Organellar proteomics: the prizes and pitfalls of opening the nuclear envelope

Proteomic studies have the potential to comprehensively define the composition of organelles but are limited by the organellar cross-contamination that arises during subcellular fractionation. Comparative proteomics of organellar subfractions can mitigate these problems, as demonstrated by a recent study involving the nuclear envelope.     

Immunoisolation and subfractionation of synaptic vesicle proteins

Within recent years, the advances in proteomics techniques have resulted in considerable novel insights into the protein expression patterns of specific tissues, cells, and organelles. The information acquired from large-scale proteomics approaches indicated, however, that the proteomic analysis of whole cells or tissues is often not suited to fully unravel the proteomes of individual organellar constituents or to identify proteins that are present at low copy numbers. In addition, the identification of hydrophobic proteins is still a challenge. Therefore, the development of techniques applicable for the enrichment of low-abundance membrane proteins is essential for a comprehensive proteomic analysis. In addition to the enrichment of particular subcellular structures by subcellular fractionation, the spectrum of techniques applicable for proteomics research can be extended toward the separation of integral and peripheral membrane proteins using organic solvents, detergents, and detergent-based aqueous two-phase systems with water-soluble polymers. Here, we discuss the efficacy of a number of experimental protocols. We demonstrate that the appropriate selection of physicochemical conditions results in the isolation of synaptic vesicles of high purity whose proteome can be subfractionated into integral membrane proteins and soluble proteins by several phase separation techniques.     

Sample preparation project for the subcellular proteome of mouse liver

Organelle proteome has become one of the most important fields of proteomics, and the subcellular fractionation with high purity and yield has always been a challenge for cell biologists and also for the Human Liver Proteome Project (HLPP). The liver of a C57BL/6J mouse was chosen as the model to find the optimum method for subcellular preparation. The method we selected could obtain the multiple fractions including plasma membrane, mitochondria, nucleus, ER, and cytosol from a single homogenate. With the same procedure, it is for the first time that the preparation method of frozen homogenized livers was compared with that of the fresh livers and frozen livers. We systematically evaluated the purity, efficiency, and integrity by protein yield, immunoblotting, and transmission electron microscopy. Taken together, the method of multiple fractions from a single tissue is effective enough for subcellular fractionation of mouse liver. We give a selective sample preparation method for frozen homogenized livers, for rare clinical samples, which cannot easily be used for subcellular separation immediately. But the frozen livers are not recommended for organelles isolation. This result is especially useful for sample preparation of human liver for subcellular fractionation of HLPP.     

Subcellular shotgun proteomics in plants: looking beyond the usual suspects

In this review we examine the current state of analytical methods used for shotgun proteomics experiments in plants. The rapid advances in this field in recent years are discussed, and contrasted with experiments performed using current widely used procedures. We also examine the use of subcellular fractionation approaches as they apply to plant proteomics, and discuss how appropriate sample preparation can produce a great increase in proteome coverage in subsequent analysis. We conclude that the conjunction of these two techniques represents a significant advance in plant proteomics, and the future of plant biology research will continue to be enriched by the ongoing development of proteomic analytical technology.     

Flow cytometry-assisted purification and proteomic analysis of the corticotropes dense-core secretory granules

The field of organellar proteomics has emerged as an attempt to minimize the complexity of the proteomics data obtained from whole cell and tissue extracts while maximizing the resolution on the protein composition of a single subcellular compartment. Standard methods involve lengthy density-based gradient and/or immunoaffinity purification steps followed by extraction, 1-DE or 2-DE, gel staining, in-gel tryptic digestion, and protein identification by MS. In this paper, we present an alternate approach to purify subcellular organelles containing a fluorescent reporter molecule. The gel-free procedure involves fluorescence-assisted sorting of the secretory granules followed by gentle extraction in a buffer compatible with tryptic digestion and MS. Once the subcellular organelle labeled, this procedure can be done in a single day, requires no major modification to any instrumentation and can be readily adapted to the study of other organelles. When applied to corticotrope secretory granules, it led to a much enriched granular fraction from which numerous proteins could be identified through MS.     

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.     

