Comparative proteomic analysis to discover potential therapeutic targets in human multiple myeloma

To clarify the molecular mechanisms that participate in the formation of multiple myeloma (MM) and to detect any tumor-related biomarkers, we performed proteomic analysis of cellular protein extracts from MM cells and normal plasma cells. Plasma cells from nine patients with newly diagnosed MM and nine healthy donors were purified by using anti-CD138 based immunomagnetic bead-positive selection. The protein profiles of purified MM and normal plasma cells were compared using 2-DE. We identified a total of 43 differentially expressed proteins, and confirmed with Western blotting six proteins. The altered proteins were analyzed using the software program Pathway Studio and the biological network can be accessed via (http://life-health.jnu.edu.cn/pathway/pathway.html). Further functional studies showed that annexin A1 knock down modestly induces lethality alone and potentiates the effects of dexamethasone on both dexamethasone-sensitive and dexamethasone-resistant MM cells. By correlating the proteomic data with these functional studies, the current results provide not only new insights into the pathogenesis of MM but also direct implications for the development of novel anti-MM therapeutic strategies and could lead to the discovery of potential therapeutic targets. Future molecular and functional studies would provide novel insights into the roles of these dysregulated proteins in the molecular etiology of MM.     

Proteomics in the investigation of HIV-1 interactions with host proteins

Productive HIV-1 infection depends on host machinery, including a broad array of cellular proteins. Proteomics has played a significant role in the discovery of HIV-1 host proteins. In this review, after a brief survey of the HIV-1 host proteins that were discovered by proteomic analyses, I focus on analyzing the interactions between the virion and host proteins, as well as the technologies and strategies used in those proteomic studies. With the help of proteomics, the identification and characterization of HIV-1 host proteins can be translated into novel antiretroviral therapeutics.     

Human pathogenic streptococcal proteomics and vaccine development

Gram-positive streptococci are non-motile, chain-forming bacteria commonly found in the normal oral and bowel flora of warm-blooded animals. Over the past decade, a proteomic approach combining 2-DE and MS has been used to systematically map the cellular, surface-associated and secreted proteins of human pathogenic streptococcal species. The public availability of complete streptococcal genomic sequences and the amalgamation of proteomic, genomic and bioinformatic technologies have recently facilitated the identification of novel streptococcal vaccine candidate antigens and therapeutic agents. The objective of this review is to examine the constituents of the streptococcal cell wall and secreted proteome, the mechanisms of transport of surface and secreted proteins, and describe the current methodologies employed for the identification of novel surface-displayed proteins and potential vaccine antigens.     

Proteomic analysis of serum, plasma, and lymph for the identification of biomarkers

Probably no topic has generated more excitement in the world of proteomics than the search for biomarkers. This excitement has been generated by two realities: the constant need for better biomarkers that can be used for disease diagnosis and prognosis, and the recent developments in proteomic technologies that are capable of scanning the individual proteins within varying complex clinical samples. Ideally a biomarker would be assayable from a noninvasively collected sample, therefore, much of the focus in proteomics has been on the analysis of biofluids such as serum, plasma, urine, cerebrospinal fluid, lymph, etc. While the discovery of biomarkers has been elusive, there have been many advances made in the understanding of the proteome content of various biofluids, and in the technologies used for their analysis, that continues to point the research community toward new methods for achieving the ultimate goal of identifying novel disease-specific biomarkers. In this review, we will describe and discuss many of the proteomic approaches taken in an attempt to find novel biomarkers in serum, plasma, and lymph.     

