High‐Throughput Proteomics Enabled by a Photocleavable Surfactant

Mass spectrometry (MS)‐based proteomics provides unprecedented opportunities for understanding the structure and function of proteins in complex biological systems; however, protein solubility and sample preparation before MS remain a bottleneck preventing high‐throughput proteomics. Herein, we report a high‐throughput bottom‐up proteomic method enabled by a newly developed MS‐compatible photocleavable surfactant, 4‐hexylphenylazosulfonate (Azo) that facilitates robust protein extraction, rapid enzymatic digestion (30 min compared to overnight), and subsequent MS‐analysis following UV degradation. Moreover, we developed an Azo‐aided bottom‐up method for analysis of integral membrane proteins, which are key drug targets and are generally underrepresented in global proteomic studies. Furthermore, we demonstrated the ability of Azo to serve as an “all‐in‐one” MS‐compatible surfactant for both top‐down and bottom‐up proteomics, with streamlined workflows for high‐throughput proteomics amenable to clinical applications.     

高通量蛋白质组学分析研究进展

基于质谱的蛋白质组学技术已经日趋成熟,可以对细胞和组织中的成千上万种蛋白质进行全面的定性和定量分析,逐步实现“深度覆盖”。随着生物医学日益增长的大队列蛋白质组学分析需求,如何在保持较为理想的覆盖深度下实现短时间、快速的“高通量”蛋白质组学分析已成为当前亟需解决的关键问题之一。常规的蛋白质组学分析流程通常包括样品前处理、色谱分离、质谱检测和数据分析。该文从以上4个方面展开介绍近10年以来高通量蛋白质组学分析技术取得的一系列研究进展,主要包括:(1)基于高通量、自动化移液工作站的蛋白质组样品前处理方法;(2)基于微升流速液相色谱与质谱联用的高通量蛋白质组检测方法;(3)利用灵敏度高、扫描速度快的质谱仪实现短色谱梯度分离下蛋白质组深度覆盖的分析方法;(4)基于人工智能、深度神经网络、机器学习等的蛋白质组学大数据分析方法。此外,对高通量蛋白质组学面临的挑战及其发展进行展望。总而言之,预期在不久的将来高通量蛋白质组学技术将会逐步“落地转化”,成为大队列蛋白质组学分析的利器。     

Interrogation of MS/MS search data with an pI Filter algorithm to increase protein identification success

In high-throughput proteomics, the bottom-up approach has become a widely used method for the identification of proteins that is based on tryptic peptide MS/MS analysis. Separation methodologies that use IEF of tryptic peptides have recently been introduced and provide an extra dimension of peptide separation. In addition to its great fractionation capability, tryptic peptide prefractionation by IEF can also increase the protein identification success. The pI information of the peptide gained can be successfully used in a post-database search filtering step. We introduce a filtering algorithm that is based on the comparison of the experimental and theoretical pI's to validate peptide identifications by MS/MS data search engines.     

毛细管电泳-质谱联用技术及其在蛋白质组学中的应用

蛋白质组学研究在生物学、精准医学等方面发挥着重要的作用。然而研究面临的巨大挑战来自生物样品的复杂性,因此在质谱（MS）鉴定技术不断革新的同时,发展分离技术以降低样品复杂度尤为重要。毛细管电泳（CE）技术具有上样体积小、分离效率高、分离速度快等优势,其与质谱的联用在蛋白质组学研究中越来越受到关注。低流速鞘流液和无鞘流液接口的发展及商品化推动了CE-MS技术的发展。目前毛细管区带电泳（CZE）、毛细管等电聚焦（CIEF）、毛细管电色谱（CEC）等分离模式已与质谱联用,其中CZE-MS应用最广泛。目前被广泛采用的蛋白质组学研究策略主要是基于酶解肽段分离鉴定的"自下而上（bottom-up）"策略。首先,CE-MS技术对酶解肽段的检测灵敏度高达1 zmol,已成功应用于单细胞蛋白质组学;其次,毛细管电泳技术与反相液相色谱互补,为疏水性质相近的肽段（尤其是翻译后修饰肽段）的分离鉴定提供了新的途径。基于整体蛋白质分离鉴定的自上而下"top-down"策略可以直接获得更精准、更完整的蛋白质信息。CE技术在蛋白质大分子的分离方面具有分离效率高、回收率高的优势,其与质谱的联用提高了整体蛋白质的鉴定灵敏度和覆盖度。非变性质谱（native MS）是一种在近生理条件下从完整蛋白质复合物水平上进行分析的质谱技术。CE与非变性质谱联用已被尝试用于蛋白质复合体的分离鉴定。该文引用了与CE-MS和蛋白质组学应用相关的93篇文献,综述了以上介绍的CE-MS的研究进展以及在蛋白质组学分析中的应用优势,并总结和展望了其应用前景。     