Protein identification and quantification from riverbank grape,Vitis riparia: Comparing SDS‐PAGE and FASP‐GPF techniques for shotgun proteomic analysis

Protein sample preparation optimisation is critical for establishing reproducible high throughput proteomic analysis. In this study, two different fractionation sample preparation techniques (in‐gel digestion and in‐solution digestion) for shotgun proteomics were used to quantitatively compare proteins identified in Vitis riparia leaf samples. The total number of proteins and peptides identified were compared between filter aided sample preparation (FASP) coupled with gas phase fractionation (GPF) and SDS‐PAGE methods. There was a 24% increase in the total number of reproducibly identified proteins when FASP‐GPF was used. FASP‐GPF is more reproducible, less expensive and a better method than SDS‐PAGE for shotgun proteomics of grapevine samples as it significantly increases protein identification across biological replicates. Total peptide and protein information from the two fractionation techniques is available in PRIDE with the identifier PXD001399 ( http://proteomecentral.proteomexchange.org/dataset/PXD001399 ).     

Generation of transgenic Arabidopsis plants expressing mcherry-fused organelle marker proteins

In eukaryotic cells, a major proportion of the cellular proteins localize to various subcellular organelles where they are involved in organelle-specific cellular processes. Thus, the localization of a particular protein in the cell is an important part of understanding the physiological role of the protein in the cell. Various approaches such as subcellular fractionation, immunolocalization and live imaging have been used to define the localization of organellar proteins. Of these various approaches, the most powerful one is the live imaging because it can show in vivo dynamics of protein localization depending on cellular and environmental conditions without disturbing cellular structures. However, the live imaging requires the ability to detect the organelles in live cells. In this study, we report generation of a new set of transgenic Arabidopsis plants using various organelle marker proteins fused to a fluorescence protein, monomeric Cherry (mCherry). All these markers representing different subcellular organelles such as chloroplasts, mitochondria, peroxisomes, endoplasmic reticulum (ER) and lytic vacuole showed clear and specific signals regardless of the cell types and tissues. These marker lines can be used to determine localization of organellar proteins by colocalization and also to study the dynamics of organelles under various developmental and environmental conditions.     

亚细胞蛋白质组研究进展

运用蛋白质组学方法对亚细胞组分进行研究是目前的研究热点。传统的亚细胞分离技术结合质谱鉴定技术为亚细胞蛋白质组学的研究提供了技术平台。本文对快速发展的亚细胞蛋白质组研究进展及其所揭示的生物学意义进行了综述。     

叶绿体蛋白质组研究进展

亚细胞蛋白质组学是近年来蛋白组学研究中的一个热点。通过细胞器的纯化和亚细胞组分的分离,降低了样品的复杂性,增大了相应蛋白质组分的富集,有利于由此分离获得的蛋白质的序列分析及功能鉴定。叶绿体蛋白质组为植物亚细胞蛋白质组学研究中相对全面的一部分,利用亚细胞分离结合双向电泳技术系统地鉴定叶绿体中蛋白质组分是获取叶绿体蛋白质信息、确定其功能的重要技术手段。本文就近年来植物叶绿体蛋白质组涵盖的叶绿体内、外被膜、叶绿体基质、类囊体膜和类囊体腔蛋白的研究进行综述,以全面认识叶绿体蛋白的组成、特点及其在叶绿体生理生化代谢网络中的作用。     

Subcellular fractionation methods and strategies for proteomics

Developments in subcellular fractionation strategies have provided the means to profile and analyze the protein composition of organelles and cellular structures by proteomics. Here, we review the application of classical (e.g. density gradient centrifugation) and emerging sophisticated techniques (fluorescent-assisted organelle sorting) in the fractionation, and statistical/bioinformatics tools for the prediction of protein localization in subcellular proteomics. We also review the validation methods currently used (such as microscopy, RNA interference and multiple reaction monitoring) and discuss the importance of verification of the results obtained in subcellular proteomics. Finally, the numerous challenges facing subcellular proteomics including the dynamics of organelles are being examined. However, complementary approaches such as modern statistics, bioinformatics and large-scale integrative analysis are beginning to emerge as powerful tools to proteomics for analyzing subcellular organelles and structures.     

