detected in smokers susceptible to a progression of COPD disease, before disease is clinically evident.     

Differential proteomic analysis of bronchoalveolar lavage fluid from lung transplant patients with and without chronic graft dysfunction

BackgroundBiomarkers are urgently needed for diagnosis, prognosis and monitoring of lung transplant chronic graft dysfunction. Bronchoalveolar lavage fluid (BAL) has been used in the past as proximal fluid for biomarker discovery in various lung diseases including chronic graft dysfunction (CGD). The current study describes the proteomic analysis of BAL fluids collected from 4 asymptomatic post-transplant patients and 3 patients with symptoms of CGD.MethodsBAL proteome was fractionated by size-exclusion chromatography at protein level and reverse-phase-chromatography at peptide level followed by Orbitrap® mass spectrometry detection.ResultsOur in-depth proteomic analysis identified 531 proteins, the largest catalog of BAL proteins reported to date in the context of CGD. A total of 30 and 39 proteins detected exclusively in CGD and non-CGD samples, respectively, are potential candidates for verification phase.ConclusionsA new protocol was developed to enhance the sensitivity of detecting less abundant proteins in BAL.     

Direct-tissue SELDI-TOF mass spectrometry analysis: a new application for clinical proteomics

BACKGROUND: New molecular profiling technologies can aid in analysis of small pathologic samples obtained by minimally invasive biopsy and may enable the discovery of key biomarkers synergistic with anatomopathologic analysis related to prognosis, therapeutic response, and innovative target validation. Thus proteomic analysis at the histologic level in healthy and pathologic settings is a major issue in the field of clinical proteomics. METHODS: We used surface-enhanced laser desorption ionization-time-of-flight mass spectrometry (SELDI-TOF MS) technology with surface chromatographic subproteome enrichment and preservation of the spatial distribution of proteomic patterns to detect discrete modifications of protein expression. We performed in situ proteomic profiling of mouse tissue and samples of human cancer tissue, including brain and lung cancer. RESULTS: This approach permitted the discrimination of glioblastomas from oligodendrogliomas and led to the identification of 3 potential markers. CONCLUSION: Direct tissue proteomic analysis is an original application of SELDI-TOF MS technology that can expand the use of clinical proteomics as a complement to the anatomopathological diagnosis.     

iTRAQ技术鉴定小鼠睾丸发育差异表达蛋白的研究

目的 用同量异位素标记的相对和绝对定量(isobaric tags for relative and absolute quantitation,iTRAQ)技术结合液相色谱质谱技术构建3周龄小鼠与成年小鼠睾丸蛋白差异表达谱系。 方法 分别抽提3周龄小鼠与成年小鼠睾丸总蛋白质,iTRAQ标记,液相色谱（LC）分离蛋白质,LTQ Orbitrap^{TM质谱仪进行蛋白质鉴定。 结果 鉴定出20个差异表达蛋白质,其中18个为3周龄小鼠睾丸高表达蛋白质,2个为成年睾丸高表达蛋白质。差异表达蛋白质主要参与细胞的有丝分裂、减数分裂、细胞增殖、分化、凋亡及精子发生等重要细胞事件。 结论 用目前最新的定量蛋白质组技术,构建的3周龄小鼠睾丸与成年睾丸差异表达蛋白谱系对研究睾丸功能、精子发生有一定理论价值,为定量蛋白组学技术在雄性生殖领域的应用奠定了基础。}     

FTICR mass spectrometry in proteomics

Analytical tools that allow rapid screening, low sample consumption and accurate protein identification are of great importance in studies of complex biological samples. Today, mass spectrometry (MS) is a key analytical tools with applications in a wide variety of fields, reaching from the analysis of elemental compositions in various materials to the identification of large protein complexes. One of the fastest growing fields of MS applications is proteomics, or the study of protein expression in an organism. In the traditional proteomic approach, two-dimensional sodium dodecylsulfate polyacrylamide gel electrophoresis is applied for the separation and visualization of proteins. In this review, the use of high resolution Fourier transform ion cyclotron resonance mass spectrometry, including up-front multidimensional liquid separations for 'top down' or 'bottom up' proteomic approaches, are presented.     

