Errata

Abstract

Mass spectrometry (MS)-based proteomics is an unrivaled tool for studying complex biological systems and diseases in the post-genomic era. In recent years, MS has emerged as a powerful structural biological tool to characterize protein conformation and conformational dynamics. The advantages of MS in structural studies are most evident for membrane proteins such as GPCRs (G protein-coupled receptors), where other well-established structural methods such as X-ray crystallography and NMR remain challenging. For proteins with available high-resolution structures, MS-based structural strategies can provide valuable, previously inaccessible information on protein conformational changes and dynamics, protein motion/flexibility, ligand–protein binding, and protein–protein interfaces. In the past several years, we have developed and adapted a number of MS-based structural approaches, such as CDSiL-MS (Conformational changes and Dynamics using Stable-isotope Labeling and MS), CXMS (Crosslinking/MS) and HDXMS (Hydrogen-Deuterium Exchange MS), to study protein structures and conformational dynamics in human β2-adrenegic receptor (β2AR) signaling. In this mini-review, we will highlight several examples demonstrating the power of MS in structural analysis to better elucidate the structural basis of GPCR signaling, particularly through the β-arrestin-mediated GPCR signaling pathway.     

Experimental evaluation of protein identification by an LC/MALDI/on-target digestion approach

Tryptic digestion of proteins continues to be a workhorse of proteomics. Traditional tryptic digestion requires several hours to generate an adequate protein digest. A number of enhanced accelerated digestion protocols have been developed in recent years. Nonetheless, a need still exists for new digestion strategies that meet the demands of proteomics for high-throughput and rapid detection and identification of proteins. We performed an evaluation of direct tryptic digestion of proteins on a MALDI target plate and the potential for integrating RP HPLC separation of protein with on-target tryptic digestion in order to achieve a rapid and effective identification of proteins in complex biological samples. To this end, we used a Tempo HPLC/MALDI target plate deposition hybrid instrument (ABI). The technique was evaluated using a number of soluble and membrane proteins and an MRC5 cell lysate. We demonstrated that direct deposition of proteins on a MALDI target plate after reverse-phase HPLC separation and subsequent tryptic digestion of the proteins on the target followed by MALDI TOF/TOF analysis provided substantial data (intact protein mass, peptide mass and peptide fragment mass) that allowed a rapid and unambiguous identification of proteins. The rapid protein separation and direct deposition of fractions on a MALDI target plate provided by the RP HPLC combined with off-line interfacing with the MALDI MS is a unique platform for rapid protein identification with improved sequence coverage. This simple and robust approach significantly reduces the sample handling and potential loss in large-scale proteomics experiments. This approach allows combination of peptide mass fingerprinting (PMF), MS/MS peptide fragment fingerprinting (PPF) and whole protein MS for both protein identification and structural analysis of proteins.     

Proteomics characterization of protein adsorption onto hemodialysis membranes

Protein-adsorptive properties are a key feature of membranes used for hemodialysis treatment. Protein adsorption is vital to the biocompatibility of a membrane material and influences membrane's performance. The object of the present study is to investigate membrane biocompatibility by correlating the adsorbed proteome repertoire with structural feature of the membrane surfaces. Minidialyzers of identical structural characteristics composed of either cellulose diacetate or ethylenevinyl alcohol materials were employed to develop an ex vivo apparatus to investigate protein adsorption. Adsorbed proteins were eluted by a strong chaotropic buffer condition and investigated by 2-DE coupled to both MALDI-TOF mass spectrometry (MS) mass fingerprinting and fragmentation analysis on a nanoLC-MS/MS hybrid instrument. Membrane surface characterization included evaluation of roughness (atomic force microscopy), elemental chemical composition (X-ray-photoelectron-spectroscopy), and hydrophilicity (pulsed nuclear magnetic resonance). The present study identifies a number of different proteins as common or characteristic of filter material interaction, showing that proteomic techniques are a promising approach for the investigation of proteins surface-adsorbed onto hemodialysis membrane. Proteomic analysis enables the characterization of protein layers of unknown composition.     

