The application of proteomic technologies for the analysis of muscle proteins of farm animals used in the meat industry (Review)

The review briefly summarizes the data on the development of proteomic technologies that became actively used in studies of the muscular proteins of farm animals used in the meat industry in 2006–2013. It has been noted that the main research trends are connected with the detection of changes in muscle proteins during post-mortem autolysis and the search for species-specific and other protein biomarkers. Particular publications regarding the development of methods based on proteomic technologies for monitoring the state of muscle proteins are considered. According to the analyzed data, we can conclude that the field is promising for the solution of a number of pressing problems in applied biochemistry.     

"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.     

Proteomic analysis and comparison of the biopsy and autopsy specimen of human brain temporal lobe

The proteomic study on human temporal lobe can help us to understand the physiological function of CNS in normal as well as in pathological state. Proteomic tools are potent for the assessment of protein stability post mortem. In this pilot study, the human temporal lobe biopsy specimen with chronic pharmacoresistant temporal lobe epilepsy (TLE) and autopsy specimen in control were separated by 2-DE. Using MALDI-TOF-MS and MS/MS, 375 protein spots were identified which were the products of 267 genes. Six down-regulated and 23 up-regulated protein spots in the autopsy specimen were ascertained after the gel image analysis with the ImageMaster software. A number of proteins that include neurotransmitter metabolic and glycolytic enzymes, cytoprotective proteins and cytoskeleton were found decreased while the precursor of apolipoprotein A-I increased in the TLE brain. We tried several methods to prepare the protein samples and found that DNase and RNase treatment, ultracentrifugation and Amersham clean-up kit purification can improve gel separation quality. This work optimized the sample preparation method and constructed a primary protein database of human temporal lobe and found some proteins with remarkable level change probably involved in the post-mortem process and chronic pharmacoresistant TLE pathogenesis.     

Poor reliability of immunocytochemical localization of IgG in immersion-fixed tissue from the central nervous system.

The effect of fixation technique and post mortem-to-fixation interval in immersion-fixed tissue from the central nervous system on immunocytochemical staining for the presence of an immunoglobulin was determined in mice. Immersion-fixed tissue was found to be inferior to perfusion-fixed tissue for immunocytochemical staining of this serum protein. Unlike what has been observed for other antigens, the quality of staining for IgG in immersion-fixed tissue decreased to unacceptable levels if the post mortem-to-fixation interval was increased to more than a few hours. This effect may be secondary to the rapid post-mortem disintegration of the blood-brain barrier and a resulting diffusion of serum proteins into surrounding tissue from the vasculature.     

厌氧产氢颗粒污泥蛋白质组分析样品的高效制备方法

【背景】厌氧产氢颗粒污泥比絮状产氢污泥具有更高的生物量、沉降性与反应效率,对颗粒污泥进行蛋白质组学研究,有助于揭示其代谢调控的分子机制,从而对厌氧代谢过程进行优化调控。目前关于产氢颗粒污泥蛋白质组分析样品制备方法的研究尚未见文献报道。革兰氏阳性菌Ethanoligenens harbinense YUAN-3是自凝集产氢发酵细菌,在间歇和连续流培养中可形成自聚集的厌氧颗粒,由于其全基因组信息清楚,可作为模式研究材料对制备方法进行评估。【目的】针对厌氧产氢颗粒污泥的蛋白质组学研究,比较不同蛋白质提取方法进行优化。【方法】分别利用液氮研磨、超声破碎、匀浆破碎对产氢颗粒污泥破碎,比较这3种方法对总蛋白提取量的影响;通过双向电泳比较三氯乙酸(Trichloroacetic acid,TCA)-丙酮沉淀法与苯酚抽提法对总蛋白提取效果的影响;对总蛋白样品分别进行同位素标记相对和绝对定量标记(Isobarictagsforrelativeandabsolutequantification,i TRAQ)、串联质谱标签(Tandemmasstag,TMT)标记以及质谱鉴定。【结果】液氮研磨、超声破碎、匀浆破碎3种破碎方法下总蛋白的提取量分别是对照样品的2.0、3.9与5.2倍。与TCA-丙酮沉淀法相比,苯酚抽提法总蛋白样品在双向电泳图谱上的蛋白质点明显增多,分布均匀,同时其在碱性蛋白端与小分子量蛋白端的蛋白质点也明显增多。质谱分析发现,iTRAQ标记样品与TMT标记样品中分别鉴定到1797个与1644个蛋白,在分子量、等电点、亚细胞定位的各个分布范围内,这些蛋白良好地覆盖了E.harbinenseYUAN-3中各个类型的蛋白。【结论】匀浆破碎与苯酚抽提法联用的总蛋白制备方法更适用于厌氧产氢颗粒污泥,该方法有利于后续的蛋白质双向电泳和定量蛋白质组质谱分析,可作为产氢颗粒污泥以及革兰氏阳性菌总蛋白制备的方法参考。     