Applications of Tandem Mass Spectrometry (MS/MS) in Protein Analysis for Biomedical Research

Mass Spectrometry (MS) allows the analysis of proteins and peptides through a variety of methods, such as Electrospray Ionization-Mass Spectrometry (ESI-MS) or Matrix-Assisted Laser Desorption Ionization-Mass Spectrometry (MALDI-MS). These methods allow identification of the mass of a protein or a peptide as intact molecules or the identification of a protein through peptide-mass fingerprinting generated upon enzymatic digestion. Tandem mass spectrometry (MS/MS) allows the fragmentation of proteins and peptides to determine the amino acid sequence of proteins (top-down and middle-down proteomics) and peptides (bottom-up proteomics). Furthermore, tandem mass spectrometry also allows the identification of post-translational modifications (PTMs) of proteins and peptides. Here, we discuss the application of MS/MS in biomedical research, indicating specific examples for the identification of proteins or peptides and their PTMs as relevant biomarkers for diagnostic and therapy.     

Mass spectrometry in biodefense

Potential agents for biological attacks include both microorganisms and toxins. In mass spectrometry (MS), rapid identification of potential bioagents is achieved by detecting the masses of unique biomarkers, correlated to each agent. Currently, proteins are the most reliable biomarkers for detection and characterization of both microorganisms and toxins, and MS-based proteomics is particularly well suited for biodefense applications. Confident identification of an organism can be achieved by top-down proteomics following identification of individual protein biomarkers from their tandem mass spectra. In bottom-up proteomics, rapid digestion of intact protein biomarkers is again followed by MS/MS to provide unambiguous bioagent identification and characterization. Bioinformatics obviates the need for culturing and rigorous control of experimental variables to create and use MS fingerprint libraries for various classes of bioweapons. For specific applications, MS methods, instruments and algorithms have also been developed for identification based on biomarkers other than proteins and peptides.     

An integrated digital microfluidic chip for multiplexed proteomic sample preparation and analysis by MALDI-MS

To realize multiplexed sample preparation on a digital microfluidic chip for high-throughput Matrix Assisted Laser Desorption/Ionization Mass Spectrometry (MALDI-MS), several fluidic functions need to be integrated. These include the generation of multiple droplets from a reservoir and parallel in-line sample purification. In this paper, we develop two critical new functions in handling protein solutions and standard proteomic reagents with electrowetting-on-dielectric (EWOD) actuation, leading to an integrated chip for multiplexed sample preparation for MALDI-MS. The first is a voltage sequence designed to generate a series of droplets from each of the three reservoirs--proteomic sample, rinsing fluid, and MALDI reagents. It is the first time that proteomic reagents have been dispensed using EWOD in an air (as opposed to oil) environment. The second is a box-in-box electrode pattern developed to allow droplet passing over dried sample spots, making the process of in-line sample purification robust for parallel processing. As a result, parallel processing of multiple sample droplets is demonstrated on the integrated EWOD-MALDI-MS chip, an important step towards high-throughput MALDI-MS. The MS results, collected directly from the integrated devices, are of good quality, suggesting that the tedious process of sample preparation can be automated on-chip for MALDI-MS applications as well as other high-throughput proteomics applications.     

