Proteomic approach to aging research

The scope of the current paper is to review existing and potential applications of proteomic analysis to aging research. The focus will lie on the unique opportunities of high-throughput studies for uncovering specific alterations in protein expression, protein complexes or protein modifications caused by biological aging. The result of such studies will outline aging phenotypes and potentially indicate pathways involved in the pathogenesis of age-associated disfunctions. Specific attention is paid to the illustrations of successful applications of proteomic technologies and potential applications of new proteomic concepts to biogerontological studies.     

Accumulation of “Old Proteins” and the Critical Need for MS‐based Protein Turnover Measurements in Aging and Longevity

Aging and age‐related diseases are accompanied by proteome remodeling and progressive declines in cellular machinery required to maintain protein homeostasis (proteostasis), such as autophagy, ubiquitin‐mediated degradation, and protein synthesis. While many studies have focused on capturing changes in proteostasis, the identification of proteins that evade these cellular processes has recently emerged as an approach to studying the aging proteome. With advances in proteomic technology, it is possible to monitor protein half‐lives and protein turnover at the level of individual proteins in vivo. For large‐scale studies, these technologies typically include the use of stable isotope labeling coupled with MS and comprehensive assessment of protein turnover rates. Protein turnover studies have revealed groups of highly relevant long‐lived proteins (LLPs), such as the nuclear pore complexes, extracellular matrix proteins, and protein aggregates. Here, the role of LLPs during aging and age‐related diseases and the methods used to identify and quantify their changes are reviewed. The methods available to conduct studies of protein turnover, used in combination with traditional proteomic methods, will enable the field to perform studies in a systems biology context, as changes in proteostasis may not be revealed in studies that solely measure differential protein abundances.     

Advanced proteomic technologies for cancer biomarker discovery

Proteomic technologies have experienced major improvements in recent years. Such advances have facilitated the discovery of potential tumor markers with improved sensitivities and specificities for the diagnosis, prognosis and treatment monitoring of cancer patients. This review will focus on four state-of-the-art proteomic technologies, namely 2D difference gel electrophoresis, MALDI imaging mass spectrometry, electron transfer dissociation mass spectrometry and reverse-phase protein array. The major advancements these techniques have brought about and examples of their applications in cancer biomarker discovery will be presented in this review, so that readers can appreciate the immense progress in proteomic technologies from 1997 to 2008. Finally, a summary will be presented that discusses current hurdles faced by proteomic researchers, such as the wide dynamic range of protein abundance, standardization of protocols and validation of cancer biomarkers, and a 5-year view of potential solutions to such problems will be provided.     

"Muscle to meat" molecular events and technological transformations: the proteomics insight

Cellular death is characterized by a complex pattern of molecular events that depend on cell type. Specifically, muscle cells first undergo rigor mortis due to ATP depletion, and later, on the time scale of days, muscle fiber degradation due to proteolytic enzyme activity. In the present review, we will refer to proteomic investigations on the post-mortem evolution of the protein patterns of animal muscle cells. These studies, carried out with the application of either bottom-up or top-down methods, are relevant for understanding the biochemical reactions that i) convert muscle to meat, ii) are associated with meat aging and iii) impact on meat tenderness, a feature of significant commercial value. We also report on the proteomic investigations that have been made to analyze the transformation of meat in industrial processes. These studies are primarily aimed at identifying protein patterns and/or individual proteins diagnostic of the quality of the final product.     

Proteomics for Protein Expression Profiling in Neuroscience

As the technology of proteomics moves from a theoretical approach to a practical reality, neuroscientists will have to determine the most appropriate applications for this technology. Neuroscientists will have to surmount difficulties particular to their research, such as limited sample amounts, heterogeneous cellular compositions in samples, and the fact that many proteins of interest are rare, hydrophobic proteins. This review examines protein isolation and protein fractionation and separation using two-dimensional electrophoresis (2-DE) and mass spectrometry proteomic methods. Methods for quantifying relative protein expression between samples (e.g., 2-DIGE, and ICAT) are also described. The coverage of the proteome, ability to detect membrane proteins, resource requirements, and quantitative reliability of different approaches is also discussed. Although there are many challenges in proteomic neuroscience, this field promises many rewards in the future.     

Enhanced detectability in proteome studies

The discovery of candidate biomarkers from biological materials coupled with the development of detection methods holds both incredible clinical potential as well as significant challenges. However, the proteomic techniques still provide the low dynamic range of protein detection at lower abundances. This review describes the current development of potential methods to enhance the detection and quantification in proteome studies. It also includes the bioinformatics tools that are helpfully used for data mining of protein ontology. Therefore, we believe that this review provided many proteomic approaches, which would be very potent and useful for proteome studies and for further diagnostic and therapeutic applications.     