The use of proteomics for the assessment of clinical samples in research

Proteomics, the analysis of expressed proteins, has been an important developing area of research for the past two decades [Anderson, NG, Anderson, NL. Twenty years of two-dimensional electrophoresis: past, present and future. Electrophoresis 1996;17:443-453]. Advances in technology have led to a rapid increase in applications to a wide range of samples; from initial experiments using cell lines, more complex tissues and biological fluids are now being assessed to establish changes in protein expression. A primary aim of clinical proteomics is the identification of biomarkers for diagnosis and therapeutic intervention of disease, by comparing the proteomic profiles of control and disease, and differing physiological states. This expansion into clinical samples has not been without difficulties owing to the complexity and dynamic range in plasma and human tissues including tissue biopsies. The most widely used techniques for analysis of clinical samples are surface-enhanced laser desorption/ionisation mass spectrometry (SELDI-MS) and 2-dimensional gel electrophoresis (2-DE) coupled to matrix-assisted laser desorption ionisation [Person, MD, Monks, TJ, Lau, SS. An integrated approach to identifying chemically induced posttranslational modifications using comparative MALDI-MS and targeted HPLC-ESI-MS/MS. Chem. Res. Toxicol. 2003;16:598-608]-mass spectroscopy (MALDI-MS). This review aims to summarise the findings of studies that have used proteomic research methods to analyse samples from clinical studies and to assess the impact that proteomic techniques have had in assessing clinical samples.     

Effects of the incubation in vitro with sorbents on serum proteomic pattern and cytokine concentration in cancer patients during chemotherapy--preliminary results.

INTRODUCTION: Cancer and treatment by chemotherapy often produce abnormalities in endogenous cytokine, chemokine, and inflammatory mediator production. Sorbent-based adsorption therapies have been used to remove cytokines in diverse human diseases. AIM: The aim of this study was to evaluate the effects of chemotherapy on serum proteomic pattern and cytokine concentration, and to evaluate the ex vivo feasibility of using sorbents to remove cytokines, chemokines and other proteins in adult cancer patients undergoing chemotherapy with fluorouracil or carboplatin-taxane combinations. PATIENTS AND METHODS: Serum samples of three female adult patients (one affected by rectal cancer and two by ovarian cancer) were examined before and on the fourth day of the first cycle of chemotherapy with fluorouracil (rectal cancer patient) or carboplatin-taxane combination (ovarian cancer patients). The analysis was performed, by means of luminex technology and with a proteomic approach, on native serum samples, and on the same sera after 2 hours of in vitro incubation with a synthetic based styrenic divinylbenzene resin. RESULTS: Chemotherapy determined variable effects on serum concentration of cytokines, while the incubation in vitro of patients serum samples with the resin induced a significant decrease (>80%) in serum concentration of different chemokines, cytokines, growth factors and proteins. The proteomic approach, using SDS PAGE and 2-DE highlighted differences in protein expression between sera from healthy controls and cancer patients. Proteomic analysis demonstrated also variations in the expression of proteins, particularly those with low-molecular weight, due to chemotherapy. Finally, the incubation in vitro of serum samples with sorbents induced a general reduction of protein expression. Within the cancer patients maps, 10 spots were chosen for identification with MALDI-TOF analysis. CONCLUSION: The incubation in vitro with sorbents normalized the over-expression of different proteins and cytokines induced by chemotherapy, suggesting further evaluation as a possible adjuvant treatment.     

Eicosanoid levels in bronchoalveolar lavage fluid of young female smokers and non-smokers

