Clinical proteomics in chronic inflammatory diseases: A review

There is a need for better markers for the diagnosis and prognosis of chronic inflammatory diseases. Proteomic strategies can be helpful to detect new biomarkers. Proteomic analyses are performed to characterize the behaviour of the system rather than the behaviour of any single component. Since the genome has been unravelled, the proteome received more attention. Aim of this review was to focus on the use of different proteomics techniques to detect potential and/or common biomarkers in chronic inflammatory diseases. The identified and validated proteins detected in the different studies are compared and discussed to conclude if there are some common markers which can be used in the diagnosis and prognosis of the three chronic inflammatory diseases described in this study; multiple sclerosis, rheumatic diseases and lung inflammatory diseases. The heat shock protein family (hsp) was entitled as biomarkers with potential for further research in multiple sclerosis. Myeloid-related protein 8 (MRP-8) was found in three different rheumatoid arthritis (RA) studies with different sample materials and could be a potential marker for RA. α1-Antitrypsin was validated in two studies as a marker for sarcoidosis and α1-antitrypsin was also found to be a marker for cystic fibrosis (CF), together with myeloperoxidase and IgG.     

Differential expression of red cell proteins in hemoglobinopathy

Red blood cell proteome has not been studied well until recently, as the large abundance of hemoglobin posed challenge to the detection of other cytosolic proteins in the linear dynamic range. However, in the last couple of years, due to emergence of various novel hemoglobin depletion strategies and more state-of-the-art detection techniques, a number of works on erythrocyte proteome have appeared in the literature. As a result, we now have much deeper information about both the membrane as well as the cytosolic proteins of erythrocytes. In this review, we have discussed the role of red cell proteome on the two most well-studied hemoglobin disorders, sickle cell disease and thalassemia, emphasizing on the differential expression of the redox regulator proteins and chaperones, in particular. We have also touched upon the importance of the association of the varying levels of hemoglobin variants, particularly HbE on the clinical manifestation of composite diseases like HbEβ thalassemia.     

Elucidation of the molecular mechanisms of preeclampsia using proteomic technologies

Preeclampsia, a disease of pregnancy, is a multisystem disorder associated with elevated maternal blood pressure, proteinurea, oedema, and fetal abnormalities. It is a major cause of mortality, morbidity, perinatal death, and premature delivery. Despite active research in the past decade, there is yet no definitive cure for preeclampsia. The disease has been treated symptomatically with antihypertensives, antieclamptics, bed rest, and a whole gamut of isolated therapies. In an attempt to understand the molecular basis of this disease and many other fatal diseases including cancer and heart disease, the scientific community has been turning to understanding the genome and more lately the "proteome". Proteomics enables researchers to identify all proteins expressed in a cell or organ and detect any PTM in the protein expression patterns. Deciphering the placental proteome and studying the differences in protein expression patterns in the normal as against the preeclamptic proteome might possibly in future lead to early detection and therapeutic targeting of preeclampsia.     

Comparison of two-dimensional gel electrophoresis based and shotgun strategies in the study of plasma membrane proteome

Membrane proteins play important roles in various plasma membrane (PM) activities such as signal transduction and cell recognition. However, a comprehensive proteomic study of membrane proteins remains difficult. Different strategies have been employed to study PM proteome, but little effort has been made to systematically evaluate them. In the present work, liver PM was prepared by subcellular fractionation and an aliquot was washed by sodium carbonate. After evaluation of the PM fraction by electron microscopy and Western blotting, proteins in both original and carbonate washed PM were identified by either 2-DE coupled MALDI-TOF-MS or shotgun strategies. Then protein characteristics such as molecular weight, pI, grand average hydrophobicity, subcellular location, and transmembrane domains were systematically compared. The comparative analysis showed that shotgun strategies were more suitable to identify membrane proteins, while 2-DE-based strategies may serve as a complement. Furthermore, carbonate washing obviously enriched the integral membrane proteins. All the results suggested that the strategy combining carbonate washing and shotgun identification was the optimum strategy to study human liver PM proteome. Using this strategy, 260 high-confidence proteins were identified, wherein 139 were integral membrane proteins which had 1-17 transmembrane domains.     