Proteomic applications of protein quantification by isotope-dilution mass spectrometry

Over the decades, isotope-dilution mass spectrometry (IDMS) has been implemented extensively for accurate quantification of drugs, metabolites and peptides in body fluids and tissues. More recently, it has been extended for quantifying specific proteins in complex mixtures. In this extended methodology, proteins are subjected to endoprotease action and specific resultant peptides are quantified by using synthetic stable isotope-labeled standard (SIS) peptides and IDMS. This article outlines the utilities and applications of quantifying proteins by IDMS, emphasizing its complementary value to global survey-based proteomic studies. The potential of SIS peptides to provide quantitative insights into cell signaling is also highlighted, with specific examples. Finally, we propose several novel mass spectrometric data acquisition strategies for large-scale applications of IDMS and SIS peptides in systems biology and protein biomarker validation studies.     

Protein microarrays and their applications

In recent years, the importance of proteomic works, such as protein expression, detection and identification, has grown in the fields of proteomic and diagnostic research. This is because complete genome sequences of humans, and other organisms, progress as cellular processing and controlling are performed by proteins as well as DNA or RNA. However, conventional protein analyses are time-consuming; therefore, high throughput protein analysis methods, which allow fast, direct and quantitative detection, are needed. These are so-called protein microarrays or protein chips, which have been developed to fulfill the need for high-throughput protein analyses. Although protein arrays are still in their infancy, technical development in immobilizing proteins in their native conformation on arrays, and the development of more sensitive detection methods, will facilitate the rapid deployment of protein arrays as high-throughput protein assay tools in proteomics and diagnostics. This review summarizes the basic technologies that are needed in the fabrication of protein arrays and their recent applications.     

The New Face of the Lipid Droplet: Lipid Droplet Proteins

The lipid droplet (LD) is an organelle with vital functions found in nearly all organisms. LD proteomic research has provided fundamentally important insights into this organelle's functions. The review provides a summary of LD proteomic studies conducted across diverse organisms and cell and tissue types. The accumulated proteomic data are reviewed for evidence of a protein targeting mechanism for the organelle. The hypotheses for several specific localization mechanisms based on what is known about targeting mechanisms for other organelles and vesicles are provided. Although the nature of the mechanism is not known, the functional data demonstrate that the targeting mechanism and, indeed, the organelle itself, is conserved from prokaryotes to eukaryotes. It is hoped that the review will help inspire further research leading to novel discoveries in the field.     

Protein signatures of centenarians and their offspring suggest centenarians age slower than other humans

Using samples from the New England Centenarian Study (NECS), we sought to characterize the serum proteome of 77 centenarians, 82 centenarians'' offspring, and 65 age‐matched controls of the offspring (mean ages: 105, 80, and 79 years). We identified 1312 proteins that significantly differ between centenarians and their offspring and controls (FDR < 1%), and two different protein signatures that predict longer survival in centenarians and in younger people. By comparing the centenarian signature with 2 independent proteomic studies of aging, we replicated the association of 484 proteins of aging and we identified two serum protein signatures that are specific of extreme old age. The data suggest that centenarians acquire similar aging signatures as seen in younger cohorts that have short survival periods, suggesting that they do not escape normal aging markers, but rather acquire them much later than usual. For example, centenarian signatures are significantly enriched for senescence‐associated secretory phenotypes, consistent with those seen with younger aged individuals, and from this finding, we provide a new list of serum proteins that can be used to measure cellular senescence. Protein co‐expression network analysis suggests that a small number of biological drivers may regulate aging and extreme longevity, and that changes in gene regulation may be important to reach extreme old age. This centenarian study thus provides additional signatures that can be used to measure aging and provides specific circulating biomarkers of healthy aging and longevity, suggesting potential mechanisms that could help prolong health and support longevity.     

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.     

Proteomic analysis of native metabotropic glutamate receptor 5 protein complexes reveals novel molecular constituents

We used a proteomic approach to identify novel proteins that may regulate metabotropic glutamate receptor 5 (mGluR5) responses by direct or indirect protein interactions. This approach does not rely on the heterologous expression of proteins and offers the advantage of identifying protein interactions in a native environment. The mGluR5 protein was immunoprecipitated from rat brain lysates; co-immunoprecipitating proteins were analyzed by mass spectrometry and identified peptides were matched to protein databases to determine the correlating parent proteins. This proteomic approach revealed the interaction of mGluR5 with known regulatory proteins, as well as novel proteins that reflect previously unidentified molecular constituents of the mGluR5-signaling complex. Immunoblot analysis confirmed the interaction of high confidence proteins, such as phosphofurin acidic cluster sorting protein 1, microtubule-associated protein 2a and dynamin 1, as mGluR5-interacting proteins. These studies show that a proteomic approach can be used to identify candidate interacting proteins. This approach may be particularly useful for neurobiology applications where distinct protein interactions within a signaling complex can dramatically alter the outcome of the response to neurotransmitter release, or the disruption of normal protein interactions can lead to severe neurological and psychiatric disorders.     