Identification of brain cell death associated proteins in human post-mortem cerebrospinal fluid

Following any form of brain insult, proteins are released from damaged tissues into the cerebrospinal fluid (CSF). This body fluid is therefore an ideal sample to use in the search for biomarkers of neurodegenerative disorders and brain damage. In this study, we used human post-mortem CSF as a model of massive brain injury and cell death for the identification of such protein markers. Pooled post-mortem CSF samples were analyzed using a protocol that combined immunoaffinity depletion of abundant CSF proteins, off-gel electrophoresis, SDS-PAGE and protein identification by LC-MS/MS. A total of 299 proteins were identified, of which 172 proteins were not previously described to be present in CSF. Of these 172 proteins, more than 75% have been described as intracellular proteins suggesting that they were released from damaged cells. Immunoblots of a number of proteins were performed on individual post-mortem CSF samples and confirmed elevated concentrations in post-mortem CSF compared to ante-mortem CSF. Interestingly, among the proteins specifically identified in the post-mortem CSF, several have been previously described as biochemical markers of brain damage.     

The proteomes of feedstocks used for the production of second‐generation ethanol: a lacuna in the biofuel era

Maize, sorghum, sugarcane, switchgrass and miscanthus are the main crops suggested as potential sources of lignocellulosic biomass for the production of second‐generation ethanol. The attention these crops have received has been concentrated in the field of genomics, and very little research has been performed in the field of proteomics, particularly in the cell wall proteomic, despite the importance of these crops in biofuel production. New mass spectrometry‐based proteomic methods allow the identification and quantification of thousands of proteins in complex mixtures, as well as the detection of post‐translational changes in complex proteomes, providing important insight into the downstream consequences of gene expression. Together with other ‘omic’ approaches, proteomic might be decisive to bring new information in the study of cell wall formation. Here, we briefly highlight proteomic techniques and review the research that has been completed on the proteomes of these five crops.     

Partitioning the proteome: phase separation for targeted analysis of membrane proteins in human post-mortem brain

Neuroproteomics is a powerful platform for targeted and hypothesis driven research, providing comprehensive insights into cellular and sub-cellular disease states, Gene × Environmental effects, and cellular response to medication effects in human, animal, and cell culture models. Analysis of sub-proteomes is becoming increasingly important in clinical proteomics, enriching for otherwise undetectable proteins that are possible markers for disease. Membrane proteins are one such sub-proteome class that merit in-depth targeted analysis, particularly in psychiatric disorders. As membrane proteins are notoriously difficult to analyse using traditional proteomics methods, we evaluate a paradigm to enrich for and study membrane proteins from human post-mortem brain tissue. This is the first study to extensively characterise the integral trans-membrane spanning proteins present in human brain. Using Triton X-114 phase separation and LC-MS/MS analysis, we enriched for and identified 494 membrane proteins, with 194 trans-membrane helices present, ranging from 1 to 21 helices per protein. Isolated proteins included glutamate receptors, G proteins, voltage gated and calcium channels, synaptic proteins, and myelin proteins, all of which warrant quantitative proteomic investigation in psychiatric and neurological disorders. Overall, our sub-proteome analysis reduced sample complexity and enriched for integral membrane proteins by 2.3 fold, thus allowing for more manageable, reproducible, and targeted proteomics in case vs. control biomarker studies. This study provides a valuable reference for future neuroproteomic investigations of membrane proteins, and validates the use Triton X-114 detergent phase extraction on human post mortem brain.     