Comparison of Golgi apparatus and endoplasmic reticulum proteins from livers of juvenile and aged rats using a novel technique for separation and enrichment of organelles.

The broad dynamic range of protein abundances, which can vary from about 10(6) for cells to 10(10) for tissues in complex proteomes, continues to challenge proteomics research. Proteome analysis, in particular organelle proteomics, using current approaches, requires extensive fractionation, separation, and enrichment. Over the years, organelle separation was achieved through the use of differential and density-gradient ultracentrifugation. However, the traditional fixed-volume process is a time-consuming and labor-intensive method, especially with large quantities of sample. Here, we present a novel tool for subcellular fractionation of biologically complex mixtures: continuous-flow ultracentrifugation of tissue homogenates to obtain both organelle separation and extensive organelle enrichment at the same time. In this study, rat liver tissues from two different age groups (3-8 wk and greater than 1 y old) were homogenized by blending. After removing nuclei, the resulting homogenates were further fractionated at the subcellular level by the use of sucrose gradient continuous-flow ultracentrifugation. Each organelle's enriched fractions were identified by Western blot analysis. To study the possible effects of aging on the endoplasmic reticulum and Golgi apparatus, we compared the organelle protein profiles of the two groups of rat liver tissues using two-dimensional gel electrophoresis, digitized imaging of two-dimensional gel electrophoresis, and mass spectrometry. Significant differences in the protein profiles of both organelles were observed between the two groups of rat tissues. The technique described here for fractionation and enrichment of organelles demonstrated a useful tool for proteomics research, including identification of low-abundance proteins and post-translational modifications.     

Labeling, detection and identification of newly synthesized proteomes with bioorthogonal non-canonical amino-acid tagging

A major aim of proteomics is the identification of proteins in a given proteome at a given metabolic state. This protocol describes the step-by-step labeling, purification and detection of newly synthesized proteins in mammalian cells using the non-canonical amino acid azidohomoalanine (AHA). In this method, metabolic labeling of newly synthesized proteins with AHA endows them with the unique chemical functionality of the azide group. In the subsequent click chemistry tagging reaction, azide-labeled proteins are covalently coupled to an alkyne-bearing affinity tag. After avidin-based affinity purification and on-resin trypsinization, the resulting peptide mixture is subjected to tandem mass spectrometry for identification. In combination with deuterated leucine-based metabolic colabeling, candidate proteins can be immediately validated. Bioorthogonal non-canonical amino-acid tagging can be combined with any subcellular fractionation, immunopurification or other proteomic method to identify specific subproteomes, thereby reducing sample complexity and enabling the identification of subtle changes in a proteome. This protocol can be completed in 5 days.     

Zooming in: fractionation strategies in proteomics

The recent development of mass spectrometry, i.e., high sensitivity, automation of protein identification and some post-translational modifications (PTMs) significantly increased the number of large-scale proteomics projects. However, there are still considerable limitations as none of the currently available proteomics techniques allows the analysis of an entire proteome in a single step procedure. On the other hand, there are several successful studies analyzing well defined groups of proteins, e.g., proteins of purified organelles, membrane microdomains or isolated proteins with certain PTMs. Coupling of advanced separation methodologies (different prefractionation strategies, such as subcellular fractionation, affinity purification, fractionation of proteins and peptides according to their physicochemical properties) to highly sensitive mass spectrometers provides powerful means to detect and analyze dynamic changes of low abundant regulatory proteins in eukaryotic cells on the subcellular level. This review summarizes and discusses recent strategies in proteomics approaches where different fractionation strategies were successfully applied.     