Monolith-based immobilized enzyme reactors: recent developments and applications for proteome analysis

In the current proteome study, protein digestion is indispensable before proteins could be identified by MS/MS, no matter based on top-down or bottom-up strategies. Compared to the traditional digestion performed in free solution, immobilized enzyme shows great advantages in digestion speed, stability, and longevity, especially with monolithic materials as the supports. Besides the improved digestion capacity, the immobilized enzyme reactors (IMERs) could be further coupled with the separation and detection systems, enabling high-throughput protein identification. In this paper, the latest advances in the monolith-based IMERs and their applications in proteomic study are briefly summarized. By reviewing these achievements, it could be seen that monilith-based IMERs have very bright future in proteome analysis.     

基于质谱的单细胞蛋白质组学分析方法及应用

细胞是生命体的最小组成单位,遗传及外部环境等因素使单细胞异质性广泛存在于众多生物体中。传统的生物学实验获得的结果多是大量细胞的平均测量值,因此在单细胞层面开展研究对于精确理解细胞的生长发育以及疾病的诊断与治疗至关重要。而作为重要的细胞和生命活动的执行者,蛋白质由于其不具备扩增特性,且种类繁多、丰度低、动态分布范围宽,与核酸等其他生物大分子相比,其单细胞组学研究相对滞后。而在所有的检测手段中,荧光检测以及电化学分析方法具有极高的灵敏度,但是囿于其研究通量有限,以及电化学活性依赖,很难成为普适性的单细胞蛋白质组学研究方法。质谱分析作为传统蛋白质组学中最为核心的研究技术,由于其高灵敏、高通量、结构信息丰富等特点,在单细胞蛋白质组学研究中独树一帜。该文综述了近年来基于质谱的单细胞蛋白质组学研究中的代表性方法,根据质谱分析前蛋白质分离方式的差异,将其分为基于毛细管电泳分离、液相色谱分离和无分离手段的直接检测3类方法,在介绍研究现状的同时对这些方法在细胞通量、蛋白质鉴定数目、灵敏度以及方法应用方面进行了总结与比较。最后,基于目前研究中面临的挑战以及发展趋势对基于质谱的单细胞蛋白质组学的研究前景进行了展望。     

The comparison between 2DE-MS and bottom-up LC-MS demands high-end techniques for both technologies

In contrast to bottom-up LC-MS only 2DE-MS can separate and detect a huge number of human protein species. Kwiatkowski et al. (in this issue) established parameters to estimate the amount of protein speciation for each human protein. Proteins identified in 2DE-MS approaches showed more protein speciation than in bottom-up LC-MS. The authors state that protein speciation is likely to increase the chance of proteins to be determined in 2-DE/MS, though admitting that low-sensitivity 2DE-MS methods were used in this study. In agreement with Kwiatkowski et al., we are convinced that the difference between 2DE-MS and bottom-up LC-MS will disappear, if high-resolution 2DE is combined with identification by a high-sensitivity LC-Orbitrap-MS. Meta-analysis of proteomic data is surely a promising tool, though the technological progress in 2DE and MS has to reach a plateau to enable useful comparisons.     

Intact protein separation by chromatographic and/or electrophoretic techniques for top-down proteomics

Mass spectrometry used in combination with a wide variety of separation methods is the principal methodology for proteomics. In bottom-up approach, proteins are cleaved with a specific proteolytic enzyme, followed by peptide separation and MS identification. In top-down approach intact proteins are introduced into the mass spectrometer. The ions generated by electrospray ionization are then subjected to gas-phase separation, fragmentation, fragment separation, and automated interpretation of mass spectrometric and chromatographic data yielding both the molecular weight of the intact protein and the protein fragmentation pattern. This approach requires high accuracy mass measurement analysers capable of separating the multi-charged isotopic cluster of proteins, such as hybrid ion trap-Fourier transform instruments (LTQ-FTICR, LTQ-Orbitrap). Front-end separation technologies tailored for proteins are of primary importance to implement top-down proteomics. This review intends to provide the state of art of protein chromatographic and electrophoretic separation methods suitable for MS coupling, and to illustrate both monodimensional and multidimensional approaches used for LC-MS top-down proteomics. In addition, some recent progresses in protein chromatography that may provide an alternative to those currently employed are also discussed.     