Large scale protein profiling by combination of protein fractionation and multidimensional protein identification technology (MudPIT)

In the past decade, shotgun proteomic analysis has been utilized extensively to answer complex biological questions. New challenges arise in large scale proteomic profiling when dealing with complex biological mixtures such as the mammalian cell lysate. In this study, we explored the approach of protein separation prior to the shotgun multidimensional protein identification technology (MudPIT) analysis. We fractionated the mammalian cancer cell lysate using the PF 2D ProteomeLab system and analyzed the distribution of molecular weight, isoelectric point, and cellular localization of the eluted proteins. As a result, we were able to reduce sample complexity by protein fractionation and increase the possibility of detecting proteins with lower abundance in the complex protein mixture.     

Altered Functional Protein Networks in the Prefrontal Cortex and Amygdala of Victims of Suicide

Probing molecular brain mechanisms related to increased suicide risk is an important issue in biological psychiatry research. Gene expression studies on post mortem brains indicate extensive changes prior to a successful suicide attempt; however, proteomic studies are scarce. Thus, we performed a DIGE proteomic analysis of post mortem tissue samples from the prefrontal cortex and amygdala of suicide victims to identify protein changes and biomarker candidates of suicide. Among our matched spots we found 46 and 16 significant differences in the prefrontal cortex and amygdala, respectively; by using the industry standard t test and 1.3 fold change as cut off for significance. Because of the risk of false discoveries (FDR) in these data, we also made FDR adjustment by calculating the q-values for all the t tests performed and by using 0.06 and 0.4 as alpha thresholds we reduced the number of significant spots to 27 and 9 respectively. From these we identified 59 proteins in the cortex and 11 proteins in the amygdala. These proteins are related to biological functions and structures such as metabolism, the redox system, the cytoskeleton, synaptic function, and proteolysis. Thirteen of these proteins (CBR1, DPYSL2, EFHD2, FKBP4, GFAP, GLUL, HSPA8, NEFL, NEFM, PGAM1, PRDX6, SELENBP1 and VIM,) have already been suggested to be biomarkers of psychiatric disorders at protein or genome level. We also pointed out 9 proteins that changed in both the amygdala and the cortex, and from these, GFAP, INA, NEFL, NEFM and TUBA1 are interacting cytoskeletal proteins that have a functional connection to glutamate, GABA, and serotonin receptors. Moreover, ACTB, CTSD and GFAP displayed opposite changes in the two examined brain structures that might be a suitable characteristic for brain imaging studies. The opposite changes of ACTB, CTSD and GFAP in the two brain structures were validated by western blot analysis.     

A novel approach and protocol for discovering extremely low-abundance proteins in serum

The proteomic analysis of serum (plasma) has been a major approach to determining biomarkers essential for early disease diagnoses and drug discoveries. The determination of these biomarkers, however, is analytically challenging since the dynamic concentration range of serum proteins/peptides is extremely wide (more than 10 orders of magnitude). Thus, the reduction in sample complexity prior to proteomic analyses is essential, particularly in analyzing low-abundance protein biomarkers. Here, we demonstrate a novel approach to the proteomic analyses of human serum that uses an originally developed serum protein separation device and a sequentially linked 3-D-LC-MS/MS system. Our hollow-fiber-membrane-based serum pretreatment device can efficiently deplete high-molecular weight proteins and concentrate low-molecular weight proteins/peptides automatically within 1 h. Four independent analyses of healthy human sera pretreated using this unique device, followed by the 3-D-LC-MS/MS successfully produced 12 000-13 000 MS/MS spectra and hit around 1800 proteins (>95% reliability) and 2300 proteins (>80% reliability). We believe that the unique serum pretreatment device and proteomic analysis protocol reported here could be a powerful tool for searching physiological biomarkers by its high throughput (3.7 days per one sample analysis) and high performance of finding low abundant proteins from serum or plasma samples.     

