Mass Spectrometry and Proteomics 2018: the Mass Spectrometry Society of Japan,Japanese Proteomics Society,and Asia-Oceania Human Proteome Organization

ABSTRACT

Introduction: The mass spectrometry society of Japan, Japanese proteomics society, and Asia–Oceania human proteome organization held the conference ‘Mass Spectrometry and Proteomics 2018’ in Osaka, Japan, on May 15–18, 2018. This international conference focused on cutting edge technologies and their applications in a variety of research fields such as agriculture, material science, environmental factors, and clinical applications. An overview of the conference and a summary of the major lectures are reported here.

Expert commentary: The meeting will facilitate the development of fundamental technologies and the multi-disciplinary applications of proteomics.     

Cross-linking/mass spectrometry as a new field and the proteomics information mountain of tomorrow

The European Proteomics Association (EuPA) 2012 Scientific Congress ‘New Horizons and Applications for Proteomics’, hosted by the British Society for Proteome Research (BSPR)

Glasgow, Scotland, UK, 12 July 2012

Cross-linking/mass spectrometry ended decades of method developments and entered the era of applications at this year’s European Proteomics Association meeting. The train has started moving, with successful applications of this tool by multiple pioneering laboratories addressing biological and structural problems. Proteomics, on the other side, sees ever increasing data volumes, leading to questions as to how to store the data mountain publically, use it and convert it into testable hypotheses. The European Proteomics Association meeting has been complementary to the American Society for Mass Spectrometry meeting in many ways, also thanks to its more manageable size and the vision of the organizers in inviting some of Europe’s best emerging minds.     

Integral membrane proteins: bottom-up,top-down and structural proteomics

Introduction: Integral membrane proteins and lipids constitute the bilayer membranes that surround cells and sub-cellular compartments, and modulate movements of molecules and information between them. Since membrane protein drug targets represent a disproportionately large segment of the proteome, technical developments need timely review.

Areas covered: Publically available resources such as Pubmed were surveyed. Bottom-up proteomics analyses now allow efficient extraction and digestion such that membrane protein coverage is essentially complete, making up around one third of the proteome. However, this coverage relies upon hydrophilic loop regions while transmembrane domains are generally poorly covered in peptide-based strategies. Top-down mass spectrometry where the intact membrane protein is fragmented in the gas phase gives good coverage in transmembrane regions, and membrane fractions are yielding to high-throughput top-down proteomics. Exciting progress in native mass spectrometry of membrane protein complexes is providing insights into subunit stoichiometry and lipid binding, and cross-linking strategies are contributing critical in-vivo information.

Expert commentary: It is clear from the literature that integral membrane proteins have yielded to advanced techniques in protein chemistry and mass spectrometry, with applications limited only by the imagination of investigators. Key advances toward translation to the clinic are emphasized.     

Methodologies for characterizing phosphoproteins by mass spectrometry

Posttranslational regulation of proteins via protein phosphorylation is one of the major means of protein regulation. Phosphorylation is a very rapid and reversible method of changing the function of proteins. Detection of phosphorylated proteins and the identification of phosphorylation sites are necessary to molecularly link specific phosphorylated events with change in phosphoprotein function. Mass Spectrometry (MS) has become the methodology of choice for phosphosite identification. Here we review current approaches including sample separation and enrichment techniques (SDS-PAGE, immunoprecipitation, metal-assisted enrichment, strong cation exchange, dendrimer capture), quantitative MS analysis methods (SILAC, iTRAQ, AQUA), and the application of recently developed methods including electron transfer dissociation ionization and "top-down" proteomics to phosphoprotein analysis.     

Top-down Proteomics: Technology Advancements and Applications to Heart Diseases

Introduction: Heart diseases are a leading cause of morbidity and mortality for both men and women worldwide, and impose significant economic burdens on the healthcare systems. Despite substantial effort over the last several decades, the molecular mechanisms underlying diseases of the heart remain poorly understood.