Proteomic analysis of circulating immune complexes in juvenile idiopathic arthritis reveals disease-associated proteins

Juvenile idiopathic arthritis reflects a group of clinically heterogeneous arthritides hallmarked by elevated concentrations of circulating immune complexes. In this study, the circulating immune complex proteome was examined to elucidate disease-associated proteins that are overexpressed in patients with an aggressive, and at times destructive, disease phenotype. To solve this proteome, circulating immune complexes were isolated from the sera of patients with chronic, erosive or early-onset, aggressive disease and from patients in medical remission or healthy controls subsequent to protein separation by 2-DE. Thirty-seven protein spots were overexpressed in the circulating immune complexes of the aggressive disease groups as compared to controls, 28 of which have been confidently identified to date. Proteolytic fragments of glyceraldehyde-3-phosphate dehydrogenase, serotransferrin, and α-1-antitrypsin have been identified among others. In total, these 28 putative disease-associated proteins most definitely contribute to immune complex formation and likely have a significant role in disease etiology and pathogenesis. Moreover, these proteins represent markers of aggressive disease, which could aid in diagnosis and management strategies, and potential therapeutic targets to prevent or control disease outcome. This is the first in-depth analysis of the circulating immune complex proteome in juvenile idiopathic arthritis.     

Exploration of the normal human bronchoalveolar lavage fluid proteome

We obtained insight into normal lung function by proteome analysis of bronchoalveolar lavage fluid (BALF) from six normal human subjects using a "Lyse-N-Go' shotgun proteomic protocol. Intra-sample variation was calculated using three different label-free methods, (i) protein sequence coverage; (ii) peptide spectral counts and (iii) peptide single-ion current areas (PICA), which generates protein expression data by summation of the area under the curve for a given peptide single-ion current trace and then adding values for all peptides from that same parent protein. PICA gave the least intra-subject variability and was used to calculate differences in protein expression between the six subjects. We observed an average threefold inter-sample variability, which affects analysis of changes in protein expression that occur in different diseases. We detected 167 unique proteins with >100 proteins detected in each of the six individual BAL samples, 42 of which were common to all six subjects. Gene ontology analysis demonstrated enrichment of several biological processes in the lung, reflecting its expected role in gas exchange and host defense as an immune organ. The same biological processes were enriched compared to either plasma or total genome proteome, suggesting an active enrichment of plasma proteins in the lung rather than passive capillary leak.     

Non-invasive biomarker sampling and analysis of the exhaled breath proteome

Exhaled breath condensate (EBC) is a biological fluid that contains trace amounts of secreted pulmonary proteins, and is emerging as a potentially valuable and non-invasively obtained source of disease biomarkers. Proteome analysis of these samples could lead to the identification of prognostic indicators of airway diseases. The objective of this study was to develop a protocol for proteome analysis of EBC samples. In this report, an improved procedure for EBC sample preparation and concentration is presented, together with a method for comparison of the protein profiles between two groups. The presented approach enabled to study the condensed exhaled breath proteome for biomarker analysis, and revealed proteins not previously identified in an EBC proteomics approach. In a comparative pilot study, EBC protein profiles obtained from smokers and non-smokers showed distinct differences and are illustrative for its potential in clinical studies. EBC from smokers contained higher concentrations of the more abundant proteins, such as cytokeratins, compared to non-smokers, and calgranulin B was identified uniquely in EBC samples from smokers.     

Gastrointestinal fluids proteomics

Seventy million people suffer from diseases of the gastrointestinal tract annually in US, translating to US$85.5 billion in direct healthcare costs. The debilitating effects of these gastrointestinal (GI) diseases can be circumvented with good biomarkers for early detection of these disorders, which will greatly increase the success of curative treatments. GI fluids represent a potential reservoir of biomarkers for early diagnosis of various GI and systemic diseases since these fluids are the most proximal fluid bathing diseased cells. They are anticipated to have proteomes that closely reflect the ensemble of proteins secreted from the respective GI tissues. Most importantly, the disease markers present in GI fluids should be present in higher concentrations than in sera, thus offering greater sensitivity in their detection. However, proteome analysis of GI fluids can be complex mainly due to the dynamic range of protein content and the numerous PTMs of proteins in each specialized GI compartment. This review attempts to discuss the physiology of the various GI fluids, the special technical considerations required for proteome analysis of each fluid, as well as to summarize the current state of knowledge of biomarker discoveries and clinical utility of GI fluids such as salivary, gastric, pancreatic, and biliary secretions.     

On-line analytical framework for the 2-DE based proteome information

The integration of proteome information is one of the key issues in proteomics research. There are currently several proteome databases which provide proteome information such as Swiss-Prot, PDB, and SRS. However, each proteome database system supports only simple inquiries on the proteome information of its database. In order to enhance the analysis support capability of proteome information, this paper proposes a data warehouse system which constructs proteome data by integrating diverse protein information along with clinical and experiment information produced in various methods in order to enhance the analysis support capability of proteome information. Based on the proteome data warehouse, OLAP and exception discovery queries are carried out. Therefore, complex multidimensional analysis is feasible for highly systematized proteome data in a proteome data warehouse. Furthermore, various analysis results which integrate experiment information, clinical information, image information, and spot information of proteins are provided.     

