Concepts and strategies of soybean seed proteomics using the shotgun proteomics approach

ABSTRACT

Introduction: The last decade has yielded significant developments in the field of proteomics, especially in mass spectrometry (MS) and data analysis tools. In particular, a shift from gel-based to MS-based proteomics has been observed, thereby providing a platform with which to construct proteome atlases for all life forms. Nevertheless, the analysis of plant proteomes, especially those of samples that contain high-abundance proteins (HAPs), such as soybean seeds, remains challenging.

Areas covered: Here, we review recent progress in soybean seed proteomics and highlight advances in HAPs depletion methods and peptide pre-fractionation, identification, and quantification methods. We also suggest a pipeline for future proteomic analysis, in order to increase the dynamic coverage of the soybean seed proteome.

Expert opinion: Because HAPs limit the dynamic resolution of the soybean seed proteome, the depletion of HAPs is a prerequisite of high-throughput proteome analysis, and owing to the use of two-dimensional gel electrophoresis-based proteomic approaches, few soybean seed proteins have been identified or characterized. Recent advances in proteomic technologies, which have significantly increased the proteome coverage of other plants, could be used to overcome the current complexity and limitation of soybean seed proteomics.     

Proteomics and the dynamic plasma membrane: Quo Vadis?

Quo Vadis: where are you going? Advances in MS-based proteomics have enabled research to move from obtaining the basic protein inventory of cells and organelles to the ability of monitoring their dynamics, including changes in abundance, location and various PTMs. In this respect, the cellular plasma membrane is of particular interest, by not only serving as a barrier between the "cell interior" and the external environment, but moreover by organizing and clustering essential components to enable dynamic responses to internal and external stimuli. Defining and characterizing the dynamic plasma membrane proteome is crucial for understanding fundamental biological processes, disease mechanisms and for finding drug targets. Protein identification, characterization of dynamic PTMs and protein-ligand interactions, and determination of transient changes in protein expression and composition are among the challenges in functional proteomic studies of the plasma membrane. We review the recent progress in MS-based plasma membrane proteomics by presenting key examples from eukaryotic systems, including mammals, yeast and plants. We highlight the importance of enrichment and quantification technologies required for detailed functional and comparative analysis of the dynamic plasma membrane proteome.     

种子蛋白质组的研究进展

蛋白质组学是通过对全套蛋白质动态的研究,来阐明生物体、组织、细胞和亚细胞全部蛋白质的表达模式及功能模式。大量可用的核苷酸序列信息和灵敏高速的质谱鉴定技术,使得蛋白质组学方法为分析模式植物和农作物的复杂功能开辟了新的途径。目前,种子蛋白质组研究主要集中在两个方面：一方面是鉴定尽可能多的蛋白,以创建种子特定生命时期的蛋白质组参照图谱;另一方面主要集中在差异蛋白质组,通过比较分析不同蛋白质组,以探明关键功能蛋白。该文综述了近年来种子蛋白质组的研究进展,内容包括种子发育过程中蛋白质组的变化,与种子休眠/萌发相关的蛋白质组、翻译后修饰蛋白质组、细胞与亚细胞差异蛋白质组以及环境因子对种子蛋白质组的影响;并对种子蛋白质组研究的热点问题进行了展望。     

种子蛋白质组的研究进展

蛋白质组学是通过对全套蛋白质动态的研究, 来阐明生物体、组织、细胞和亚细胞全部蛋白质的表达模式及功能模式。大量可用的核苷酸序列信息和灵敏高速的质谱鉴定技术, 使得蛋白质组学方法为分析模式植物和农作物的复杂功能开辟了新的途径。目前, 种子蛋白质组研究主要集中在两个方面: 一方面是鉴定尽可能多的蛋白, 以创建种子特定生命时期的蛋白质组参照图谱; 另一方面主要集中在差异蛋白质组, 通过比较分析不同蛋白质组, 以探明关键功能蛋白。该文综述了近年来种子蛋白质组的研究进展, 内容包括种子发育过程中蛋白质组的变化, 与种子休眠/萌发相关的蛋白质组、翻译后修饰蛋白质组、细胞与亚细胞差异蛋白质组以及环境因子对种子蛋白质组的影响; 并对种子蛋白质组研究的热点问题进行了展望。     

Quantitative proteomic analyses of crop seedlings subjected to stress conditions; a commentary

Quantitative proteomics is one of the analytical approaches used to clarify crop responses to stress conditions. Recent remarkable advances in proteomics technologies allow for the identification of a wider range of proteins than was previously possible. Current proteomic methods fall into roughly two categories: gel-based quantification methods, including conventional two-dimensional gel electrophoresis and two-dimensional fluorescence difference gel electrophoresis, and MS-based quantification methods consists of label-based and label-free protein quantification approaches. Although MS-based quantification methods have become mainstream in recent years, gel-based quantification methods are still useful for proteomic analyses. Previous studies examining crop responses to stress conditions reveal that each method has both advantages and disadvantages in regard to protein quantification in comparative proteomic analyses. Furthermore, one proteomics approach cannot be fully substituted by another technique. In this review, we discuss and highlight the basis and applications of quantitative proteomic analysis approaches in crop seedlings in response to flooding and osmotic stress as two environmental stresses.     