Areas covered: Altered protein post-translational modifications (PTMs) and protein isoform switching are increasingly recognized as important disease mechanisms. Top-down high-resolution mass spectrometry (MS)-based proteomics has emerged as the most powerful method for the comprehensive analysis of PTMs and protein isoforms. Here, we will review recent technology developments in the field of top-down proteomics, as well as highlight recent studies utilizing top-down proteomics to decipher the cardiac proteome for the understanding of the molecular mechanisms underlying diseases of the heart.

Expert commentary: Top-down proteomics is a premier method for the global and comprehensive study of protein isoforms and their PTMs, enabling the identification of novel protein isoforms and PTMs, characterization of sequence variations, and quantification of disease-associated alterations. Despite significant challenges, continuous development of top-down proteomics technology will greatly aid the dissection of the molecular mechanisms underlying diseases of the hearts for the identification of novel biomarkers and therapeutic targets.     

Evaluation of the compact high-field orbitrap for top-down proteomics of human cells

Mass spectrometry based proteomics generally seeks to identify and fully characterize protein species with high accuracy and throughput. Recent improvements in protein separation have greatly expanded the capacity of top-down proteomics (TDP) to identify a large number of intact proteins. To date, TDP has been most tightly associated with Fourier transform ion cyclotron resonance (FT-ICR) mass spectrometry. Here, we couple the improved separations to a Fourier-transform instrument based not on ICR but using the Orbitrap Elite mass analyzer. Application of this platform to H1299 human lung cancer cells resulted in the unambiguous identification of 690 unique proteins and over 2000 proteoforms identified from proteins with intact masses <50 kDa. This is an early demonstration of high throughput TDP (>500 identifications) in an Orbitrap mass spectrometer and exemplifies an accessible platform for whole protein mass spectrometry.     

Further advances in the development of a data interchange standard for proteomics data

The Protein Standards Initiative (PSI) aims to define community standards for data representation in proteomics and to facilitate data comparison, exchange and verification. Significant progress was made in advancing the design and implementation of a draft standard for exchanging experimental data from proteomics experiments involving mass spectrometry at the 51st Annual Conference of the American Society for Mass Spectrometry. In collaboration with the American Society for Tests and Measurements, the PSI propose to publish this first draft at the forthcoming HUPO 2nd World Congress in Montreal, 8-11 October 2003.     

植物蛋白质组学研究进展Ⅰ. 蛋白质组关键技术

随着模式植物拟南芥和水稻基因组测序相继完成, 使植物基因组学研究成功迈入到功能基因组学研究的时代。这为蛋白质组学产生及其发展奠定了坚实的基础。文章重点介绍了蛋白质组学的概念、产生背景和蛋白质组学的关键技术。蛋白质组学的关键技术包括双向电泳、高效液相色谱、蛋白芯片、质谱技术、蛋白质组学的相关数据库、定量蛋白组技术、蛋白复合体标签亲和纯化技术和酵母双杂交系统。同时对当前蛋白质组技术面临的挑战和发展前景进行了讨论。     

Mass spectrometry in plant proteomic analysis

Abstract

The current revolution in proteomics has been generated by the combination of very sensitive mass spectrometers coupled to microcapillary liquid chromatography, specific proteolysis of protein mixtures and software that is capable of searching vast numbers of mass measurements against predicted peptides from sequenced genomes. The challenges of post‐genomic plant biology include characterization of protein function, post‐translational modifications and composition of protein complexes as well as deciphering protein complements in intracellular compartments – proteomes of cell organelles. In this review we summarize the current mass spectrometry methods currently being used in plant proteomics and discuss the various tagging strategies that are being used for purification and proteomic analysis of plant protein complexes.

