Proteomics and transfusion medicine: future perspectives

Limited number of important discoveries have greatly contributed to the progresses achieved in the blood transfusion; ABO histo-blood groups, citrate as anticoagulant, fractionation of plasma proteins, plastic bags and apheresis machines. Three major types of blood products are transfused to patients: red cell concentrates, platelet concentrates and fresh frozen plasma. Several parameters of these products change during storage process and they have been well studied over the years. However, several aspects have completely been ignored; in particular those related to peptide and protein changes. This review presents what has been done using proteomic tools and the potentials of proteomics for transfusion medicine.     

Stored red blood cells: A changing universe waiting for its map(s)

The availability of stored red blood cells (RBCs) for transfusion remains an important aspect of the treatment of polytrauma, acute anemia or major bleedings. RBCs are prepared by blood banks from whole blood donations and stored in the cold in additive solutions for typically six weeks. These far from physiological storage conditions result in the so-called red cell storage lesion that is of importance both to blood bankers and to clinical practitioners. Here we review the current state of knowledge about the red cell storage lesion from a proteomic perspective. In particular, we describe the current models accounting for RBC aging and response to lethal stresses, review the published proteomic studies carried out to uncover the molecular basis of the RBC storage lesion, and conclude by suggesting a few possible proteomic studies that would provide further knowledge of the molecular alterations carried by RBCs stored in the cold for six weeks.     

Transfusion medicine in the era of proteomics

Blood components (BCs) are highly complex mixtures of plasma proteins and cells. At present, BC and blood derivatives (BDs) quality control is mainly focused on standardized quantitative assessment, providing relatively limited information about products. Unfortunately, during the production, inactivation, and storage processes there is the risk of changes in their integrity, especially at the protein level, which could cause negative effects on transfusion. It is therefore a major challenge to identify significant alterations of these products, and, in this context, proteomics can play a potentially relevant role in transfusion medicine (TM) to assess the protein composition of blood-derived therapeutics, particularly for identifying modified proteins. It can provide comprehensive information about changes occurring during processing and storage of BCs and BDs and can be applied to assess or improve them, therefore potentially enabling a global assessment of processing, inactivation and storage methods, as well as of possible contaminants and neoantigens that may influence the immunogenic capacity of blood-derived therapeutics. Thus, proteomics could become a relevant part of quality-control process to verify the identity, purity, safety, and potency of various blood therapeutics. A more detailed understanding of the proteins found in blood and blood products, and the identification of their interactions, may also yield important information for the design of new small molecule therapeutics and also for future improvements in TM.

Proteomics, together with genomics in the near future, will presumably have an impact on disease diagnosis and prognosis as well as on further advances in the production, pathogen inactivation and storage processes of blood-based therapeutics.     

Recent advances in platelet proteomics

Platelets are the fundamental players in primary hemostasis, but are also involved in several pathological conditions. The remarkable advances in proteomic methodologies have allowed a better understanding of the basic physiological pathways underlying platelet biology. In addition, recent platelet proteomics focused on disease conditions, helping to elucidate the molecular mechanisms of complex and/or unknown human disorders and to find novel biomarkers for early diagnosis and drug targets. The most common and innovative proteomic techniques, both gel-based and gel-free, used in platelet proteomics will be reviewed here. A particular focus will be given to studies that used a subproteomic strategy to analyze specific platelet conditions (resting or activated), compartments (membrane, granules and microparticles) or fractions (phosphoproteome or glycoproteome). The thousands of platelet proteins and interactions discovered so far by these different powerful proteomic approaches represent a precious source of information for both basic science and clinical applications in the field of platelet biology.     

Blood-related proteomics

Blood-related proteomics is an emerging field, recently gaining momentum. Indeed, a wealth of data is now available and a plethora of groups has contributed to add pieces to the jigsaw puzzle of protein complexity within plasma and blood cells.In this review article we purported to sail across the mare magnum of the actual knowledge in this research endeavour. The main strides in proteomic investigations on red blood cells, platelets, plasma and white blood cells are hereby presented in a chronological order.Moreover, a glance is given at prospective studies which promise to shift the focus of attention from the end product to its provider, the donor, in a sort of Kantian “Copernican revolution”.A well-rounded portrait of the usefulness of proteomics in blood-related research is accurately given. In particular, proteomic tools could be adopted to follow the main steps of the blood-banking production processes (a comparison of collection methods, pathogen inactivation techniques, storage protocols). Thus proteomics has been recently transformed from a mere basic-research extremely-expensive toy into a dramatically-sensitive and efficient eye-lens to either delve into the depths of the molecular mechanisms of blood and blood components or to establish quality parameters in the blood-banking production chain totally anew.     