Gel-free mass spectrometry-based high throughput proteomics: tools for studying biological response of proteins and proteomes

Revolutionary advances in biological mass spectrometry (MS) have provided a basic tool to make possible comprehensive proteomic analysis. Traditionally, two-dimensional gel electrophoresis has been used as a separation method coupled with MS to facilitate analysis of complex protein mixtures. Despite the utility of this method, the many challenges of comprehensive proteomic analysis has motivated the development of gel-free MS-based strategies to obtain information not accessible using two-dimensional gel separations. These advanced strategies have enabled researchers to dig deeper into complex proteomes, gaining insights into the composition, quantitative response, covalent modifications and macromolecular interactions of proteins that collectively drive cellular function. This review describes the current state of gel-free, high throughput proteomic strategies using MS, including (i) the separation approaches commonly used for complex mixture analysis; (ii) strategies for large-scale quantitative analysis; (iii) analysis of post-translational modifications; and (iv) recent advances and future directions. The use of these strategies to make new discoveries at the proteome level into the effects of disease or other cellular perturbations is discussed in a variety of contexts, providing information on the potential of these tools in electromagnetic field research.     

Catch me if you can: mass spectrometry-based phosphoproteomics and quantification strategies

Phosphorylation of proteins is one of the most prominent PTMs and for instance a key regulator of signal transduction. In order to improve our understanding of cellular phosphorylation events, considerable effort has been devoted to improving the analysis of phosphorylation by MS-based proteomics. Different enrichment strategies for phosphorylated peptides/proteins, such as immunoaffinity chromatography (IMAC) or titanium dioxide, have been established and constantly optimized for subsequent MS analysis. Concurrently, specific MS techniques were developed for more confident identification and phosphorylation site localization. In addition, more attention is paid to the LC-MS instrumentation to avoid premature loss of phosphorylated peptides within the analytical system. Despite major advances in all of these fields, the analysis of phosphopeptides still remains far from being routine in proteomics. However, to reveal cellular regulation by phosphorylation events, not only qualitative information about the phosphorylation status of proteins but also, in particular, quantitative information about distinct changes in phosphorylation patterns upon specific stimulation is mandatory. Thus, yielded insights are of outstanding importance for the emerging field of systems biology. In this review, we will give an insight into the historical development of phosphoproteome analysis and discuss its recent progress particularly regarding phosphopeptide quantification and assessment of phosphorylation stoichiometry.     

Proteomics strategies for protein identification

The information from genome sequencing provides new approaches for systems-wide understanding of protein networks and cellular function. DNA microarray technologies have advanced to the point where nearly complete monitoring of gene expression is feasible in several organisms. An equally important goal is to comprehensive survey cellular proteomes and profile protein changes under different cellular states. This presents a complex analytical problem, due to the chemical variability between proteins and peptides. Here, we discuss strategies to improve accuracy and sensitivity of peptide identification, distinguish represented protein isoforms, and quantify relative changes in protein abundance.     

Data-independent acquisition proteomics methods for analyzing post-translational modifications

Protein post-translational modifications (PTMs) increase the functional diversity of the cellular proteome. Accurate and high throughput identification and quantification of protein PTMs is a key task in proteomics research. Recent advancements in data-independent acquisition (DIA) mass spectrometry (MS) technology have achieved deep coverage and accurate quantification of proteins and PTMs. This review provides an overview of DIA data processing methods that cover three aspects of PTMs analysis, that is, detection of PTMs, site localization, and characterization of complex modification moieties, such as glycosylation. In addition, a survey of deep learning methods that boost DIA-based PTMs analysis is presented, including in silico spectral library generation, as well as feature scoring and error rate control. The limitations and future directions of DIA methods for PTMs analysis are also discussed. Novel data analysis methods will take advantage of advanced MS instrumentation techniques to empower DIA MS for in-depth and accurate PTMs measurements.     

Recent progress in mass spectrometry proteomics for biomedical research

Proteins are the key players in many cellular processes. Their composition, trafficking, and interactions underlie the dynamic processes of life. Furthermore, diseases are frequently accompanied by malfunction of proteins at multiple levels. Understanding how biological processes are regulated at the protein level is critically important to understanding the molecular basis for diseases and often shed light on disease prevention, diagnosis, and treatment. With rapid advances in mass spectrometry (MS) instruments and experimental methodologies, MS-based proteomics has become a reliable and essential tool for elucidating biological processes at the protein level. Over the past decade, we have witnessed great expansion of knowledge of human diseases with the application of MS-based proteomic technologies, which has led to many exciting discoveries. Herein we review the recent progress in MS-based proteomics in biomedical research, including that in establishing disease-related proteomes and interactomes. We also discuss how this progress will benefit biomedical research and clinical diagnosis and treatment of disease.     

Proteomics approaches to biomarker detection.