Abbreviations: BCCD, biotin carboxyl carrier protein domain; CBP, calmodulin‐binding protein; CID, collision‐induced dissociation; ESI, electrospray ionization; EST, expressed sequence tag; FT‐ICR, Fourier transform ion cyclotron resonance; GFP, green fluorescent protein; GST, glutathione S‐transferase; HA, haemagglutinin; HILEP, hydroponic isotope labelling of entire plants; His, histidine; HPB, HA–PreScission–Biotin; HPLC, high‐performance liquid chromatography; ICAT, isotope‐coded affinity tags; ICPL, isotope‐coded protein label; iTRAQ, isobaric tag for relative and absolute quantification; LC, liquid chromatography; MALDI, matrix‐assisted laser desorption ionization; MBP, maltose‐binding protein; MS, mass spectrometry; SDS‐PAGE, sodium dodecyl sulphate‐polyacrylamide gel electrophoresis; SILAC, stable isotope labelling with amino acids in cell culture; SILIP, stable isotope labelling in planta; Strep, streptavidin; TAP, tandem affinity purification; TBP, TATA‐box‐binding protein; TOF, time‐of‐flight; UPLC, ultraperformance liquid chromatography     

Recent developments and applications of electron transfer dissociation mass spectrometry in proteomics

Electron transfer dissociation (ETD) has been developed recently as an efficient ion fragmentation technique in mass spectrometry (MS), being presently considered a step forward in proteomics with real perspectives for improvement, upgrade and application. Available also on affordable ion trap mass spectrometers, ETD induces specific N–Cα bond cleavages of the peptide backbone with the preservation of the post-translational modifications and generation of product ions that are diagnostic for the modification site(s). In addition, in the last few years ETD contributed significantly to the development of top-down approaches which enable tandem MS of intact protein ions. The present review, covering the last 5 years highlights concisely the major achievements and the current applications of ETD fragmentation technique in proteomics. An ample part of the review is dedicated to ETD contribution in the elucidation of the most common posttranslational modifications, such as phosphorylation and glycosylation. Further, a brief section is devoted to top-down by ETD method applied to intact proteins. As the last few years have witnessed a major expansion of the microfluidics systems, a few considerations on ETD in combination with chip-based nanoelectrospray (nanoESI) as a platform for high throughput top-down proteomics are also presented.     

Global views of proteasome-mediated degradation by mass spectrometry

Introduction: Degradation of proteins by cellular proteasomes is critical for the fidelity of protein homeostasis and proper cell function. Indeed, perturbations in proteasome function, as well as the degradation of specific substrates, are associated with a variety of human diseases. Yet, monitoring and analyzing protein degradation in a high throughput manner in physiology and pathology remains limited.

Areas covered: Here we discuss several of the recently developed mass spectrometry-based methods for studying proteasome-mediated cellular degradation and discuss their advantages and limitations. We highlight Mass Spectrometry Analysis of Proteolytic Peptides (MAPP), a method designed to purify and identify proteasome-cleaved cellular proteins as a novel approach in molecular and clinical profiling of human disease.

Expert opinion: The recent improvement of proteomics technologies now offers an unprecedented ability to study disease in clinical settings. Expanding clinical studies to include the degradation landscape will provide a new resolution to complement the cellular proteome. In turn, this holds promise to provide both new disease targets and novel peptide biomarkers which will further enhance personalized proteomics.     

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.     

Characterization and Quantification of Intact 26S Proteasome Proteins by Real-Time Measurement of Intrinsic Fluorescence Prior to Top-down Mass Spectrometry

Quantification of gas-phase intact protein ions by mass spectrometry (MS) is impeded by highly-variable ionization, ion transmission, and ion detection efficiencies. Therefore, quantification of proteins using MS-associated techniques is almost exclusively done after proteolysis where peptides serve as proxies for estimating protein abundance. Advances in instrumentation, protein separations, and informatics have made large-scale sequencing of intact proteins using top-down proteomics accessible to the proteomics community; yet quantification of proteins using a top-down workflow has largely been unaddressed. Here we describe a label-free approach to determine the abundance of intact proteins separated by nanoflow liquid chromatography prior to MS analysis by using solution-phase measurements of ultraviolet light-induced intrinsic fluorescence (UV-IF). UV-IF is measured directly at the electrospray interface just prior to the capillary exit where proteins containing at least one tryptophan residue are readily detected. UV-IF quantification was demonstrated using commercially available protein standards and provided more accurate and precise protein quantification than MS ion current. We evaluated the parallel use of UV-IF and top-down tandem MS for quantification and identification of protein subunits and associated proteins from an affinity-purified 26S proteasome sample from Arabidopsis thaliana. We identified 26 unique proteins and quantified 13 tryptophan-containing species. Our analyses discovered previously unidentified N-terminal processing of the β6 (PBF1) and β7 (PBG1) subunit - such processing of PBG1 may generate a heretofore unknown additional protease active site upon cleavage. In addition, our approach permitted the unambiguous identification and quantification both isoforms of the proteasome-associated protein DSS1.     