Activation of platelet concentrate during preparation and storage.

This review will discuss how stored platelets become activated and will examine their ability to function and survive in vivo, posttransfusion. Experimental methods which have been shown to alter platelets during storage will be detailed. Using beta-thromboglobulin (beta-TG) and surface adhesion receptors as markers, investigators have examined the activation changes in platelet concentrates during preparation and storage. Resuspension of the platelet pellet after isolation of platelet-rich plasma appears to play a major role in producing platelet activation and beta-TG release during preparation. However, there is a significant amount of interdonor variability in platelet activation even at this early stage of storage. Over 5 days of storage, platelets release approximately 50% of their beta-TG contents. Furthermore, between 40% and 60% of the platelets express the alpha-granule membrane protein, P-selectin (GMP-140), during storage, which is also indicative of platelet activation. These activation changes correlate to some degree with platelet recovery posttransfusion but clearly do not explain the full lesion of platelet storage. The surface density of two platelet membrane receptors, glycoproteins (GP) Ib and IIb/IIIa, also change with activation, although in opposite directions. Platelet surface GPIb decreases initially with storage and then recovers, perhaps due to its relocation to the platelet surface from an intracellular pool. In contrast to GPIb, mean platelet surface GPIIb/IIIa increases slightly during storage, probably as a consequence of platelet activation and release of alpha-granule GPIIb/IIIa to the surface. Some hypotheses are offered regarding how these activated platelets can continue to circulate after transfusion. Further exploration of the platelet storage lesion will hopefully provide needed answers and thus permit better treatment of hemostatic disorders in the future.     

Red Blood Cell (RBC) membrane proteomics — Part II: Comparative proteomics and RBC patho-physiology

Membrane proteomics offers unprecedented possibilities to compare protein expression in health and disease leading potentially to the identification of markers, of targets for therapeutics and to a better understanding of disease mechanisms. From transfusion medicine to infectious diseases, from cardiovascular affections to diabetes, comparative proteomics has made a contribution to the identification of proteins unique to RBCs of patients with specific illnesses shedding light on possible RBC markers for systemic diseases.In this review we will provide a short overview of some of the main achievements obtained by comparative proteomics in the field of RBC-related local and systemic diseases and suggest some additional areas of RBCs research to which comparative proteomics approaches could be fruitfully applied or extended in combination with biochemical techniques.     

Human Proteinpedia as a resource for clinical proteomics

Clinical proteomics is an emerging field that deals with the use of proteomic technologies for medical applications. With a major objective of identifying proteins involved in pathological processes and as potential biomarkers, this field is already gaining momentum. Consequently, clinical proteomics data are being generated at a rapid pace, although mechanisms of sharing such data with the biomedical community lag far behind. Most of these data are either provided as supplementary information through journal web sites or directly made available by the authors through their own web resources. Integration of these data within a single resource that displays information in the context of individual proteins is likely to enhance the use of proteomic data in biomedical research. Human Proteinpedia is one such portal that unifies human proteomic data under a single banner. The goal of this resource is to ultimately capture and integrate all proteomic data obtained from individual studies on normal and diseased tissues. We anticipate that harnessing of these data will help prioritize experiments related to protein targets and also permit meta-analysis to uncover molecular signatures of disease. Finally, we encourage all biomedical investigators to maximize dissemination of their valuable proteomic data to rest of the community by active participation in existing repositories such as Human Proteinpedia.     

Proteomics of blood and derived products: what’s next?

Proteomics has changed the way proteins are analyzed in living systems. This approach has been applied to blood products and protein profiling has evolved in parallel with the development of techniques. The identification of proteins belonging to red blood cell, platelets or plasma was achieved at the end of the last century. Then, the questions on the applications emerged. Hence, several studies have focused on problems related to blood banking and products, such as the aging of blood products, identification of biomarkers, related diseases and the protein–protein interactions. More recently, a mass spectrometry-based proteomics approach to quality control has been applied in order to offer solutions and improve the quality of blood products. The current challenge we face is developing a closer relationship between transfusion medicine and proteomics. In this article, these issues will be approached by focusing first on the proteome identification of blood products and then on the applications and future developments within the field of proteomics and blood products.     

Mass spectrometry-based proteomic approaches to study pathogenic bacteria-host interactions

Elucidation of molecular mechanisms underlying hostpathogen interactions is important for control and treatment of infectious diseases worldwide. Within the last decade, mass spectrometry (MS)-based proteomics has become a powerful and effective approach to better understand complex and dynamic host-pathogen interactions at the protein level. Herein we will review the recent progress in proteomic analyses towards bacterial infection of their mammalian host with a particular focus on enteric pathogens. Large-scale studies of dynamic proteomic alterations during infection will be discussed from the perspective of both pathogenic bacteria and host cells.     