Simple But Efficacious Enrichment of Integral Membrane Proteins and Their Interactions for In-Depth Membrane Proteomics

Membrane proteins play essential roles in various cellular processes, such as nutrient transport, bioenergetic processes, cell adhesion, and signal transduction. Proteomics is one of the key approaches to exploring membrane proteins comprehensively. Bottom–up proteomics using LC–MS/MS has been widely used in membrane proteomics. However, the low abundance and hydrophobic features of membrane proteins, especially integral membrane proteins, make it difficult to handle the proteins and are the bottleneck for identification by LC–MS/MS. Herein, to improve the identification and quantification of membrane proteins, we have stepwisely evaluated methods of membrane enrichment for the sample preparation. The enrichment methods of membranes consisted of precipitation by ultracentrifugation and treatment by urea or alkaline solutions. The best enrichment method in the study, washing with urea after isolation of the membranes, resulted in the identification of almost twice as many membrane proteins compared with samples without the enrichment. Notably, the method significantly enhances the identified numbers of multispanning transmembrane proteins, such as solute carrier transporters, ABC transporters, and G-protein–coupled receptors, by almost sixfold. Using this method, we revealed the profiles of amino acid transport systems with the validation by functional assays and found more protein–protein interactions, including membrane protein complexes and clusters. Our protocol uses standard procedures in biochemistry, but the method was efficient for the in-depth analysis of membrane proteome in a wide range of samples.     

Applications of sequence coevolution in membrane protein biochemistry

Recently, protein sequence coevolution analysis has matured into a predictive powerhouse for protein structure and function. Direct methods, which use global statistical models of sequence coevolution, have enabled the prediction of membrane and disordered protein structures, protein complex architectures, and the functional effects of mutations in proteins. The field of membrane protein biochemistry and structural biology has embraced these computational techniques, which provide functional and structural information in an otherwise experimentally-challenging field. Here we review recent applications of protein sequence coevolution analysis to membrane protein structure and function and highlight the promising directions and future obstacles in these fields. We provide insights and guidelines for membrane protein biochemists who wish to apply sequence coevolution analysis to a given experimental system.     

Solution NMR study of integral membrane proteins

Signals between a cell and its environment are often transmitted through membrane proteins; therefore, many membrane proteins, including G protein-coupled receptors (GPCRs) and ion channels, are important drug targets. Structural information about membrane proteins remains limited owing to challenges in protein expression, purification and the selection of membrane-mimicking systems that will retain protein structure and function. This review describes recent advances in solution NMR applied to the structural study of integral membrane proteins. The examples herein demonstrate that solution NMR spectroscopy will play a unique role not only in structural analysis, but also drug discovery of membrane proteins.     

Membrane proteins in their native habitat as seen by solid-state NMR spectroscopy

Membrane proteins play many critical roles in cells, mediating flow of material and information across cell membranes. They have evolved to perform these functions in the environment of a cell membrane, whose physicochemical properties are often different from those of common cell membrane mimetics used for structure determination. As a result, membrane proteins are difficult to study by traditional methods of structural biology, and they are significantly underrepresented in the protein structure databank. Solid-state Nuclear Magnetic Resonance (SSNMR) has long been considered as an attractive alternative because it allows for studies of membrane proteins in both native-like membranes composed of synthetic lipids and in cell membranes. Over the past decade, SSNMR has been rapidly developing into a major structural method, and a growing number of membrane protein structures obtained by this technique highlights its potential. Here we discuss membrane protein sample requirements, review recent progress in SSNMR methodologies, and describe recent advances in characterizing membrane proteins in the environment of a cellular membrane.     

Recent technological developments for native mass spectrometry

Native mass spectrometry (MS), the analysis of proteins and protein complexes from solutions that stabilize native solution structures, is a rapidly expanding area. There is strong evidence supporting the retention of proteins' native folds in the absence of solvent under the experimental timescales of MS experiments. Therefore, instrumentation has been developed to use gas-phase native-like protein ions to exploit the speed, sensitivity, and selectivity of mass spectrometry approaches to solve emerging problems in structural biology. This article reviews some of the recent advances and applications in gas-phase instrumentation for structural proteomics.     

