Chloroform-Assisted Phenol Extraction Improving Proteome Profiling of Maize Embryos through Selective Depletion of High-Abundance Storage Proteins

The presence of abundant storage proteins in plant embryos greatly impedes seed proteomics analysis. Vicilin (or globulin-1) is the most abundant storage protein in maize embryo. There is a need to deplete the vicilins from maize embryo extracts for enhanced proteomics analysis. We here reported a chloroform-assisted phenol extraction (CAPE) method for vicilin depletion. By CAPE, maize embryo proteins were first extracted in an aqueous buffer, denatured by chloroform and then subjected to phenol extraction. We found that CAPE can effectively deplete the vicilins from maize embryo extract, allowing the detection of low-abundance proteins that were masked by vicilins in 2-DE gel. The novelty of CAPE is that it selectively depletes abundant storage proteins from embryo extracts of both monocot (maize) and dicot (soybean and pea) seeds, whereas other embryo proteins were not depleted. CAPE can significantly improve proteome profiling of embryos and extends the application of chloroform and phenol extraction in plant proteomics. In addition, the rationale behind CAPE depletion of abundant storage proteins was explored.     

A comparison of the bovine uterine and plasma proteome using iTRAQ proteomics

Early embryo loss is a key factor affecting fertility in dairy and beef herds. Prior to implantation, the bovine embryo spends around 16 days free-floating in the uterine environment and is dependent on the composition of uterine fluid for normal growth and development. However, there is a lack of information regarding the protein composition of the bovine uterus and how it relates to plasma. In this study, uterine flushings (UF) (n = 6) and blood plasma (n = 4) were collected from beef heifers on day 7 of the oestrous cycle, albumin depleted and compared using iTRAQ proteomics. A total of 35 proteins were higher and 18 were lower in UF including metabolic enzymes, proteins with anti-oxidant activity and those involved in modulation of the immune response. This study confirms the dynamic nature of the bovine uterine proteome and that it differs from plasma. Factors affecting the uterine proteome and how it impacts on embryo survival warrant further study.     

The utility of nuclear magnetic resonance spectroscopy in assisted reproduction

Infertility affects approximately 15–20% of individuals of reproductive age worldwide. Over the last 40 years, assisted reproductive technology (ART) has helped millions of childless couples. However, ART is limited by a low success rate and risk of multiple gestations. Devising methods for selecting the best gamete or embryo that increases the ART success rate and prevention of multiple gestation has become one of the key goals in ART today. Special emphasis has been placed on the development of non-invasive approaches, which do not require perturbing the embryonic cells, as the current morphology-based embryo selection approach has shortcomings in predicting the implantation potential of embryos. An observed association between embryo metabolism and viability has prompted researchers to develop metabolomics-based biomarkers. Nuclear magnetic resonance (NMR) spectroscopy provides a non-invasive approach for the metabolic profiling of tissues, gametes and embryos, with the key advantage of having a minimal sample preparation procedure. Using NMR spectroscopy, biologically important molecules can be identified and quantified in intact cells, extracts or secretomes. This, in turn, helps to map out the active metabolic pathways in a system. The present review covers the contribution of NMR spectroscopy in assisted reproduction at various stages of the process.     

Human Endometrial Extracellular Vesicles Functionally Prepare Human Trophectoderm Model for Implantation: Understanding Bidirectional Maternal‐Embryo Communication

Embryo implantation into maternal endometrium is critical for initiation and establishment of pregnancy, requiring developmental synchrony between endometrium and blastocyst. However, factors regulating human endometrial–embryo cross talk and facilitate implantation remain largely unknown. Extracellular vesicles (EVs) are emerging as important mediators of this process. Here, a trophectoderm spheroid‐based in vitro model mimicking the pre‐implantation human embryo is used to recapitulate important functional aspects of blastocyst implantation. Functionally, human endometrial EVs, derived from hormonally treated cells synchronous with implantation, are readily internalized by trophectoderm cells, regulating adhesive and invasive capacity of human trophectoderm spheroids. To gain molecular insights into mechanisms underpinning endometrial EV‐mediated enhancement of implantation, quantitative proteomics reveal critical alterations in trophectoderm cellular adhesion networks (cell adhesion molecule binding, cell–cell adhesion mediator activity, and cell adherens junctions) and metabolic and gene expression networks, and the soluble secretome from human trophectodermal spheroids. Importantly, transfer of endometrial EV cargo proteins to trophectoderm to mediate changes in trophectoderm function is demonstrated. This is highlighted by correlation among endometrial EVs, the trophectodermal proteome following EV uptake, and EV‐mediated trophectodermal cellular proteome, important for implantation. This work provides an understanding into molecular mechanisms of endometrial EV‐mediated regulation of human trophectoderm functions—fundamental in understanding human endometrium–embryo signaling during implantation.     