Proteomic identification of proteins in the human brain: Towards a more comprehensive understanding of neurodegenerative disease

Proteomics has revealed itself as a powerful tool in the identification and determination of proteins and their biological significance. More recently, several groups have taken advantage of the high-throughput nature of proteomics in order to gain a more in-depth understanding of the human brain. In turn, this information has provided researchers with invaluable insight into the potential pathways and mechanisms involved in the pathogenesis of several neurodegenerative disorders, e.g., Alzheimer and Parkinson disease. Furthermore, these findings likely will improve methods to diagnose disease and monitor disease progression as well as generate novel targets for therapeutic intervention. Despite these advances, comprehensive understanding of the human brain proteome remains challenging, and requires development of improved sample enrichment, better instrumentation, and innovative analytic techniques. In this review, we will focus on the most recent progress related to identification of proteins in the human brain under normal as well as pathological conditions, mainly Alzheimer and Parkinson disease, their potential application in biomarker discovery, and discuss current advances in protein identification aimed at providing a more comprehensive understanding of the brain.     

Proteomics in human cancer research

Proteomics is now widely employed in the study of cancer. Many laboratories are applying the rapidly emerging technologies to elucidate the underlying mechanisms associated with cancer development, progression, and severity in addition to developing drugs and identifying patients who will benefit most from molecular targeted compounds. Various proteomic approaches are now available for protein separation and identification, and for characterization of the function and structure of candidate proteins. In spite of significant challenges that still exist, proteomics has rapidly expanded to include the discovery of novel biomarkers for early detection, diagnosis and prognostication (clinical application), and for the identification of novel drug targets (pharmaceutical application). To achieve these goals, several innovative technologies including 2-D-difference gel electrophoresis, SELDI, multidimensional protein identification technology, isotope-coded affinity tag, solid-state and suspension protein array technologies, X-ray crystallography, NMR spectroscopy, and computational methods such as comparative and de novo structure prediction and molecular dynamics simulation have evolved, and are being used in different combinations. This review provides an overview of the field of proteomics and discusses the key proteomic technologies available to researchers. It also describes some of the important challenges and highlights the current pharmaceutical and clinical applications of proteomics in human cancer research.     

Proteomics: A new perspective for the understanding of pterygia

Pterygia is a common ocular surface disease bothering both the patient because of its unsightly appearance and the surgeon because of its tendency to recur. The pathogenesis of pterygia is complex and the exact mechanism(s), especially at the molecular level, remains unknown. The use of modern proteomic techniques such as iTRAQ may yield new knowledge on the underlying pathogenesis of pterygia. In this issue of Proteomics Clinical Applications, Linghu et al. (article number 1600094) identified a total of 156 proteins that expressed differently between the pterygia and healthy conjunctiva using the isobaric tags for relative and absolute quantification based quantitative proteomic analysis. Most significantly, western blotting confirmed that two candidate proteins matrix metalloproteinase 10 (MMP‐10) and CD34 were significantly upregulated in pterygia, suggesting that they have potential roles in the pathogenesis of pterygia. The findings in Linhu's study may provide a new perspective for the understanding of pterygia and develop a new therapeutic target.     

Urinary proteomics in renal pathophysiology: Impact of proteinuria

Urinary differential proteomics is used to study renal pathophysiological mechanisms, find novel markers of biological processes and renal diseases, and stratify patients according to proteomic profiles. The proteomic procedure determines the pathophysiological meaning and clinical relevance of results. Urine samples for differential proteomic studies are usually normalized by protein content, regardless of its pathophysiological characteristics. In the field of nephrology, this approach translates into the comparison of a different fraction of the total daily urine output between proteinuric and nonproteinuric samples. Accordingly, alterations in the level of specific proteins found by this method reflect the relative presence of individual proteins in the urine; but they do not necessarily show alterations in their daily excretion, which is a key parameter for the understanding of the pathophysiological meaning of urinary components. For renal pathophysiology studies and clinical biomarker identification or determination, an alternative proteomic concept providing complementary information is based on sample normalization by daily urine output, which directly informs on changes in the daily excretion of individual proteins. This is clinically important because daily excretion (rather than absolute or relative concentration) is the only self-normalized way to evaluate the real meaning of urinary parameters, which is also independent of urine concentration.     