Liquid chromatography MALDI MS/MS for membrane proteome analysis

Membrane proteins play critical roles in many biological functions and are often the molecular targets for drug discovery. However, their analysis presents a special challenge largely due to their highly hydrophobic nature. We present a surfactant-aided shotgun proteomics approach for membrane proteome analysis. In this approach, membrane proteins were solubilized and digested in the presence of SDS followed by newly developed auto-offline liquid chromatography/matrix-assisted laser desorption ionization (LC/MALDI) tandem MS analysis. Because of high tolerance of MALDI to SDS, one-dimensional (1D) LC separation can be combined with MALDI for direct analysis of protein digests containing SDS, without the need for extensive sample cleanup. In addition, the heated droplet interface used in LC/MALDI can work with high flow LC separations, allowing a relatively large amount of protein digest to be used for 1D LC/MALDI which facilitates the detection of low abundance proteins. The proteome identification results obtained by LC/MALDI are compared to the gel electrophoresis/MS method as well as the shotgun proteomics method using 2D LC/electrospray ionization MS. It is demonstrated that, while LC/MALDI provides more extensive proteome coverage compared to the other two methods, these three methods are complementary to each other and a combination of these methods should provide a more comprehensive membrane proteome analysis.     

Optimised two-dimensional electrophoresis procedures for the protein characterisation of structural tissues

The protein analysis of structural tissues is typically highly problematic. Amniotic membrane displays unique wound healing and anti-scarring properties; however, little is known concerning its active protein content. The structural nature of amniotic membrane necessitated development and extensive optimisation of the entire two-dimensional (2-D) workflow. Proteins were extracted using powerful solubilisation buffers and analysis carried out using 2-D electrophoresis followed by mass spectrometry (MS) identification. Preservation and processing resulted in prefractionation of soluble from structural and membrane-associated proteins. Enhanced protein solubility was achieved by cysteine blocking using both N,N-dimethylacrylamide (DMA) alkylation and bis(2-hydroxyethyl) disulphide (HED); an alternative procedure for the effective application of HED is demonstrated. The benefits of precipitation and cup-loading versus in-gel rehydration were also assessed, with procedures for the employment of HED with the latter described. Following optimisation, a representative sample 21 proteins were identified from amniotic membrane using MS verify procedures were MS-compatible. Our results demonstrate that techniques for the reproducible separation of proteins from a proteinaceous structural tissue have been optimised. Briefly, proteins are extracted using a thiourea/urea extraction buffer containing carrier ampholytes, dithiothreitol (DTT), and 3-(cyclohexylamino)-1-propanesulfonic acid (CHAPS). After DMA alkylation, proteins were precipitated (using the 2-D clean-up kit from Amersham Biosciences) and resolubilised in extraction buffer containing a lower concentration of DTT. Samples were either cup-loaded onto rehydrated HED-containing strips or rebuffered into HED-containing buffer followed by in-gel rehydration.     

Comprehensive proteomic analysis of membrane proteins in Toxoplasma gondii

Toxoplasma gondii (T. gondii) is an obligate intracellular protozoan parasite that is an important human and animal pathogen. Experimental information on T. gondii membrane proteins is limited, and the majority of gene predictions with predicted transmembrane motifs are of unknown function. A systematic analysis of the membrane proteome of T. gondii is important not only for understanding this parasite's invasion mechanism(s), but also for the discovery of potential drug targets and new preventative and therapeutic strategies. Here we report a comprehensive analysis of the membrane proteome of T. gondii, employing three proteomics strategies: one-dimensional gel liquid chromatography-tandem MS analysis (one-dimensional gel electrophoresis LC-MS/MS), biotin labeling in conjunction with one-dimensional gel LC-MS/MS analysis, and a novel strategy that combines three-layer "sandwich" gel electrophoresis with multidimensional protein identification technology. A total of 2241 T. gondii proteins with at least one predicted transmembrane segment were identified and grouped into 841 sequentially nonredundant protein clusters, which account for 21.8% of the predicted transmembrane protein clusters in the T. gondii genome. A large portion (42%) of the identified T. gondii membrane proteins are hypothetical proteins. Furthermore, many of the membrane proteins validated by mass spectrometry are unique to T. gondii or to the Apicomplexa, providing a set of gene predictions ripe for experimental investigation, and potentially suitable targets for the development of therapeutic strategies.     