To evaluate indicators of inflammatory changes in the airways of young smokers we have measured the levels of several eicosanoids in bronchoalveolar lavage (BAL) fluid of 18 female smokers (age 33 +/- 2 years) and 9 female non-smokers (age 29 +/- 2 years) who were hospitalized for treatment not related to any pulmonary disease. In each BAL specimen the following eicosanoids were determined by radioimmunoassay: prostaglandin (PG) E2; PGF2 alpha; 9 alpha, 11 beta-PGF2, a metabolite of PGD2; 6-keto PGF1 alpha, a metabolite of prostacyclin; thromboxane (Tx) B2, a metabolite of TxA2; the 5-lipoxygenase products 5-hydroxy-eicosa-tetraenoic acid (HETE), leukotriene (LT) B4 and LTC4; the 12-lipoxygenase product 12-HETE; and the 15-lipoxygenase product 15-HETE. The concentrations of the cyclooxygenase products (pg ml-1) in the BAL fluid of the non-smokers were: PGE2 15.4 +/- 1.9, PGF2 alpha 7.6 +/- 1.0, 9 alpha, 11 beta-PGF2 8.7 +/- 1.8, TxB2 8.8 +/- 1.3, and 6-keto PGF1 alpha only 1.5 +/- 0.8. The concentration of the lipoxygenase products were: 15-HETE 781 +/- 200, 12-HETE 193 +/- 33, 5-HETE 14.0 +/- 3.1, LTC4 9.5 +/- 3.1, LTB4 6.2 +/- 1.4. BAL fluid from smokers contained two- to three-fold higher levels of TxB2 and PGF2 alpha (P less than 0.05). The levels of TxB2 and PGF2 alpha were positively correlated to the number of package years (rs = 0.55 and rs = 0.65, P less than 0.02). The concentrations of 5-, 12- and 15-HETE tended to be higher in BAL fluid from smokers, but this was not significant.(ABSTRACT TRUNCATED AT 250 WORDS)     

Monomeric calgranulins measured by SELDI-TOF mass spectrometry and calprotectin measured by ELISA as biomarkers in arthritis

BACKGROUND: SELDI-TOF mass spectrometry (MS) is a high-throughput proteomic approach with potential for identifying novel forms of serum biomarkers of arthritis. METHODS: We used SELDI-TOF MS to analyze serum samples from patients with various forms of inflammatory arthritis. Several protein profiles were collected on different Bio-Rad Laboratories ProteinChip arrays (CM10 and IMAC-Cu(2+)) and were evaluated statistically to select potential biomarkers. RESULTS: SELDI-TOF MS analyses identified several calgranulin proteins [S100A8 (calgranulin A), S100A9 (calgranulin B), S100A9*, and S100A12 (calgranulin C)], serum amyloid A (SAA), SAA des-Arg (SAA-R), and SAA des-Arg/des-Ser (SAA-RS) as biomarkers and confirmed the results with other techniques, such as western blotting, immunoprecipitation, and nano-LC-MS/MS. The S100 proteins were all able to significantly differentiate samples from patients with rheumatoid arthritis (RA), psoriatic arthritis (PsA), and ankylosing spondylitis (AS) from those of patients with inflammatory bowel diseases used as an inflammatory control (IC) group, whereas the SAA, SAA-R, and SAA-RS proteins were not, with the exception of AS. The 4 S100 proteins were coproduced in all of the pathologies and were significantly correlated with the plasma calprotectin concentration; however, these S100 proteins were correlated with the SAA peak intensities only in the RA and IC patient groups. In RA, these S100 proteins (except for S100A12) were significantly correlated with the serum concentrations of C-reactive protein, matrix metalloproteinase 3, and anti-cyclic citrullinated peptide and with the Disease Activity Score (DAS(28)). CONCLUSIONS: The SELDI-TOF MS technology is a powerful approach for analyzing the status of monomeric, truncated, or posttranslationally modified forms of arthritis biomarkers, such as the S100A8, S100A9, S100A12, and SAA proteins. The fact that the SELDI-TOF MS data were correlated with results obtained with the classic calprotectin ELISA test supports the reliability of this new proteomic technique.     

Bronchoalveolar lavage fluid characteristics of early intermediate and late phases of ARDS