Immobilized trypsin on hydrophobic cellulose decorated nanoparticles shows good stability and reusability for protein digestion

The preparation of biocatalysts based on immobilized trypsin is of great importance for both proteomic research and industrial applications. Here, we have developed a facile method to immobilize trypsin on hydrophobic cellulose-coated silica nanoparticles by surface adsorption. The immobilization conditions for the trypsin enzyme were optimized. The as-prepared biocatalyst was characterized by Fourier transform infrared spectroscopy, transmission electron microscopy, and elemental analysis. In comparison with free enzyme, the immobilized trypsin exhibited greater resistances against thermal inactivation and denaturants. In addition, the immobilized trypsin showed good durability for multiple recycling. The general applicability of the immobilized trypsin for proteomic studies was confirmed by enzymatic digestion of two widely used protein substrates: bovine serum albumin (BSA) and cytochrome c. The surface adsorption protocols for trypsin immobilization may provide a promising strategy for enzyme immobilization in general, with great potential for a range of applications in proteomic studies.     

Host–pathogen interactions: a proteomic view

Host–pathogen interactions reflect the balance of host defenses and pathogen virulence mechanisms. Advances in proteomic technologies now afford opportunities to compare protein content between complex biologic systems ranging from cells to animals and clinical samples. Thus, it is now possible to characterize host–pathogen interactions from a global proteomic view. Most reports to date focus on cataloging protein content of pathogens and identifying virulence-associated proteins or proteomic alterations in host response. A more in-depth understanding of host–pathogen interactions has the potential to improve our mechanistic understanding of pathogenicity and virulence, thereby defining novel therapeutic and vaccine targets. In addition, proteomic characterization of the host response can provide pathogen-specific host biomarkers for rapid pathogen detection and characterization, as well as for early and specific detection of infectious diseases. A review of host–pathogen interactions focusing on proteomic analyses of both pathogen and host will be presented. Relevant genomic studies and host model systems will be also be discussed.     

Host-pathogen interactions: a proteomic view

Host-pathogen interactions reflect the balance of host defenses and pathogen virulence mechanisms. Advances in proteomic technologies now afford opportunities to compare protein content between complex biologic systems ranging from cells to animals and clinical samples. Thus, it is now possible to characterize host-pathogen interactions from a global proteomic view. Most reports to date focus on cataloging protein content of pathogens and identifying virulence-associated proteins or proteomic alterations in host response. A more in-depth understanding of host-pathogen interactions has the potential to improve our mechanistic understanding of pathogenicity and virulence, thereby defining novel therapeutic and vaccine targets. In addition, proteomic characterization of the host response can provide pathogen-specific host biomarkers for rapid pathogen detection and characterization, as well as for early and specific detection of infectious diseases. A review of host-pathogen interactions focusing on proteomic analyses of both pathogen and host will be presented. Relevant genomic studies and host model systems will be also be discussed.     

活性蛋白质表达谱分析与病毒学研究

活性蛋白质表达谱（activity-based protein profiling, ABPP）分析技术是功能蛋白质组学的一种策略,属于化学蛋白质组学的一部分.它借助化学小分子从功能角度直接切入蛋白质组的研究,能够直接对蛋白质组中感兴趣的靶酶蛋白的活性进行检测,为药物的发现提供强有力的支持.因此,ABPP技术被认为是基于功能的新一代蛋白质组学技术.随着ABPP分析技术和方法的不断成熟,其应用领域也不断扩展.最近一系列研究表明, 今后ABPP分析技术可能成为病毒学研究的又一重要武器.本文综述了ABPP分析技术的基本原理及其在病毒学研究中的应用.     

Walking through the protein sequence space: towards new generation of the homology modeling

A new method is proposed to reveal apparent evolutionary relationships between protein fragments with similar 3D structures by finding "intermediate" sequences in the proteomic database. Instead of looking for homologies and intermediates for a whole protein domain, we build a chain of intermediate short sequences, which allows one to link similar structural modules of proteins belonging to the same or different families. Several such chains of intermediates can be combined into an evolutionary tree of structural protein modules. All calculations were made for protein fragments of 20 aa residues. Three evolutionary trees for different module structures are described. The aim of the paper is to introduce the new method and to demonstrate its potential for protein structural predictions. The approach also opens new perspectives for protein evolution studies.     

Protein oxidation and degradation during aging: Role in skin aging and neurodegeneration

During aging, the products of oxidative processes accumulate and might disturb cellular metabolism. Among them are oxidized proteins and protein aggregates. On the other hand, in a functioning metabolic system oxidized proteins are degraded, mainly by the proteasome. During aging, however, proteasome activity declines. Therefore, the ability to degrade oxidized proteins is attenuated. The following review summarises the accumulation of oxidized proteins and the decline of the proteasomal system during skin and brain aging including some age-related neurodegenerative processes. The role of protein aggregates will be discussed as a potential reason for the accelerated dysfunction of tissue during aging.