Mass spectrometric analysis of integral membrane proteins: application to complete mapping of bacteriorhodopsins and rhodopsin.

Integral membrane proteins have not been readily amenable to the general methods developed for mass spectrometric (or internal Edman degradation) analysis of soluble proteins. We present here a sample preparation method and high performance liquid chromatography (HPLC) separation system which permits online HPLC-electrospray ionization mass spectrometry (ESI-MS) and -tandem mass spectrometry (MS/MS) analysis of cyanogen bromide cleavage fragments of integral membrane proteins. This method has been applied to wild type (WT) bacteriorhodopsin (bR), cysteine containing mutants of bR, and the prototypical G-protein coupled receptor, rhodopsin (Rh). In the described method, the protein is reduced and the cysteine residues pyridylethylated prior to separating the protein from the membrane. Following delipidation, the pyridylethylated protein is cleaved with cyanogen bromide. The cleavage fragments are separated by reversed phase HPLC using an isopropanol/acetonitrile/aqueous TFA solvent system and the effluent peptides analyzed online with a Finnigan LCQ Ion Trap Mass Spectrometer. With the exception of single amino acid fragments and the glycosylated fragment of Rh, which is observable by matrix assisted laser desorption ionization (MALDI)-MS, this system permits analysis of the entire protein in a single HPLC run. This methodology will enable pursuit of chemical modification and crosslinking studies designed to probe the three dimensional structures and functional conformational changes in these proteins. The approach should also be generally applicable to analysis of other integral membrane proteins.     

低温电子显微技术在膜蛋白结构研究中的应用和展望

介绍了蛋白质电子晶体学和单颗粒分析技术这两种低温电子显微技术在膜蛋白和膜蛋白复合体结构研究中的具体方法和近10~20年来的实际应用,并分别分析了这两种方法的优势和瓶颈。此外,还介绍了Amphipol替代、Streptavidin二维晶体锚定脂质体和纳米球包被脂质体等近两年来出现的新的用于低温电镜成像的膜蛋白样品制备方法。最后对膜蛋白的低温电子显微研究的未来发展做了展望。     

膜蛋白结晶方法学研究进展

膜蛋白执行着物质运输、能量转换和信号转导等重要生物学功能,其分子的三维结构解析对阐述其功能及开展理性药物设计有着十分重要的意义．目前膜蛋白结构解析以X射线单晶衍射技术为主,该技术需要高质量晶体作为衍射对象．然而由于膜蛋白具有两亲性,难以得到高度有序的三维晶体,进而导致其结构解析十分困难．针对此问题,研究者们发展了一些专门面向膜蛋白的结晶方法,如基于去垢剂的方法,基于脂类的方法等．本文回顾了这些方法,并对未来膜蛋白的结晶研究进行了展望．     

Structural Characterization of Carbohydrates by Fourier Transform Tandem Mass Spectrometry

Fourier transform tandem mass spectrometry (MS/MS) provides high mass accuracy, high sensitivity, and analytical versatility and has therefore emerged as an indispensable tool for structural elucidation of biomolecules. Glycosylation is one of the most common posttranslational modifications, occurring in ~50% of proteins. However, due to the structural diversity of carbohydrates, arising from non-template driven biosynthesis, achievement of detailed structural insight is highly challenging. This review briefly discusses carbohydrate sample preparation and ionization methods, and highlights recent developments in alternative high-resolution MS/MS strategies, including infrared multiphoton dissociation (IRMPD), electron capture dissociation (ECD), and electron detachment dissociation (EDD), for carbohydrates with a focus on glycans and proteoglycans from mammalian glycoproteins.     