Proteomics of endometrial cancer diagnosis,treatment, and prognosis

This review discusses the current status of proteomics technology in endometrial cancer diagnosis, treatment and prognosis. The first part of this review focuses on recently identified biomarkers for endometrial cancer, their importance in clinical use as well as the proteomic methods used in their discovery. The second part highlights some of the emerging mass spectrometry based proteomic technologies that promise to contribute to a better understanding of endometrial cancer by comparing the abundance of hundreds or thousands of proteins simultaneously.     

Application of quantitative proteomics technologies to the biomarker discovery pipeline for multiple sclerosis

Multiple sclerosis is an inflammatory-mediated demyelinating disorder most prevalent in young Caucasian adults. The various clinical manifestations of the disease present several challenges in the clinic in terms of diagnosis, monitoring disease progression and response to treatment. Advances in MS-based proteomic technologies have revolutionized the field of biomarker research and paved the way for the identification and validation of disease-specific markers. This review focuses on the novel candidates discovered by the application of quantitative proteomics to relevant disease-affected tissues in both the human context and within the animal model of the disease known as experimental autoimmune encephalomyelitis. The role of targeted MS approaches for biomarker validation studies, such as multiple reaction monitoring will also be discussed.     

Proteomic profiling of animal models mimicking skeletal muscle disorders

Over the last few decades of biomedical research, animal models of neuromuscular diseases have been widely used for determining pathological mechanisms and for testing new therapeutic strategies. With the emergence of high-throughput proteomics technology, the identification of novel protein factors involved in disease processes has been decisively improved. This review outlines the usefulness of the proteomic profiling of animal disease models for the discovery of new reliable biomarkers, for the optimization of diagnostic procedures and the development of new treatment options for skeletal muscle disorders. Since inbred animal strains show genetically much less interindividual differences as compared to human patients, considerably lower experimental repeats are capable of producing meaningful proteomic data. Thus, animal model proteomics can be conveniently employed for both studying basic mechanisms of molecular pathogenesis and the effects of drugs, genetic modifications or cell-based therapies on disease progression. Based on the results from comparative animal proteomics, a more informed decision on the design of clinical proteomics studies could be reached. Since no one animal model represents a perfect pathobiochemical replica of all of the symptoms seen in complex human disorders, the proteomic screening of novel animal models can also be employed for swift and enhanced protein biochemical phenotyping.     

Urine proteomic studies in preeclampsia

Preeclampsia (PE) is a multisystem disorder of pregnancy that develops after 20 wk of gestation in previously normotensive women and complicates 5–8% of pregnancies. This rapidly progressive syndrome is usually diagnosed when the mother develops hypertension and proteinuria. The only effective treatment is delivery of the baby although early low-dose aspirin has been shown to significantly reduce the risk for PE. Recent advances in proteomic methods of protein separation, identification, and quantitation may allow for the identification of proteins and peptides that could facilitate early detection of disease, improve assessment of prognosis, and allow closer monitoring of women at risk for PE. This review summarizes all currently available markers for prediction and diagnosis of PE and presents urine proteomic studies performed for the identification of novel biomarkers.     

Customising an antibody leukocyte capture microarray for systemic lupus erythematosus: beyond biomarker discovery

Systemic lupus erythematosus (SLE) is a complex autoimmune disease that has heterogeneous clinical manifestation with diverse patterns of organ involvement, autoantibody profiles and varying degrees of severity of disease. Research and clinical experience indicate that different subtypes of SLE patients will likely benefit from more tailored treatment regimes, but we currently lack a fast and objective test with high enough sensitivity to enable us to perform such sub-grouping for clinical use. In this article, we review how proteomic technologies could be used as such an objective test. In particular, we extensively review many leukocyte surface markers that are known to have an association with the pathogenesis of SLE, and we discuss how these markers can be used in the further development of a novel SLE-specific antibody leukocyte capture microarray. In addition, we review some bioinformatics challenges and current methods for using the data generated by these cell-capture microarrays in clinical use. In a broader context, we hope our experience in developing a disease specific cell-capture microarray for clinical application can be a guide to other proteomic practitioners who intend to extend their technologies to develop clinical diagnostic and prognostic tests for complex diseases.     