Proteomics techniques for the development of flood tolerant crops

Proteomics is a useful analytical approach for investigating crop responses to stress. Recent remarkable advances in proteomic techniques allow for the identification of a wider range of proteins than was previously possible. The application of proteomic techniques to clarify the molecular mechanisms underlying crop responses to flooding stress may facilitate the development of flood tolerant crops. Flooding is an environmental stress found worldwide and may increase in frequency due to changes in global climate. Waterlogging resulting from flooding causes significant reductions in the growth and yield of several crops. Transient flooding displaces gases in soil pores and often causes hypoxia in plants grown on land with poor drainage. Changes in protein expression and post-translational modification of proteins occur as plants activate their defense system in response to flooding stress. In this review, we discuss the contributions that proteomic studies have made toward increasing our understanding of the well-organized cellular response to flooding in soybean and other crops. The biological relevance of the proteins identified using proteomic techniques in regard to crop stress tolerance will be discussed as well.     

Biochemical and molecular studies using human autopsy brain tissue

The use of human brain tissue obtained at autopsy for neurochemical, pharmacological and physiological analyses is reviewed. RNA and protein samples have been found suitable for expression profiling by techniques that include RT-PCR, cDNA microarrays, western blotting, immunohistochemistry and proteomics. The rapid development of molecular biological techniques has increased the impetus for this work to be applied to studies of brain disease. It has been shown that most nucleic acids and proteins are reasonably stable post-mortem. However, their abundance and integrity can exhibit marked intra- and intercase variability, making comparisons between case-groups difficult. Variability can reveal important functional and biochemical information. The correct interpretation of neurochemical data must take into account such factors as age, gender, ethnicity, medicative history, immediate ante-mortem status, agonal state and post-mortem and post-autopsy intervals. Here we consider issues associated with the sampling of DNA, RNA and proteins using human autopsy brain tissue in relation to various ante- and post-mortem factors. We conclude that valid and practical measures of a variety of parameters may be made in human brain tissue, provided that specific factors are controlled.     

Optimal replication and the importance of experimental design for gel-based quantitative proteomics

Quantitative proteomic studies, based on two-dimensional gel electrophoresis, are commonly used to find proteins that are differentially expressed between samples or groups of samples. These proteins are of interest as potential diagnostic or prognostic biomarkers, or as proteins associated with a trait. The complexity of proteomic data poses many challenges, so while experiments may reveal proteins that are differentially expressed, these are often not significant when subjected to rigorous statistical analysis. However, this can be addressed through appropriate experimental design. A good experimental design considers the impact of different sources of variation, both analytical and biological, on the statistical importance of the results. The design should address the number of samples that must be analyzed and the number of replicate gels per sample, in the context of a particular minimum difference that one is seeking to achieve. In this study, we explore the ways to improve the quality of protein expression data from 2-DE gels, and describe an approach for defining the number of samples required and the number of gels per sample. It has been developed for the simplest of situations, two groups of samples with variation at two levels: between samples and between gels. This approach will also be useful as a guide for more complex designs involving more than two groups of samples. We describe some Internet-accessible tools that can assist in the design of proteomic studies.     

Tissue-specific microdissection coupled with ProteinChip array technologies: applications in cancer research

Analysis of whole genomes to monitor specific changes in gene activation or changes in gene copy number due to perturbation has recently become possible using DNA chip technologies. It is now becoming apparent, however, that knowing the genetic sequence encoding a protein is not sufficient to predict the size or biological nature of a protein. This can be particularly important in cancer research where posttranslational modifications of a protein can specifically lead to the disease. To address this area, several proteomic tools have been developed. Currently the most widely used proteomics tool is two-dimensional polyacrylamide gel electrophoresis (2D-PAGE), which can display protein expression patterns to a high degree of resolution. However, 2D-PAGE can be time consuming; the analysis is complicated and, compared with DNA techniques, is not very sensitive. Although some of these problems can be alleviated by using high-quality homogeneous samples, such as those generated using microdissection techniques, the quantity of sample is often limited and may take several days to generate sufficient material for a single 2D-PAGE analysis. As an alternative to 2D-PAGE, a preliminary study using a new technique was used to generate protein expression patterns from either whole tissue extracts or microdissected material. Surface-enhanced laser desorption and ionization allows the retention of proteins on a solid-phase chromatographic surface or ProteinChip Array with direct detection of retained proteins by time-of-flight mass spectrometry. Using this system, we analyzed tumor and normal tissue from head and neck cancer and microdissected melanoma to determine differentially expressed proteins. In particular, comparisons of the protein expression patterns from microdissected normal and tumor tissues indicated several differences, highlighting the importance of extremely defined tissue lysates for protein profiling.     