Deepening our understanding of HDL proteome

ABSTRACT

Introduction: High-density lipoprotein (HDL) particles are heterogeneous and their proteome is complex and distinct from HDL cholesterol. However, it is largely unknown whether HDL proteins are associated with cardiovascular protection.

Areas covered: HDL isolation techniques and proteomic analyses are reviewed. A list of HDL proteins reported in 37 different studies was compiled and the effects of different isolation techniques on proteins attributed to HDL are discussed. Mass spectrometric techniques used for HDL analysis and the need for precise and robust methods for quantification of HDL proteins are discussed.

Expert opinion: Proteins associated with HDL have the potential to be used as biomarkers and/or help to understand HDL functionality. To achieve this, large cohorts must be studied using precise quantification methods. Key factors in HDL proteome quantification are the isolation methodology and the mass spectrometry technique employed. Isolation methodology affects what proteins are identified in HDL and the specificity of association with HDL particles needs to be addressed. Shotgun proteomics yields imprecise quantification, but the majority of HDL studies relied on this approach. Few recent studies used targeted tandem mass spectrometry to quantify HDL proteins, and it is imperative that future studies focus on the application of these precise techniques.     

Application of Mass Spectrometry in Proteomics

Mass spectrometry has arguably become the core technology in proteomics. The application of mass spectrometry based techniques for the qualitative and quantitative analysis of global proteome samples derived from complex mixtures has had a big impact in the understanding of cellular function. Here, we give a brief introduction to principles of mass spectrometry and instrumentation currently used in proteomics experiments. In addition, recent developments in the application of mass spectrometry in proteomics are summarised. Strategies allowing high-throughput identification of proteins from highly complex mixtures include accurate mass measurement of peptides derived from total proteome digests and multidimensional peptide separations coupled with mass spectrometry. Mass spectrometric analysis of intact proteins permits the characterisation of protein isoforms. Recent developments in stable isotope labelling techniques and chemical tagging allow the mass spectrometry based differential display and quantitation of proteins, and newly established affinity procedures enable the targeted characterisation of post-translationally modified proteins. Finally, advances in mass spectrometric imaging allow the gathering of specific information on the local molecular composition, relative abundance and spatial distribution of peptides and proteins in thin tissue sections.     

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.     

Methodologies for Characterizing Phosphoproteins by Mass Spectrometry

Posttranslational regulation of proteins via protein phosphorylation is one of the major means of protein regulation. Phosphorylation is a very rapid and reversible method of changing the function of proteins. Detection of phosphorylated proteins and the identification of phosphorylation sites are necessary to molecularly link specific phosphorylated events with change in phosphoprotein function. Mass Spectrometry (MS) has become the methodology of choice for phosphosite identification. Here we review current approaches including sample separation and enrichment techniques (SDS-PAGE, immunoprecipitation, metal-assisted enrichment, strong cation exchange, dendrimer capture), quantitative MS analysis methods (SILAC, iTRAQ, AQUA), and the application of recently developed methods including electron transfer dissociation ionization and “top-down” proteomics to phosphoprotein analysis.     

ETD fragmentation features improve algorithm

Electron transfer dissociation (ETD) is an alternative technique used in mass spectrometry-based proteomics experiments. Because it is newer, most of the protein identification algorithms for ETD are still a simple derivation of well-established collision-activated dissociation algorithms without the consideration of many unique ETD spectral features. Sridhara and coworkers recently reported removing the charge-reduced precursors and corresponding neutral loss peaks to improve ETD peptide identification with the Open Mass Spectrometry Search Algorithm (OMSSA). These peaks were also used to deduce the charge of the precursors for low resolution data. The scheme is a concrete example of implementing known ETD fragmentation features to improve a protein identification algorithm.