A combined dataset of human cerebrospinal fluid proteins identified by multi-dimensional chromatography and tandem mass spectrometry

Human cerebrospinal fluid (CSF) is an important source for studying protein biomarkers of age-related neurodegenerative diseases. Before characterizing biomarkers unique to each disease, it is necessary to categorize CSF proteins systematically and extensively. However, the enormous complexity, great dynamic range of protein concentrations, and tremendous protein heterogeneity due to post-translational modification of CSF create significant challenges to the existing proteomics technologies for an in-depth, nonbiased profiling of the human CSF proteome. To circumvent these difficulties, in the last few years, we have utilized several different separation methodologies and mass spectrometric platforms that greatly enhanced the identification coverage and the depth of protein profiling of CSF to characterize CSF proteome. In total, 2594 proteins were identified in well-characterized pooled human CSF samples using stringent proteomics criteria. This report summarizes our efforts to comprehensively characterize the human CSF proteome to date.     

A chemical proteomics approach to phosphatidylinositol 3-kinase signaling in macrophages

Prior work using lipid-based affinity matrices has been done to investigate distinct sets of lipid-binding proteins, and one series of experiments has proven successful in mammalian cells for the proteome-wide identification of lipid-binding proteins. However, most lipid-based proteomics screens require scaled up sample preparation, are often composed of multiple cell types, and are not adapted for simultaneous signal transduction studies. Herein we provide a chemical proteomics strategy that uses cleavable lipid "baits" with broad applicability to diverse biological samples. The novel baits were designed to avoid preparative steps to allow functional proteomics studies when the biological source is a limiting factor. Validation of the chemical baits was first confirmed by the selective isolation of several known endogenous phosphatidylinositol 3-kinase signaling proteins using primary bone marrow-derived macrophages. The use of this technique for cellular proteomics and MS/MS analysis was then demonstrated by the identification of known and potential novel lipid-binding proteins that was confirmed in vitro for several proteins by direct lipid-protein interactions. Further to the identification, the method is also compatible with subsequent signal transduction studies, notably for protein kinase profiling of the isolated lipid-bound protein complexes. Taken together, this integration of minimal scale proteomics, lipid chemistry, and activity-based readouts provides a significant advancement in the ability to identify and study the lipid proteome of single, relevant cell types.     

Targeted proteomics by selected reaction monitoring mass spectrometry: applications to systems biology and biomarker discovery

Mass Spectrometry-based proteomics is now considered a relatively established strategy for protein analysis, ranging from global expression profiling to the identification of protein complexes and specific post-translational modifications. Recently, Selected Reaction Monitoring Mass Spectrometry (SRM-MS) has become increasingly popular in proteome research for the targeted quantification of proteins and post-translational modifications. Using triple quadrupole instrumentation (QqQ), specific analyte molecules are targeted in a data-directed mode. Used routinely for the quantitative analysis of small molecular compounds for at least three decades, the technology is now experiencing broadened application in the proteomics community. In the current review, we will provide a detailed summary of current developments in targeted proteomics, including some of the recent applications to biological research and biomarker discovery.     