A catalogue of human saliva proteins identified by free flow electrophoresis-based peptide separation and tandem mass spectrometry

Human saliva has great potential for clinical disease diagnostics. Constructing a comprehensive catalogue of saliva proteins using proteomic approaches is a necessary first step to identifying potential protein biomarkers of disease. However, because of the challenge presented in cataloguing saliva proteins with widely varying abundance, new proteomic approaches are needed. To this end, we used a newly developed approach coupling peptide separation using free flow electrophoresis with linear ion trap tandem mass spectrometry to identify proteins in whole human saliva. We identified 437 proteins with high confidence (false positive rate below 1%), producing the largest catalogue of proteins from a single saliva sample to date and providing new information on the composition and potential diagnostic utility of this fluid. The statistically validated, transparently presented, and annotated dataset provides a model for presenting large scale proteomic data of this type, which should facilitate better dissemination and easier comparisons of proteomic datasets from future studies in saliva.     

Protein profile changes during porcine oocyte aging and effects of caffeine on protein expression patterns

It has been shown that oocyte aging critically affects reproduction and development. By using proteomic tools, in the present study, changes in protein profiles during porcine oocyte aging and effects of caffeine on oocyte aging were investigated. By comparing control MII oocytes with aging MII oocytes, we identified 23 proteins that were up-regulated and 3 proteins that were down-regulated during the aging process. In caffeine-treated oocytes, 6 proteins were identified as up-regulated and 12 proteins were identified as down-regulated. A total of 38 differentially expressed proteins grouped into 5 regulation patterns were determined to relate to the aging and anti-aging process. By using the Gene Ontology system, we found that numerous functional gene products involved in metabolism, stress response, reactive oxygen species and cell cycle regulation were differentially expressed during the oocyte aging process, and most of these proteins are for the first time reported in our study, including 2 novel proteins. In addition, several proteins were found to be modified during oocyte aging. These data contribute new information that may be useful for future research on cellular aging and for improvement of oocyte quality.     

Identification and quantification of basic and acidic proteins using solution-based two-dimensional protein fractionation and label-free or 18O-labeling mass spectrometry

Reduction in sample complexity enables more thorough proteomic analysis using mass spectrometry (MS). A solution-based two-dimensional (2D) protein fractionation system, ProteomeLab PF 2D, has recently become available for sample fractionation and complexity reduction. PF 2D resolves proteins by isoelectric point (pI) and hydrophobicity in the first and second dimensions, respectively. It offers distinctive advantages over 2D gel electrophoresis with respects to automation of the fractionation processes and characterization of proteins having extreme pIs. Besides fractionation, PF 2D is equipped with built-in UV detectors intended for relative quantification of proteins in contrasting samples using its software tools. In this study, we utilized PF 2D for the identification of basic and acidic proteins in mammalian cells, which are generally under-characterized. In addition, mass spectrometric methods (label-free and 18O-labeling) were employed to complement protein quantification based on UV absorbance. Our studies indicate that the selection of chromatographic fractions could impact protein identification and that the UV-based quantification for contrasting complex proteomes is constrained by coelution or partial coelution of proteins. In contrast, the quantification post PF 2D chromatography based on label-free or 18O-labeling mass spectrometry provides an alternative platform for basic/acidic protein identification and quantification. With the use of HCT116 colon carcinoma cells, a total of 305 basic and 183 acidic proteins was identified. Quantitative proteomics revealed that 17 of these proteins were differentially expressed in HCT116 p53-/- cells.