Exploring the potential of platelet proteomics in children

Proteomics is a rapidly evolving ‘‘post-genomic’’ science utilizing advanced technologies in protein separation, identification, quantitation and heavily relying on bioinformatics. Proteomic research in pediatrics is important and most of the successes thus far are seen in research that utilize samples that require less invasive procedures and focus on prevailing childhood diseases such as acute lymphoblastic leukaemia and neuroblastoma. Recent advances in proteomics are helping to elucidate platelet processes that are relevant to bleeding and clotting disorders, as well as other important roles of platelets such as in angiogenesis and inflammation. Nevertheless, most of platelet proteome data obtained to date are derived from the adult population and the potential of platelet proteomic application in children has not yet been explored. As it happens in all research fields, there are additional challenges in studying children such as procuring sufficient biological samples and access to less common disease cohorts as compared to in adults. Furthermore, many of the prevalent platelet-mediated diseases in adults, such as coronary heart disease and atherosclerotic lesions, are believed to have origins during childhood. Hence, platelet proteomic research in children may reveal some important information on how platelet plays a role in the pathogenesis of disease. In this article, we refer to the current knowledge from platelet proteomic research strategies in adults and address the specific concerns in the study of pediatric samples.     

Proteome of human endometrium: Identification of differentially expressed proteins in proliferative and secretory phase endometrium

Purpose: To exploit the potential of proteomics to identify and study additional yet‐unidentified important proteins present in human endometrium. Experimental design: The proteome of human endometrium would be established using 2‐DE and MALDI and the data analyzed to identify differential protein expression in the proliferative and secretory phase of the menstrual cycle using PDQuest software and MALDI. Results: In the present work, 2‐DE of human endometrium protein led to the resolution of over 200 spots. Subsequent MALDI analysis of 215 spots allowed the identification of 194 proteins. A total of 57 out of the 215 spots were found to be differentially expressed, out of which 49 could be identified using MALDI. These differentially expressed proteins included structural proteins, molecular chaperones, signaling proteins, metabolic proteins, proteins related to immunity, RNA biogenesis, protein biosynthesis and others. The differential expressions of seven representative proteins in secretory and proliferative phase endometrium tissue were confirmed by immunoblot analysis. Conclusion and clinical relevance: This study establishes the 2‐D proteome of human endometrium represented by 194 identified protein spots. The present data provides an important clue towards determining the function of these proteins with respect to endometrium related diseases.     

Reverse-phase protein arrays for application-orientated cancer research

A detailed and quantitative analysis of disease-relevant signaling will greatly contribute to our understanding of tumorigenesis and cancer progression, and thus open new strategies for drug discovery. However, throughput and sensitivity of currently established methods available for proteome profiling do not comply with the needs of clinical research such as high sample capacity and low sample consumption. Protein microarrays emerged as a promising alternative to analyze the abundance of proteins and their phosphorylation status on a high-throughput level. Here we summarize recent methodological advancements in the field of reverse-phase protein arrays and demonstrate their potential for clinical research as well as for in vitro applications.     

Proteomics of human cerebrospinal fluid - the good, the bad, and the ugly

The development of MALDI ESI in the late 1980s has revolutionized the biological sciences and facilitated the emergence of a new discipline called proteomics. Application of proteomics to human cerebrospinal fluid (CSF) has greatly hastened the advancement of characterizing the CSF proteome as well as revealing novel protein biomarkers that are diagnostic of various neurological diseases. While impressive progressions have been made in this field, it has become increasingly clear that proteomics results generated by various laboratories are highly variable. The underlying issues are vast, including limitations and complications with heterogeneity of patients/testing subjects, experimental design, sample processing, as well as current proteomics technology. Accordingly, this review not only summarizes the current status of characterization of the human CSF proteome and biomarker discovery for major neurodegenerative disorders, i.e., Alzheimer's disease and Parkinson's disease, but also addresses a few essential caveats involved in several steps of CSF proteomics that may contribute to the variable/contradicting results reported by different laboratories. The potential future directions of CSF proteomics are also discussed with this analysis.     

Proteomic profiling of heat shock proteins: An emerging molecular approach with direct pathophysiological and clinical implications

The HSP family is one of the most ancient and evolutionarily conserved protective protein families found in nature. Originally discovered as inducible molecules capable of maintaining cellular homeostasis against abrupt temperature changes, HSPs were later determined to represent an adaptive physiological response that copes with a variety of different cellular proteotoxic stresses. These physiological molecular chaperones facilitate the synthesis, folding, assembly, trafficking and secretion of specific proteins in various cellular compartments. Most importantly, these proteins guard the whole cell proteome against misfolding and inappropriate aggregation. A series of diversified proteotoxic stresses, including heat, hypoxia/ischemia, free radicals, acidosis, ATP depletion and toxins are capable of inducing a typical cellular stress response characterised by rapid inhibition of overall protein synthesis, with a concomitant dramatic increase in HSP expression. From a pathophysiological point of view, HSP induction has been observed in a wide spectrum of inflammatory and degenerative diseases (from cancer to prion disease by passing to infective and autoimmune diseases) and, intriguingly, overexpression monitoring seems to have potential implications in terms of diagnosis, prognosis and, above all, therapy. Proteomics studies, identifying a series of modification of HSP expression patterns in different diseases, are confirming these promising clinical applications.     