Oocyte spindle proteomics analysis leading to rescue of chromosome congression defects in cloned embryos

Embryos produced by somatic cell nuclear transfer (SCNT) display low term developmental potential. This is associated with deficiencies in spindle composition prior to activation and at early mitotic divisions, including failure to assemble certain proteins on the spindle. The protein-deficient spindles are accompanied by chromosome congression defects prior to activation and during the first mitotic divisions of the embryo. The molecular basis for these deficiencies and how they might be avoided are unknown. Proteomic analyses of spindles isolated from normal metaphase II (MII) stage oocytes and SCNT constructs, along with a systematic immunofluorescent survey of known spindle-associated proteins were undertaken. This was the first proteomics study of mammalian oocyte spindles. The study revealed four proteins as being deficient in spindles of SCNT embryos in addition to those previously identified; these were clathrin heavy chain (CLTC), aurora B kinase, dynactin 4, and casein kinase 1 alpha. Due to substantial reduction in CLTC abundance after spindle removal, we undertook functional studies to explore the importance of CLTC in oocyte spindle function and in chromosome congression defects of cloned embryos. Using siRNA knockdown, we demonstrated an essential role for CLTC in chromosome congression during oocyte maturation. We also demonstrated rescue of chromosome congression defects in SCNT embryos at the first mitosis using CLTC mRNA injection. These studies are the first to employ proteomics analyses coupled to functional interventions to rescue a specific molecular defect in cloned embryos.     

Proteome Oxidative Modifications and Impairment of Specific Metabolic Pathways During Cellular Senescence and Aging

Accumulation of oxidatively modified proteins is a hallmark of organismal aging in vivo and of cellular replicative senescence in vitro. Failure of protein maintenance is a major contributor to the age‐associated accumulation of damaged proteins that is believed to participate to the age‐related decline in cellular function. In this context, quantitative proteomics approaches, including 2‐D gel electrophoresis (2‐DE)‐based methods, represent powerful tools for monitoring the extent of protein oxidative modifications at the proteome level and for identifying the targeted proteins, also referred as to the “oxi‐proteome.” Previous studies have identified proteins targeted by oxidative modifications during replicative senescence of human WI‐38 fibroblasts and myoblasts and have been shown to represent a restricted set within the total cellular proteome that fall in key functional categories, such as energy metabolism, protein quality control, and cellular morphology. To provide mechanistic support into the role of oxidized proteins in the development of the senescent phenotype, untargeted metabolomic profiling is also performed for young and senescent myoblasts and fibroblasts. Metabolomic profiling is indicative of energy metabolism impairment in both senescent myoblasts and fibroblasts, suggesting a link between oxidative protein modifications and the altered cellular metabolism associated with the senescent phenotype of human myoblasts and fibroblasts.     

2014蛋白质组学专刊序言

蛋白质组学研究是后基因组学时代最重要的功能基因组学研究之一,与医学生物学、化学、物理学、信息学以及现代技术等关系十分密切。为了检阅近年来国内外蛋白质组学某些重要研究进展,探索其可能的应用范围,讨论其存在的问题,展望其发展前景,特组织出版"蛋白质组学专刊"。本期专刊包括综述和研究论文两部分,内容主要涉及不同物种(包括人类、哺乳类动物、原核生物、放线菌等)蛋白质组学研究、蛋白质组学重要方法学与技术研究(包括串联质谱分析、尿蛋白膜保存法、定量蛋白质组学分折、meta分析等)和蛋白质组功能与应用研究(包括蜘蛛毒素蛋白质组、磷酸化蛋白质组、卵母细胞和早期胚胎蛋白质组、肝脏纤维化蛋白质组、分枝杆菌耐药的蛋白质组等)。     

Multidimensional fractionation of the bovine skeletal muscle proteome.

The ultimate goal of proteomics is to understand complex biological systems. The first step toward this end is the discovery of protein differences by profiling a given proteome. One approach to proteome profiling is to fractionate it into intact proteins, with subsequent identification and quantitation. In this work, lysates of bovine skeletal muscle were prepared. Reproducible proteome profiles were generated by an automatic two-dimensional protein fractionation system. Proteins were separated by isoelectric point and then by hydrophobicity. The data collected from both separations were used to generate proteome profiles. A high protein content fraction with pl above 8.5 was digested with trypsin, and its main protein component was identified as lysozyme C by matrix assisted laser desorption/ionization-time of flight mass spectrometry.     

The past, present, and future of embryo selection in in vitro fertilization: Frontiers in Reproduction Conference

Assisted reproductive technology (ART) has been recognized for its success in treating infertility, a condition that affects 15 percent of couples in the United States. The most popular option is in vitro fertilization (IVF), which relies on embryo culture, selection, and transfer for implantation, with the ultimate aim of pregnancy. Previous embryo selection methods relied on morphological factors to select for greatest viability. At Yale's Frontiers in Reproduction Conference on April 29, 2011, at the New Haven Lawn Club, Dr. Denny Sakkas of Yale's Department of Obstetrics, Gynecology, and Reproductive Sciences presented a paradigm shift: using morphological factors along with metabolic, protein, and genetic markers in culture media to enhance embryo selection and IVF success rates.     