Objective: To determine the concentration of proteins and phospholipids, markers of inflammatory reaction such as platelet-activating factor (PAF), and cell alterations in bronchoalveolar lavage (BAL) fluid during the evolution of the acute respiratory distress syndrome (ARDS). Design: Prospective controlled study. Setting: 14-bed medical-surgical intensive care unit in a 750-bed university teaching hospital. Patients: 19 mechanically ventilated patients, 9 patients with ARDS and 10 patients without cardiopulmonary disease (controls), were eligible for this study. Interventions: BAL was performed during the early, intermediate, and late phases of ARDS. Measurements and results: Total phospholipids and individual phospholipid classes of the surfactant, proteins, PAF, and cells were measured. High levels of PAF, an increase in neutrophils and proteins, and quantitative as well as qualitative alterations in phospholipids in BAL fluid were observed in ARDS patients compared to the control group. PAF, proteins, and neutrophils were higher in early ARDS than in intermediate or late ARDS. The surfactant pool increased in the early phase and decreased in the intermediate or late phase of the syndrome. The qualitative alterations of surfactant consist of reduced phospholipid content in the surfactant structures with good surface properties; moreover, there was a considerable decrease in the percentage of phosphatidylcholine and phosphatidylglycerol, followed by an increase in phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, and sphingomyelin in all three phases of ARDS compared to the control group. Lyso-phosphatidylcholine was detectable only in late ARDS. Conclusion: Total surfactant phospholipids, surfactant components, and inflammatory markers such as PAF, cells, and proteins were affected in patients with ARDS. These factors, undergoing quantitative alterations during the course of ARDS, could have a significant role in the pathogenesis and evolution of ARDS. Received: 8 July 1997 Accepted: 17 December 1997     

Transcriptomic and proteomic analysis of liver and muscle alterations caused by surgical stress in rats

The metabolic response to injury includes major alterations in protein metabolism; however, little is known about alterations in the synthesis of individual proteins and their role in the stress response. Our aim was to study how individual proteins in liver and muscle are altered by abdominal surgery. Changes produced in mRNA and proteins by abdominal surgery were studied in rats using RAP (random arbitrary priming)-PCR, to investigate mRNA alterations, and standard or isotopic (with in vivo radioactive labelling of proteins) two-dimensional electrophoresis/MS proteomic analyses, to study differential expression of proteins. Many of the differentially expressed proteins identified in blood were specifically synthesized by the liver to participate in the stress response. The hepatic proteins (antioxidant proteins, serine protease inhibitors, acute-phase proteins and transport proteins) were secreted into the bloodstream to produce a systemic action, indicating the central role of the liver in the stress response. Overexpressed proteins identified in liver were associated with the glycolytic processes and the folding of nascent proteins, confirming the high metabolic activity of the liver after surgery. The role of skeletal muscle protein as an amino acid donor to fuel the processes involved in the stress response was shown by the decrease in high-molecular-mass myofibrillar proteins. Combined use of the three techniques studied, differential RAP-PCR and standard and isotopic proteome analysis, provided complementary information on the differentially expressed proteins in a rat model of surgical stress.     

Preanalytic influence of sample handling on SELDI-TOF serum protein profiles

BACKGROUND: High-throughput proteomic methods for disease biomarker discovery in human serum are promising, but concerns exist regarding reproducibility of results and variability introduced by sample handling. This study investigated the influence of different preanalytic handling methods on surface-enhanced laser desorption/ionization time-of-flight mass spectrometry (SELDI-TOF MS) protein profiles of prefractionated serum. We investigated whether older collections with longer sample transit times yield useful protein profiles, and sought to establish the most feasible collection methods for future clinical proteomic studies. METHODS: To examine the effect of tube type, clotting time, transport/incubation time, temperature, and storage method on protein profiles, we used 6 different handling methods to collect sera from 25 healthy volunteers. We used a high-throughput, prefractionation strategy to generate anion-exchange fractions and examined their protein profiles on CM10, IMAC30-Cu, and H50 arrays by using surface-enhanced laser desorption/ionization time-of-flight mass spectrometry. RESULTS: Prolonged transport and incubation at room temperature generated low mass peaks, resulting in distinctions among the protocols. The most and least stringent methods gave the lowest overall peak variances, indicating that proteolysis in the latter may have been nearly complete. For samples transported on ice there was little effect of clotting time, storage method, or transit time. Certain proteins (TTR, ApoCI, and transferrin) were unaffected by handling, but others (ITIH4 and hemoglobin beta) displayed significant variability. CONCLUSIONS: Changes in preanalytical handling variables affect profiles of serum proteins, including proposed disease biomarkers. Proteomic analysis of samples from serum banks collected using less stringent protocols is applicable if all samples are handled identically.     