SEREX, Proteomex, AMIDA, and beyond: Serological screening technologies for target identification

Despite the great body of knowledge about the aetiology, pathogenesis, risk factors, and associated molecular processes, cancer remains a prime health concern. Over the past decades scientific and medical research focused on the identification of biomarkers and target molecules for the diagnosis and therapy of cancer. Such markers may allow for improved and early diagnosis, as well as for immunotherapeutic approaches for cancer treatment. A plethora of technologies dedicated to the identification of target molecules was developed including those relying on a humoral response against tumour-associated antigens (TAA) in diseased individuals. As for other diseases, cancers elicit immune responses that result in the induction of T and B lymphocytes specific for tumour-associated proteins, largely self-antigens, but also those comprising viral and bacterial proteins. Cancer-specific serum antibodies are of great use for the isolation and subsequent identification of their cognate antigens. The present review will concentrate on three major serological target identification methods, i.e. SEREX, Proteomex, and AMIDA, concluding with a summary of the milestones in the clinical advancement and applications of serological TAA.     

Vascular proteomics

The characterization of patients with acute coronary syndromes (ACS) at the molecular and cellular levels provides a novel vision for understanding the pathological and clinical expression of the disease. Recent advances in proteomic technologies permit the evaluation of systematic changes in protein expression in many biological systems and have been extensively applied to cardiovascular diseases (CVD). The cardiovascular system is in permanent intimate contact with blood, making blood-based biomarker discovery a particularly worthwhile approach. Thus, proteomics can potentially yield novel biomarkers reflecting CVD, establish earlier detection strategies, and monitor response to therapy. Here we review the different proteomic strategies used in the study of atherosclerosis and the novel proteins differentially expressed and secreted by atherosclerotic lesions which constitute novel potential biomarkers (HSP-27, Cathepsin D). Special attention is paid to MS-Imaging of atheroma plaque and the generation, for the first time, of 2-D images of lipids, showing the distribution of these molecules in the different areas of the atherosclerotic lesions. In addition new potential biomarkers have been identified in plasma (amyloid A1α, transtherytin), circulating cells (protein profile in monocytes from ACS patients) and individual cells constituents of atheroma plaques (endothelial, VSMC, macrophages) which provide novel insights into vascular pathophysiology.     

Phosphoproteomics for the discovery of kinases as cancer biomarkers and drug targets

Early detection and targeted therapy represent a novel regimen of cancer management. The understanding of receptor tyrosine kinases in tumorigenesis at the molecular level has led to the first generation of kinase inhibitors for anticancer therapy that targets a specific kinase or pathway. While the therapeutic advantage is obvious, targeted therapy often relapses and results in drug resistance for advanced cancers. To achieve feasible early detection and better efficacy of therapeutics targeting multiple pathways, significantly more biomarkers and drug targets are in demand, especially for individualized therapy. Recent advances in phosphoprotein enrichment and MS technologies for quantitative phosphoproteome analysis provide great opportunities in the identification and validation of kinases as drug targets. The MS-based phosphoproteomic technologies would be useful tools as well for the identification of phosphosignatures unique to a specific type or subtype of cancer and drug responsive biomarkers. This review summarizes the major kinases acting as cancer biomarkers and drug targets, the advances of MS-based phosphoproteomic technologies, and some potential values and challenges of this emerging phosphoproteomics-based biomarker and drug target discovery field. Strategies for global, targeted, and quantitative phosphoproteomics are discussed, and some recent interesting applications are also evaluated.     