Use of proteomics for the identification of novel drug targets in brain diseases

In spite of the rapid advances in the development of the new proteomic technologies, there are, to date, relatively fewer studies aiming to explore the neuronal proteome. One of the reasons is the complexity of the brain, which presents high cellular heterogeneity and a unique subcellular compartmentalization. Therefore, tissue fractionation of the brain to enrich proteins of interest will reduce the complexity of the proteomics approach leading to the production of manageable and meaningful results. In this review, general considerations and strategies of proteomics, the advantages and challenges to exploring the neuronal proteome are described and summarized. In addition, this article presents an overview of recent advances of proteomic technologies and shows that proteomics can serve as a valuable tool to globally explore the changes in brain proteome during various disease states. Understanding the molecular basis of brain function will be extremely useful in identifying novel targets for the treatment of brain diseases.     

Differential expression of CD42b and CD9 proteins in platelets and extracellular membrane vesicles during platelet-concentrate storage

Platelet concentrate is used to restore and maintain hemostasis in patients with a reduced number or activity of platelets. Platelet concentrate changes its properties when stored, which leads to a weakening of the therapeutic effect, as well as the occurrence of transfusion side effects. One of the processes that alter platelet concentrates during storage is the secretion of several types of membrane vesicles. Despite the fact that platelet-derived membrane vesicles affect homeostasis and transmit signals of intercellular communication, mechanisms of their formation, features of regulatory activity and molecular composition are still poorly understood. In this paper, the results of analysis of CD42b and CD9 membrane proteins expression in platelets and platelet-derived membrane vesicles during platelet-concentrate storage are presented. Populations of membrane vesicles of different sizes were isolated from the platelet concentrates and characterized. Aggregation and morphological alteration of platelets are observed during storage; the protein composition of platelets, as well as membrane vesicles, changes, and there is a significant increase in the levels of CD42b and CD9 proteins in fractions of membrane vesicles. The results obtained indicate that platelet concentrates contain different populations of membrane vesicles, the molecular composition of which varies during storage.     

Toward functional proteomics of alveolar macrophages

Alveolar macrophages (AM) belong to a phenotype of macrophages with distinct biological functions and important pathophysiological roles in lung health and disease. The molecular details determining AM differentiation from blood monocytes and AM roles in lung homeostasis are largely unknown. With the use of different technological platforms, advances in the field of proteomics have made it possible to search for differences in protein expression between AM and their precursor monocytes. Proteome features of each cell type provide new clues into understanding mononuclear phagocyte biology. In-depth analyses using subproteomics and subcellular proteomics offer additional information by providing greater protein resolution and detection sensitivity. With the use of proteomic techniques, large-scale mapping of phosphorylation differences between the cell types have become possible. Furthermore, two-dimensional gel proteomics can detect germline protein variants and evaluate the impact of protein polymorphisms on an individual's susceptibility to disease. Finally, surface-enhanced laser desorption and ionization (SELDI) time-of-flight mass spectrometry offers an alternative method to recognizing differences in protein patterns between AM and monocytes or between AM under different pathological conditions. This review details the current status of this field and outlines future directions in functional proteomic analyses of AM and monocytes. Furthermore, this review presents viewpoints of integrating proteomics with translational topics in lung diseases to define the mechanisms of disease and to uncover new diagnostic and therapeutic targets.     

2nd BSPR London Regional Meeting and 6th Annual Imperial Proteomics Day. November 2007, Imperial College London, London, UK

The 2(nd) BSPR London Regional Meeting held at Imperial College London focused on nanoproteomics and single cell proteomics, the solutions to many of the technical challenges in proteomics and protein based molecular diagnostics. This one day meeting included presentations from leading scientists within and outside of Imperial College who share a common interest in novel solutions for the identification and characterization of proteins from a single cell. The conclusion was that nanomaterials are delivering enhanced reagents and have been tested at the proof-of-concept level, but have yet to be incorporated into routine proteomic workflows.     

Clinical proteomics: present and future prospects

Advances in proteomics technology offer great promise in the understanding and treatment of the molecular basis of disease. The past decade of proteomics research, the study of dynamic protein expression, post-translational modifications, cellular and sub-cellular protein distribution, and protein-protein interactions, has culminated in the identification of many disease-related biomarkers and potential new drug targets. While proteomics remains the tool of choice for discovery research, new innovations in proteomic technology now offer the potential for proteomic profiling to become standard practice in the clinical laboratory. Indeed, protein profiles can serve as powerful diagnostic markers, and can predict treatment outcome in many diseases, in particular cancer. A number of technical obstacles remain before routine proteomic analysis can be achieved in the clinic; however the standardisation of methodologies and dissemination of proteomic data into publicly available databases is starting to overcome these hurdles. At present the most promising application for proteomics is in the screening of specific subsets of protein biomarkers for certain diseases, rather than large scale full protein profiling. Armed with these technologies the impending era of individualised patient-tailored therapy is imminent. This review summarises the advances in proteomics that has propelled us to this exciting age of clinical proteomics, and highlights the future work that is required for this to become a reality.     