Advances and challenges in liquid chromatography-mass spectrometry-based proteomics profiling for clinical applications

Recent advances in proteomics technologies provide tremendous opportunities for biomarker-related clinical applications; however, the distinctive characteristics of human biofluids such as the high dynamic range in protein abundances and extreme complexity of the proteomes present tremendous challenges. In this review we summarize recent advances in LC-MS-based proteomics profiling and its applications in clinical proteomics as well as discuss the major challenges associated with implementing these technologies for more effective candidate biomarker discovery. Developments in immunoaffinity depletion and various fractionation approaches in combination with substantial improvements in LC-MS platforms have enabled the plasma proteome to be profiled with considerably greater dynamic range of coverage, allowing many proteins at low ng/ml levels to be confidently identified. Despite these significant advances and efforts, major challenges associated with the dynamic range of measurements and extent of proteome coverage, confidence of peptide/protein identifications, quantitation accuracy, analysis throughput, and the robustness of present instrumentation must be addressed before a proteomics profiling platform suitable for efficient clinical applications can be routinely implemented.     

Fractionation profiling: a fast and versatile approach for mapping vesicle proteomes and protein–protein interactions

We developed “fractionation profiling,” a method for rapid proteomic analysis of membrane vesicles and protein particles. The approach combines quantitative proteomics with subcellular fractionation to generate signature protein abundance distribution profiles. Functionally associated groups of proteins are revealed through cluster analysis. To validate the method, we first profiled >3500 proteins from HeLa cells and identified known clathrin-coated vesicle proteins with >90% accuracy. We then profiled >2400 proteins from Drosophila S2 cells, and we report the first comprehensive insect clathrin-coated vesicle proteome. Of importance, the cluster analysis extends to all profiled proteins and thus identifies a diverse range of known and novel cytosolic and membrane-associated protein complexes. We show that it also allows the detailed compositional characterization of complexes, including the delineation of subcomplexes and subunit stoichiometry. Our predictions are presented in an interactive database. Fractionation profiling is a universal method for defining the clathrin-coated vesicle proteome and may be adapted for the analysis of other types of vesicles and particles. In addition, it provides a versatile tool for the rapid generation of large-scale protein interaction maps.     

Proteomics in radiation research: present status and future perspectives

Rapidly developing postgenome research has made proteins an attractive target for biological analysis. The well-established term of proteome is defined as the complete set of proteins expressed in a given cell, tissue or organism. Unlike the genome, a proteome is rapidly changing as it tends to adapt to microenvironmental signals. The systematic analysis of the proteome at a given time and state is referred to as proteomics. This technique provides information on the molecular and cellular mechanisms that regulate physiology and pathophysiology of the cell. Applications of proteome profiling in radiation research are increasing. However, the large-scale proteomics data sets generated need to be integrated into other fields of radiation biology to facilitate the interpretation of radiation-induced cellular and tissue effects. The aim of this review is to introduce the most recent developments in the field of radiation proteomics.     

Proteomic discovery of protease substrates

Elucidation of in vivo substrate degradomes of a protease is a daunting endeavor because of the large number of proteins in a proteome and often minute and transient amounts of key substrates. Proteomic substrate screens for proteases are currently experiencing impressive progress. Mass spectrometry-based global proteome analysis, interfaced with liquid-chromatography and together with stable isotope labeling strategies, has provided increased coverage and sensitivity for quantitative proteomics. ICAT and iTRAQ labeling have been used to identify a plethora of new matrix metalloproteinase substrates. Emerging techniques focus on the quantitative analysis of proteolytically generated neo amino-termini, which we call terminopes, on a system-wide basis. In vivo SILAC pulse-chase experiments have also enabled the study of individual protein turnover and global proteome dynamics in cells and whole organisms. Together with activity-based probes for the profiling of functional proteases, there is now in place an array of complementary technologies to dissect the 'protease web' and its distortion in pathology.     