Proteomics of industrial fungi: trends and insights for biotechnology

Filamentous fungi are widely known for their industrial applications, namely, the production of food-processing enzymes and metabolites such as antibiotics and organic acids. In the past decade, the full genome sequencing of filamentous fungi increased the potential to predict encoded proteins enormously, namely, hydrolytic enzymes or proteins involved in the biosynthesis of metabolites of interest. The integration of genome sequence information with possible phenotypes requires, however, the knowledge of all the proteins in the cell in a system-wise manner, given by proteomics. This review summarises the progress of proteomics and its importance for the study of biotechnological processes in filamentous fungi. A major step forward in proteomics was to couple protein separation with high-resolution mass spectrometry, allowing accurate protein quantification. Despite the fact that most fungal proteomic studies have been focused on proteins from mycelial extracts, many proteins are related to processes which are compartmentalised in the fungal cell, e.g. β-lactam antibiotic production in the microbody. For the study of such processes, a targeted approach is required, e.g. by organelle proteomics. Typical workflows for sample preparation in fungal organelle proteomics are discussed, including homogenisation and sub-cellular fractionation. Finally, examples are presented of fungal organelle proteomic studies, which have enlarged the knowledge on areas of interest to biotechnology, such as protein secretion, energy production or antibiotic biosynthesis.     

快速发展的亚细胞蛋白质组学

亚细胞蛋白质组是蛋白质组学领域中的一支新生力量 ,已成为蛋白质组学新的主流方向 ,通过多种策略和技术方法 ,一些重要的亚细胞结构的蛋白质组不断的得到分析 ,到目前为止 ,几乎所有亚细胞结构的蛋白质组学研究都有报道 ,而且已经深入到亚细胞器和复合体水平 ;另外 ,不仅局限于对亚细胞结构的蛋白组成进行简单分析 ,而且更注重功能性分析 ,将定量技术和差异分析引入亚细胞蛋白质组学 ,来观察此亚细胞结构的蛋白质组在某些生理或病理条件下的变化 ,这已经成为亚细胞蛋白质组学新的发展方向 .亚细胞蛋白质组学最大的困难在于怎样确认鉴定出来蛋白质的定位 ,是在提取过程中的污染还是真正在此亚细胞结构中有定位 ?这将是亚细胞蛋白质组学需要努力解决的挑战 .文章全面介绍了亚细胞蛋白质组学的最新研究进展 ,阐述了亚细胞蛋白质组学面临的挑战 ,并对亚细胞蛋白质组学的发展方向作了展望 .     

Trends in ultrasensitive proteomics

Here we review recent developments and trends in sample preparation, pre-fractionation, chromatography and mass spectrometry contributing towards the ultra-sensitive global analysis of proteins. Highly sensitive MS-based proteomics is not only beneficiary for the proteome analysis of single cells, an aim which is getting into reach, but also clearly relevant for the analysis of (a) subcellular organelles, (b) specific low-abundant cell-types such as adult stem cells, and (c) smaller but more homogeneous cell populations sorted or dissected from (diseased) tissue.     

Proteomic reactors and their applications in biology

Proteomic analysis requires the combination of an extensive suite of technologies including protein processing and separation, micro-flow HPLC, MS and bioinformatics. Although proteomic technologies are still in flux, approaches that bypass gel electrophoresis (gel-free approaches) are dominating the field of proteomics. Along with the development of gel-free proteomics, came the development of devices for the processing of proteomic samples termed proteomic reactors. These microfluidic devices provide rapid, robust and efficient pre-MS sample procession by performing protein sample preparation/concentration, digestion and peptide fractionation. The proteomic reactor has advanced in two major directions: immobilized enzyme reactor and ion exchange-based proteomic reactor. This review summarizes the technical developments and biological applications of the proteomic reactor over the last decade.     

植物蛋白质组学研究进展

 蛋白质组学是后基因组时代功能基因组学研究的新兴学科和热点领域。该文简要介绍了蛋白质组学产生的科学背景、研究方法和研究内容。蛋白质组学研究方法主要有双向聚丙烯酰胺凝胶电泳（2D-PAGE）、质谱(Mass-spectrometric)技术、蛋白质芯片（Protein chips）技术、酵母双杂交系统（Yeast two-hybrid system）、植物蛋白质组数据库等。其应用的范围包括植物群体遗传学、在个体水平上植物对生物和非生物环境的适应机制、植物的发育和组织器官的分化过程,以及不同亚细胞结构在生理生态过程中的作用等诸多方面。同时对植物蛋白质组学的发展前景进行了展望。