Immune response to chemically modified proteome

Both enzymatic and nonenzymatic PTMs of proteins involve chemical modifications. Some of these modifications are prerequisite for the normal functioning of cell, while other chemical modifications render the proteins as “neo-self” antigens, which are recognized as “non-self” leading to aberrant cellular and humoral immune responses. However, these modifications could be a secondary effect of autoimmune diseases, as in the case of type I diabetes, hyperglycemia leads to protein glycation. The enigma of chemical modifications and immune response is akin to the “chick-and-egg” paradox. Nevertheless, chemical modifications regulate immune response. In some of the well-known autoimmune diseases such as rheumatoid arthritis, systemic lupus erythematosus, and multiple sclerosis, chemically modified proteins act as autoantigens forming immune complexes. In some instances, chemical modifications are also involved in regulating immune response during pathogen infection. Further, the usefulness of proteomic analysis of immune complexes is briefly discussed.     

Proteomics of bacterial pathogenicity: therapeutic implications

Identification of the molecular mechanisms of host-pathogen interaction is becoming a key focus of proteomics. Analysis of these interactions holds promise for significant developments in the identification of new therapeutic strategies to combat infectious diseases, a process that will also benefit parallel improvements in molecular diagnostics, biomarker identification and drug discovery. This review highlights recent advances in functional proteomics initiatives in infectious disease with emphasis on studies undertaken within physiologically relevant parameters that enable identification of the infectious proteome rather than that of the vegetative state. Deciphering the molecular details of what constitutes physiologically relevant host-pathogen interactions remains an underdeveloped aspect of research into infectious disease. The magnitude of this deficit will be largely influenced by the ease with which model systems can be established to investigate such interactions. As the selective pressures exerted by the host on an infecting pathogen are numerous, the adequacy of certain model systems should be considered carefully.     

The salivary proteome: challenges and perspectives

We provide a brief overview of the salivary proteome but with an emphasis on the major challenges in protein identification and quantitation. Precautions are necessary to avoid proteolysis, deglycosylation and dephosphorylation of salivary proteins by microbial and host enzymes in saliva. Many proteins are differentially expressed in secretions from different salivary glands and their proportional contributions to saliva vary with the flow rate. The total protein concentration in the secretion from any one gland varies considerably, depending on factors such as flow rate, duration of stimulation, nature of the stimulus and circadian rhythms. Many plasma proteins enter saliva via gingival crevicular fluid, of which there are increased amounts in persons with gingivitis or periodontal disease. These factors must be taken into account in the identification of potential biomarkers for different oral or systemic diseases.     

Mining the low molecular weight proteome of blood

Serum and plasma are composed of highly complex protein/peptide mixtures resulting from the systemic monitoring of every biological process in a living organism. Some of these sentinel changes are extremely short-lived while others produce more stable by-products. In addition, since biological events occur simultaneously and with overlapping physiological demands, separating the desired ones from "background" changes is an exceptional challenge. In this review, we outline a definition of the "low molecular weight proteome" as a valuable subcomponent of the blood proteome. We make a case that this derivative proteome is as information rich and equally complex as the parent proteome. We discuss some of the technical challenges in the analysis of the low molecular weight proteome with an emphasis on MS-based analytical approaches. With specific example of several reported methodologies we attempt to frame the current state-of-the-art in study design as a guide to future efforts.     

Proteomic analysis of pericardial effusion: Characteristics of tuberculosis-related proteins

The aim of this study has been designed to identify the tuberculosis (TB)-related proteins in pericardial effusion by proteomic approaches. TB is one of the major infectious diseases causing pericardial effusion. This study details protein profiles in pericardial effusion from three TB patients and three heart failure patients. Pericardial effusions were analyzed using 2-DE combined with the nano-HPLC-ESI-MS/MS. Eleven protein spots with differential expression in pericardial effusion were identified between the two groups of TB and heart failure patients (the control group). Seven protein spots were upregulated and four were downregulated. The composition of the pericardial effusion proteome may reflect the pathophysiological conditions affecting the progression of tuberculous pericarditis. The proteins in the tuberculous pericardial effusion with differential expression may serve as new and direct indicators of drug treatment. A possible conclusion is indicated that fibrinogen may play an important role for fibrin assembly in tuberculous pericardial effusion.