Cotyledonary somatic embryos of Pinus pinaster Ait. most closely resemble fresh,maturing cotyledonary zygotic embryos: biological,carbohydrate and proteomic analyses

Cotyledonary somatic embryos (SEs) of maritime pine are routinely matured for 12 weeks before being germinated and converted to plantlets. Although regeneration success is highly dependent on SEs quality, the date of harvesting is currently determined mainly on the basis of morphological features. This empirical method does not provide any accurate information about embryo quality with respect to storage compounds (proteins, carbohydrates). We first analyzed SEs matured for 10, 12 and 14 weeks by carrying out biological (dry weight, water content) and biochemical measurements (total protein and carbohydrate contents). No difference could be found between collection dates, suggesting that harvesting SEs after 12 weeks is appropriate. Cotyledonary SEs were then compared to various stages, from fresh to fully desiccated, in the development of cotyledonary zygotic embryos (ZEs). We identified profiles that were similar using hierarchical ascendant cluster analysis (HCA). Fresh and dehydrated ZEs could be distinguished, and SEs clustered with fresh ZEs. Both types of embryo exhibited similar carbohydrate and protein contents and signatures. This high level of similarity (94.5 %) was further supported by proteome profiling. Highly expressed proteins included storage, stress-related, late embryogenesis abundant and energy metabolism proteins. By comparing overexpressed proteins in developing and cotyledonary SEs or ZEs, some (23 proteins) could be identified as candidate biomarkers for the late, cotyledonary stage. This is the first report of useful generic protein markers for monitoring embryo development in maritime pine. Our results also suggest that improvements of SEs quality may be achieved if the current maturation conditions are refined.     

Clinical-scale high-throughput human plasma proteome analysis: lung adenocarcinoma

Clinical proteomics requires the stable and reproducible analysis of a large number of human samples. We report a high-throughput comprehensive protein profiling system comprising a fully automated, on-line, two-dimensional microflow liquid chromatography/tandem mass spectrometry (2-D microLC-MS/MS) system for use in clinical proteomics. A linear ion-trap mass spectrometer (ITMS) also known as a 2-D ITMS instrument, which is characterized by high scan speed, was incorporated into the microLC-MS/MS system in order to obtain highly improved sensitivity and resolution in MS/MS acquisition. This system was used to evaluate bovine serum albumin and human 26S proteasome. Application of these high-throughput microLC conditions and the 2-D ITMS resulted in a 10-fold increase in sensitivity in protein identification. Additionally, peptide fragments from the 26S proteasome were identified three-fold more efficiently than by the conventional 3-D ITMS instrument. In this study, the 2-D microLC-MS/MS system that uses linear 2-D ITMS has been applied for the plasma proteome analysis of a few samples from healthy individuals and lung adenocarcinoma patients. Using the 2-D and 1-D microLC-MS/MS analyses, approximately 250 and 100 different proteins were detected, respectively, in each HSA- and IgG-depleted sample, which corresponds to only 0.4 microL of blood plasma. Automatic operation enabled the completion of a single run of the entire 1-D and 2-D microLC-MS/MS analyses within 11 h. Investigation of the data extracted from the protein identification datasets of both healthy and adenocarcinoma groups revealed that several of the group-specific proteins could be candidate protein disease markers expressed in the human blood plasma. Consequently, it was demonstrated that this high-throughput microLC-MS/MS protein profiling system would be practically applicable to the discovery of protein disease markers, which is the primary objective in clinical plasma proteome projects.     

Recent advances in quantitative and chemical proteomics for autophagy studies

Macroautophagy/autophagy is an evolutionarily well-conserved cellular degradative process with important biological functions that is closely implicated in health and disease. In recent years, quantitative mass spectrometry-based proteomics and chemical proteomics have emerged as important tools for the study of autophagy, through large-scale unbiased analysis of the proteome or through highly specific and accurate analysis of individual proteins of interest. At present, a variety of approaches have been successfully applied, including (i) expression and interaction proteomics for the study of protein post-translational modifications, (ii) investigating spatio-temporal dynamics of protein synthesis and degradation, and (iii) direct determination of protein activity and profiling molecular targets in the autophagic process. In this review, we attempted to provide an overview of principles and techniques relevant to the application of quantitative and chemical proteomics methods to autophagy, and outline the current landscape as well as future outlook of these methods in autophagy research.