Proteomics of blood and derived products: what's next?

Proteomics has changed the way proteins are analyzed in living systems. This approach has been applied to blood products and protein profiling has evolved in parallel with the development of techniques. The identification of proteins belonging to red blood cell, platelets or plasma was achieved at the end of the last century. Then, the questions on the applications emerged. Hence, several studies have focused on problems related to blood banking and products, such as the aging of blood products, identification of biomarkers, related diseases and the protein-protein interactions. More recently, a mass spectrometry-based proteomics approach to quality control has been applied in order to offer solutions and improve the quality of blood products. The current challenge we face is developing a closer relationship between transfusion medicine and proteomics. In this article, these issues will be approached by focusing first on the proteome identification of blood products and then on the applications and future developments within the field of proteomics and blood products.     

2D electrophoresis-based expression proteomics: a microbiologist's perspective

Quantitative proteomics based on 2D electrophoresis (2-DE) coupled with peptide mass fingerprinting is still one of the most widely used quantitative proteomics approaches in microbiology research. Our view on the exploitation of this global expression analysis technique and its contribution and potential to push forward the field of molecular microbial physiology towards a molecular systems microbiology perspective is discussed in this article. The advances registered in 2-DE-based quantitative proteomic analysis leading to increased protein resolution, sensitivity and accuracy, and the promising use of 2-DE to gain insights into post-translational modifications at a proteome-wide level (considering all the proteins/protein forms expressed by the genome) are focused on. Given the progress made in this field, it is foreseen that the 2-DE-based approach to quantitative proteomics will continue to be a fundamental tool for microbiologists working at a genome-wide scale. Guidelines are also provided for the exploitation of expression proteomics data, based on useful computational tools, and for the integration of these data with other genome-wide expression information. The advantages and limitations of a complete 2-DE-based expression proteomics analysis, envisaging the quantification of the global changes occurring in the proteome of a given cell depending on environmental or genetic manipulations, are discussed from the microbiologist's perspective. Particular focus is given to the emerging field of toxicoproteomics, a new systems toxicity approach that offers a powerful tool to directly monitor the earliest stages of the toxicological response by identifying critical proteins and pathways that are affected by, and respond to, a chemical stress. The experimental design and the bioinformatics analysis of data used in our laboratory to gain mechanistic insights through expression proteomics into the responses of the eukaryotic model Saccharomyces cerevisiae or of Pseudomonas strains to environmental toxicants are presented as case studies.     

2D electrophoresis-based expression proteomics: a microbiologist’s perspective

Quantitative proteomics based on 2D electrophoresis (2-DE) coupled with peptide mass fingerprinting is still one of the most widely used quantitative proteomics approaches in microbiology research. Our view on the exploitation of this global expression analysis technique and its contribution and potential to push forward the field of molecular microbial physiology towards a molecular systems microbiology perspective is discussed in this article. The advances registered in 2-DE-based quantitative proteomic analysis leading to increased protein resolution, sensitivity and accuracy, and the promising use of 2-DE to gain insights into post-translational modifications at a proteome-wide level (considering all the proteins/protein forms expressed by the genome) are focused on. Given the progress made in this field, it is foreseen that the 2-DE-based approach to quantitative proteomics will continue to be a fundamental tool for microbiologists working at a genome-wide scale. Guidelines are also provided for the exploitation of expression proteomics data, based on useful computational tools, and for the integration of these data with other genome-wide expression information. The advantages and limitations of a complete 2-DE-based expression proteomics analysis, envisaging the quantification of the global changes occurring in the proteome of a given cell depending on environmental or genetic manipulations, are discussed from the microbiologist’s perspective. Particular focus is given to the emerging field of toxicoproteomics, a new systems toxicity approach that offers a powerful tool to directly monitor the earliest stages of the toxicological response by identifying critical proteins and pathways that are affected by, and respond to, a chemical stress. The experimental design and the bioinformatics analysis of data used in our laboratory to gain mechanistic insights through expression proteomics into the responses of the eukaryotic model Saccharomyces cerevisiae or of Pseudomonas strains to environmental toxicants are presented as case studies.