Protein glycosylation analysis by liquid chromatography-mass spectrometry

Liquid chromatography (LC)-mass spectrometry (MS) has developed into an invaluable technology for the analysis of protein glycosylation. This review focuses on the recent developments in LC and combinations thereof with MS for this field of research. Recently introduced methods for the structural analysis of released glycans (native or derivatised) as well as glycopeptides, on normal phase, reverse phase and graphitized carbon LC columns with online MS(/MS) will be reviewed. Performed on nano-scale or capillary-scale, these LC-MS methods operate at femtomole sensitivity and support the further integration of glycosylation analysis in proteomics methodology.     

Exploring the membrane proteome—Challenges and analytical strategies

The analysis of proteins in biological membranes forms a major challenge in proteomics. Despite continuous improvements and the development of more sensitive analytical methods, the analysis of membrane proteins has always been hampered by their hydrophobic properties and relatively low abundance. In this review, we describe recent successful strategies that have led to in-depth analyses of the membrane proteome. To facilitate membrane proteome analysis, it is essential that biochemical enrichment procedures are combined with special analytical workflows that are all optimized to cope with hydrophobic polypeptides. These include techniques for protein solubilization, and also well-matched developments in protein separation and protein digestion procedures. Finally, we discuss approaches to target membrane–protein complexes and lipid–protein interactions, as such approaches offer unique insights into function and architecture of cellular membranes.     

An in vitro enzymatic assay coupled to proteomics analysis reveals a new DNA processing activity for Ewing sarcoma and TAF(II)68 proteins

Based on structural and functional similarities, translocated in liposarcoma/fusion (TLS/FUS) protein, Ewing sarcoma (EWS) protein and human TATA binding protein-associated factor (hTAF(II)68) have been grouped in the TLS-EWS-TAF(II)68 (TET) protein family. Translocations involving their genes lead to sarcomas. Polypyrimidine tract-binding protein-associated splicing factor (PSF), although not grouped in this family, presents structural and functional similarities with TET proteins and is involved in translocation leading to carcinoma. Beside their role in RNA metabolism, the precise cellular functions of these multifunctional proteins are not yet fully elucidated. We previously showed that both TLS/FUS and PSF display activities able to pair homologous DNA on membrane in an in vitro assay. In the present study, we address the question whether EWS and hTAF(II)68 also display pairing on membrane activities, and to a larger extent whether other proteins also exhibit such activity. We applied the pairing on membrane assay to 2-DE coupled to MS analysis for a global screening of DNA pairing on membrane activities. In addition to TLS/FUS and PSF, this test allowed us to identify EWS and hTAF(II)68, but no other proteins, indicating a feature specific to a protein family whose members share extensive structural similarities. This common activity suggests a role for TET proteins and PSF in genome plasticity control.     

Analysis of the synaptic vesicle proteome using three gel-based protein separation techniques

Synaptic vesicles are key organelles in neurotransmission. Their functions are governed by a unique set of integral and peripherally associated proteins. To obtain a complete protein inventory, we immunoisolated synaptic vesicles from rat brain to high purity and performed a gel-based analysis of the synaptic vesicle proteome. Since the high hydrophobicity of integral membrane proteins hampers their resolution by gel electrophoretic techniques, we applied in parallel three different gel electrophoretic methods for protein separation prior to MS. Synaptic vesicle proteins were subjected to either 1-D SDS-PAGE along with nano-LC ESI-MS/MS or to the 2-D gel electrophoretic techniques benzyldimethyl-n-hexadecylammonium chloride (BAC)/SDS-PAGE, and double SDS (dSDS)-PAGE in combination with MALDI-TOF-MS. We demonstrate that the combination of all three methods provides a comprehensive survey of the proteinaceous inventory of the synaptic vesicle membrane compartment. The identified synaptic vesicle proteins include transporters, soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs), synapsins, rab and rab-interacting proteins, additional guanine nucleotide triphosphate (GTP) binding proteins, cytoskeletal proteins, and proteins modulating synaptic vesicle exo- and endocytosis. In addition, we identified novel proteins of unknown function. Our results demonstrate that the parallel application of three different gel-based approaches in combination with mass spectrometry permits a comprehensive analysis of the synaptic vesicle proteome that is considerably more complex than previously anticipated.