Impact of preanalytical variables on the analysis of biological fluids in proteomic studies

Although proteomic technologies have been enthusiastically embraced in the field of biomarker discovery and particularly with biological fluids, it is increasingly being recognized that pre-analytical effects, i.e. those occurring prior to the point of actual sample analysis and including factors such as sample processing and storage, can exert marked influences on the results obtained. Such effects have been recognized already for specific analytes in clinical chemistry, but with the increasing sensitivity and resolution of the newer technologies, such effects are potentially even more marked. The challenge of translating initial findings into clinical application requires such issues to be addressed at a very early stage in study design and this paper reviews the current knowledge of the potential impact of pre-analytical factors on proteomic studies of biological fluids.     

Proteomics in paediatric cardiac surgery: Is a personalised approach feasible?

The incidence of congenital cardiac abnormalities remains high. Paediatric patients with congenital cardiac defects often require surgery at a young age. The surgeries are often long and complex, rendering this population particularly vulnerable to the deleterious effects of cardiopulmonary bypass and cardiac surgery. The search for cardioprotective strategies is ongoing in an attempt to reduce the morbidity in this population. In the post-genomic era, it is apparent that simply determining the genomic sequences holds little diagnostic potential and means to determine progression of disease and response to treatment. The field of proteomics is expanding and application of proteomic techniques in the clinical setting holds great potential to advance our understanding of the proteomic changes involved in specific disease stages. This review will assess the application of proteomic techniques in the setting of paediatric cardiac surgery and highlight the need to obtain a clear understanding of the role of various proteins in children with cardiac conditions. The success and challenges of the available proteomic technology will be discussed as well as the future potential of proteomic methods for advancing our understanding of protein changes in children requiring cardiac surgery.     

Proteomic and other analyses to determine the functional consequences of deregulated kallikrein-related peptidase (KLK) expression in prostate and ovarian cancer

Rapidly developing proteomic tools are improving detection of deregulated kallikrein-related peptidase (KLK) expression, at the protein level, in prostate and ovarian cancer, as well as facilitating the determination of functional consequences downstream. MS-driven proteomics uniquely allows for the detection, identification, and quantification of thousands of proteins in a complex protein pool, and this has served to identify certain KLKs as biomarkers for these diseases. In this review, we describe applications of this technology in KLK biomarker discovery and elucidate MS-based techniques that have been used for unbiased, global screening of KLK substrates within complex protein pools. Although MS-based KLK degradomic studies are limited to date, they helped to discover an array of novel KLK substrates. Substrates identified by MS-based degradomics are reported with improved confidence over those determined by incubating a purified or recombinant substrate and protease of interest, in vitro. We propose that these novel proteomic approaches represent the way forward for KLK research, in order to correlate proteolysis of biological substrates with tissue-related consequences, toward clinical targeting of KLK expression and function for cancer diagnosis, prognosis, and therapies.     

Colorectal cancer proteomics, molecular characterization and biomarker discovery

Colorectal cancer (CRC) is a widespread disease, whose major genetic changes and mutations have been well characterized in the sporadic form. Much less is known at the protein and proteome level. Still, CRC has been the subject of multiple proteomic studies due to the urgent necessity of finding clinically relevant markers and to elucidate the molecular mechanisms underlying the progression of the disease. These proteomic approaches have been limited by different technical issues, mainly related with sensitivity and reproducibility. However, recent advances in proteomic techniques and MS systems have rekindled the quest for new biomarkers in CRC and an improved molecular characterization. In this review, we will discuss the application of different proteomic approaches to the identification of differentially expressed proteins in CRC. In particular, we will make a critical assessment about the use of 2-D DIGE, MS and protein microarray technologies, in their different formats, to identify up- or downregulated proteins and/or autoantibodies profiles that could be useful for CRC characterization and diagnosis. Despite a wide list of potential biomarkers, it is clear that more scientific efforts and technical advances are still needed to cover the range of low-abundant proteins, which may play a key role in CRC diagnostics and progression.