The development of mass spectrometry (MS) technologies has brought the ability to gather massive amounts of data characterising the proteomes of complex mixtures. A major focus in proteomics is to leverage this data-gathering capability to conduct comparative analyses of biofluids from healthy and disease-affected patients for the identification of highly specific biomarkers and/or the development of MS-based diagnostic platforms. Much effort has gone into optimising the biofluid proteome coverage that can be obtained using these technologies, leaving proteomics poised to make an important impact in disease diagnostics in the future.     

Does water stress promote the proteome-wide adjustment of intrinsically disordered proteins in plants?

Plant response to water stress involves the activation of mechanisms expected to help them cope with water scarcity. Among these mechanisms, proteome-wide adjustment is well known. This includes actions to save energy, protect cellular and molecular components, and maintain vital functions of the cell. Intrinsically disordered proteins, which are proteins without a rigid three-dimensional structure, are seen as emerging multifunctional cellular components of proteomes. They are highly abundant in eukaryotic proteomes, and numerous functions for these proteins have been proposed. Here, we discuss several reasons why the collection of intrinsically disordered proteins in a proteome (disordome) could be subjected to an active regulation during conditions of water scarcity in plants. We also discuss the potential misinterpretations of disordome content estimations made so far due to bias-prone data and the need for reliable analysis based on experimental data in order to acknowledge the plasticity nature of the disordome.     

Immunodepletion of albumin and immunoglobulin G from bovine plasma

Current MS-based proteomics has facilitated the identification of large numbers of proteins from complex mixtures. The bovine plasma proteome has the potential to provide a wealth of information concerning the biological state of an animal. However, during MS-based experiments, higher abundance proteins such as albumin and immunoglobulin G (IgG) can hinder the identification of potentially important proteins that are present in much lower abundance. While a variety of readily available technologies exist for the depletion of multiple high-abundance proteins from human, mouse and rat samples, there are few available for bovine. In this study, we report the depletion of >97% of albumin and >92% of IgG from bovine plasma.     

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.     

The use of proteome similarity for the qualitative and quantitative profiling of reperfused myocardium

An LC-MS-based approach is presented for the identification and quantification of proteins from unsequenced organisms. The method relies on the preservation of homology across species and the similarity in detection characteristics of proteomes in general. Species related proteomes share similarity that progresses from the amino acid frequency distribution to the complete amino sequence of matured proteins. Moreover, the comparative analysis between theoretical and experimental proteome distributions can be used as a measure for the correctness of detection and identification obtained through LC-MS-based schemes. Presented are means to the identification and quantification of rabbit myocardium proteins, immediately after inducing cardiac arrest, using a data-independent LC-MS acquisition strategy. The employed method of acquisition affords accurate mass information on both the precursor and associated product ions, whilst preserving and recording the intensities of the ions. The latter facilitates label-free quantification. The experimental ion density observations obtained for the rabbit sub proteome were found to share great similarity with five other mammalian samples, including human heart, human breast tissue, human plasma, rat liver and a mouse cell line. Redundant, species-homologues peptide identifications from other mammalian organisms were used for initial protein identification, which were complemented with peptide identifications of translated gene sequences. The feasibility and accuracy of label-free quantification of the identified peptides and proteins utilizing above mentioned strategy is demonstrated for selected cardiac rabbit proteins.     

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.     

Proteomic databases and tools to decipher post-translational modifications

Post-translational modifications (PTMs) are vital cellular control mechanism, which affect protein properties, including folding, conformation, activity and consequently, their functions. As a result they play a key role in various disease conditions, including cancer and diabetes. Proteomics as a rapidly growing field has witnessed tremendous advancement during the last decade, which has led to the generation of prodigious quantity of data for various organisms' proteome. PTMs being biologically and chemically dynamic process, pose greater challenges for its study. Amidst these complexities connecting the modifications with physiological and cellular cascade of events are still very challenging. Advancement in proteomic technologies such as mass spectrometry and microarray provides HT platform to study PTMs and help to decipher role of some of the very essential biological phenomenon. To enhance our understanding of various PTMs in different organisms, and to simplify the analysis of complex PTM data, many databases, software and tools have been developed. These PTM databases and tools contain crucial information and provide a valuable resource to the research community. This article intends to provide a comprehensive overview of various PTM databases, software tools, and analyze critical information available from these resources to study PTMs in various biological organisms.     

Quantitative proteome analysis using isotope-coded affinity tags and mass spectrometry

A main objective of proteomics research is to systematically identify and quantify proteins in a given proteome (cells, subcellular fractions, protein complexes, tissues or body fluids). Protein labeling with isotope-coded affinity tags (ICAT) followed by tandem mass spectrometry allows sequence identification and accurate quantification of proteins in complex mixtures, and has been applied to the analysis of global protein expression changes, protein changes in subcellular fractions, components of protein complexes, protein secretion and body fluids. This protocol describes protein-sample labeling with ICAT reagents, chromatographic fractionation of the ICAT-labeled tryptic peptides, and protein identification and quantification using tandem mass spectrometry. The method is suitable for both large-scale analysis of complex samples including whole proteomes and small-scale analysis of subproteomes, and allows quantitative analysis of proteins, including those that are difficult to analyze by gel-based proteomics technology.