急性髓系白血病与急性淋巴系白血病的蛋白质组比较研究

本研究通过对急性髓系白血病(AML)与急性淋巴系白血病(ALL)细胞比较蛋白质组分析,寻找亚型特异性的蛋白质表达谱。应用双向电泳分别分离AML不同亚型(M1、M2、M3)、ALL患者骨髓白血病细胞蛋白质,利用PDQuest 7.4软件分析双向电泳图谱差异蛋白质点,基质辅助的激光解析飞行时间质谱和生物信息学鉴定差异蛋白质。结果显示:经过电泳图谱分析得到21个差异蛋白质点,质谱鉴定提示15种蛋白有统计学意义。在AML中高表达的蛋白质包括髓过氧化物酶(MPO)、硫氧还蛋白依赖的过氧化物还原酶(PRDX3),钙网蛋白(CALR)、烯酰辅酶A水合酶ECH1等7种,在ALL中高表达的蛋白质包括ARHGDIB、肌动蛋白抑制蛋白1(PFN1)、肌动蛋白(ACTG1)等8种。结论:AML与ALL细胞存在差异蛋白质表达谱,这将有助于发现早期诊断的分子标志物及特异性治疗靶标。     

Serum retinol binding protein 4 and galectin-3 binding protein as novel markers for postmenopausal nonalcoholic fatty liver disease

ObjectiveTo investigate the differential protein expression before and after menopause in women with nonalcoholic fatty liver disease (NAFLD) and to explore novel markers for menopausal NAFLD.MethodsEight serum samples collected from pre- or post-menopausal women with NAFLD were analysed by iTRAQ 2D-LC-MS/MS. Two protein candidates were selected and verified by enzyme-linked immunosorbent assay (ELISA) in serum samples collected from a one hundred and fifty-three female population subsequently, including 51 in post-menopausal status with NAFLD, 41 in pre-menopausal with NAFLD, 19 healthy individuals in post-menopausal status and 42 healthy pre-menopausal women.ResultsA total of one hundred and sixty-seven proteins exhibiting significant changes were characterized, among which sixty-five were up-regulated and one hundred and two were down-regulated. Of those altered proteins, the expression of serum retinol binding protein 4 (RBP4) and galectin-3 binding protein (LGALS3BP) was obviously increased in the post-menopausal patient group compared to the other. ELISA validations for the two proteins were consistent with the proteomic profiling.ConclusionsSerum RBP4 and LGAL3BP were up-regulated after menopause under NAFLD conditions, which suggested the two proteins may be potential markers as NAFLD in postmenopausal population.     

Analytical characteristics of cleavable isotope-coded affinity tag-LC-tandem mass spectrometry for quantitative proteomic studies

Quantitative proteomic studies using cleavable isotope-coded affinity tags (cICAT) in concert with tandem mass spectrometry (MS/MS) permit unbiased comparisons between biologically distinct samples. We sought to determine the analytical characteristics of cICAT-based studies by examining the cumulative results of multiple, separate cICAT-based experiments involving human lymphoma-derived cells. We found that the number of identified proteins increased with larger numbers of fractions analyzed. The majority of proteins were identified by single peptides. Only 24 to 41% of the peptides contained cysteine residues, but 85% of the cysteine-containing peptides yielded quantification data. Approximately 28% of all identified proteins yielded quantification data, with 57% of these being differentially expressed by at least 1.5-fold. The quantification ratios of peptides for proteins with multiple quantified peptides were concordant in trend in 87% of instances. cICAT-labeled peptides identified proteins in all subcellular compartments without significant bias. Analysis of the flow-through fraction did not increase the number of peptides identified per protein. Our studies indicate that cICAT-LC-MS/MS yields quantifications primarily based on single peptides, and analysis of flow-through peptides does not contribute signifi-cantly to the results. Nevertheless, identifications based on single cICAT-labeled peptides with tryptic ends provide sufficiently reliable protein identifications and quantification information in cICAT-LC-MS/MS-based proteomic studies.     

Increased levels of bombesin-like peptides in the lower respiratory tract of asymptomatic cigarette smokers.

Bombesin-related peptides are growth factors for a variety of cells, including normal human bronchial epithelial cells. An ELISA for bombesin-like peptides (BLP) has been devised using the MAb BBC353, which is specific for the biologically active carboxy-terminal fragment shared by all known BLP. Using this ELISA, we measured bronchoalveolar lavage (BAL) fluid levels of BLP in normal cigarette smokers (n = 15) and normal nonsmokers (n = 18). Smokers' BAL fluid contained increased levels of BLP, whether expressed in terms of BAL fluid volume (P = 0.0001) or protein content (P less than 0.05). BLP levels did not correlate with any cellular constituent in the BAL fluid but immunostaining of lung tissue with BBC353 revealed an intense specific staining of neuroendocrine cells, implying these as a potential source. Two peaks of bombesin-like immunoreactivity were purified using sequential reverse phase and gel filtration HPLC. Both BLP have apparent molecular weights similar to gastrin-releasing peptide on gel filtration HPLC analysis. However, the amino acid composition of these BLP is different from that of gastrin-releasing peptide or neuromedin B, the only known mammalian forms of BLP, suggesting either incomplete purification or novel peptides. Sequence analysis could not be performed due to blocking groups at the amino terminus of these peptides. Our data demonstrate that cigarette smoking is associated with increased levels of pulmonary BLP and imply a potential role for these neuropeptides in the lung's response to tobacco smoke.     

Identification of potential serum biomarkers of acute paraquat poisoning in humans using an iTRAQ quantitative proteomic

Paraquat (PQ) poisoning has high mortality rates in many countries. Due to it readily being absorbed through the gastrointestinal tract and rapidly excreted in the urine, few biomarkers possess satisfactory specificity and sensitivity in diagnostic and forensic practices. To investigate serum biomarkers in patients with PQ poisoning, pooled sera was analyzed using a proteomic approach based on iTRAQ coupled LC-MS/MS. Of the 413 proteins identified with high confidence, 81 were found to be differentially expressed (1.5-fold change) in the sera of patients with PQ poisoning. The differential expression pattern of 4 of these proteins was validated by enzyme-linked immunosorbent assay (ELISA) in clinical samples. A sera sample from a PQ poisoning patient has shown relatively increased abundance of S100A8 and S100A9. The overexpression of S100A8 and S100A9 was further validated in the lung tissue of PQ-treated rat associated with lung damage. Meanwhile, we identified another two down-expressed proteins, transferrin receptor protein 1 (TfR1) and serum amyloid P-component (SAP), which may be also practicable in human clinical samples as PQ poisoning serum biomarkers. Furthermore, receiver operating characteristic curve analysis confirmed that the expression levels of S100 alarmins, TfR1 and SAP in patient serum could provide a discriminatory diagnostic test for predicting PQ poisoning in patients. Therefore, our results suggest that increased serum levels of S100 alarmins and decreased serum levels of TfR1 and SAP may constitute potential biomarkers for the prediction of PQ poisoning in humans, and might be novel therapeutic targets in PQ poisoning.

Using an iTRAQ quantitative proteomic, S100 alarmins, TfR1 and SAP have been discovered as potential indicators to paraquat poisoning in humans.     

Identification and confirmation of haptoglobin as a potential serum biomarker in hypertrophic cardiomyopathy using proteomic approaches

Abstract

Introduction. Aiming at identifying biomarkers for hypertrophic cardiomyopathy (HCM), the serum proteome was explored through a two-dimensional gel-based proteomic approach (2D-DIGE) coupled with mass spectrometry and database interrogation.

Methods. Serum samples from 20 male HCM patients and their sex- and age-matched controls were cleaned from interfering components. Patients and controls were pooled in five matched groups with the same age, and proteins extracts from each pool were labelled with cyanine dyes. Then, gel images were analysed using a fluorescence scanner and proteins were identified. Tryptic peptides were analysed by capillary reversed-phase liquid chromatography coupled online with tandem mass spectrometry (MS/MS).

Results. Four different proteins were observed to be differentially expressed between HCM patients and their matched controls. Of them, decreases in haptoglobin levels were confirmed to be associated with HCM in an independent set of 181 consecutive HCM patients from our monographic clinic and 114 controls with similar age and sex using a nephelometer-based technique. Moreover, a significant negative correlation was observed between haptoglobin and subaortic gradient, thus highlighting the role of haptoglobin in HCM.

Conclusion. All these observations point out the utility of the 2D-DIGE proteomic strategy for the identification of serum proteins indicative of the presence of cardiac injury.