Analysis of membrane and hydrophilic proteins simultaneously derived from the mouse brain using cloud-point extraction

In this study, a temperature-induced phase fractionation known as cloud-point extraction (CPE) with the non-ionic surfactant Triton X-114 was used to simultaneously extract, concentrate, and fractionate hydrophobic and hydrophilic proteins from mouse brain tissue. Two bottom-up proteomic techniques were used to comprehensively identify the extracted proteins. The first “shotgun”-based approach included tryptic digestion of the proteins followed by reversed-phase nanoliquid chromatography (RP-nanoLC) in combination with electrospray ionization (ESI) tandem mass spectrometry (MS/MS). In the second approach, the extracted intact proteins were first separated by one-dimensional (1D) gel electrophoresis and then in-gel digested with trypsin and analyzed with nanoLC-MS/MS. In total, 1,825 proteins were unambiguously identified and the percentage of membrane proteins was 26% which is at the reported genome expression levels of 20–30%. The protein overlap between the two approaches was high. The majority (77%) of the identifications in the first approach was also found by the second method. The protein overlap between the CPE-extracted hydrophilic and hydrophobic fractions was rather small (16–23%) for both methods, which indicates a good phase separation. A quantitative evaluation of the CPE with iTRAQ labeling and nanoLC-ESI-MS/MS analysis gave iTRAQ ratios at the expected levels and an overall variation of the entire method at 17–31%. The results indicate very reproducible sample preparation and analysis methods that readily can be applied on large-scale sample sets.     

Fully automated chip-based nanoelectrospray combined with electron transfer dissociation for high throughput top-down proteomics

The conventional protocol for protein identification by electrospray ionization mass spectrometry (MS) is based on enzymatic digestion which renders peptides to be analyzed by liquid chromatography-MS and collision-induced dissociation (CID) multistage MS, in the so-called bottom-up approach. Though this method has brought a significant progress to the field, many limitations, among which, the low throughput and impossibility to characterize in detail posttranslational modifications in terms of site(s) and structure, were reported. Therefore, the research is presently focused on the development of procedures for efficient top-down fragmentation of intact protein ions. In this context, we developed here an approach combining fully automated chip-based-nanoelectrospray ionisation (nanoESI), performed on a NanoMate robot, with electron transfer dissociation (ETD) for peptide and top-down protein sequencing and identification. This advanced analytical platform, integrating robotics, microfluidics technology, ETD and alternate ETD/CID, was tested and found ideally suitable for structural investigation of peptides and modified/functionalized peptides as well as for top-down analysis of medium size proteins by tandem MS experiments of significantly increased throughput and sensitivity. The obtained results indicate that NanoMate-ETD and ETD/CID may represent a viable alternative to the current MS strategies, with potential to develop into a method of routine use for high throughput top-down proteomics.     

Development of an integrated approach for evaluation of 2-D gel image analysis: impact of multiple proteins in single spots on comparative proteomics in conventional 2-D gel/MALDI workflow

With 2-D gel mapping, it is often observed that essentially identical proteins migrate to different positions in the gel, while some seemingly well-resolved protein spots consist of multiple proteins. These observations can undermine the validity of gel-based comparative proteomic studies. Through a comparison of protein identifications using direct MALDI-TOF/TOF and LC-ESI-MS/MS analyses of 2-D gel separated proteins from cauliflower florets, we have developed an integrated approach to improve the accuracy and reliability of comparative 2-D electrophoresis. From 46 spots of interest, we identified 51 proteins by MALDI-TOF/TOF analysis and 108 proteins by LC-ESI-MS/MS. The results indicate that 75% of the analyzed spots contained multiple proteins. A comparison of hit rank for protein identifications showed that 37 out of 43 spots identified by MALDI matched the top-ranked hit from the ESI-MS/MS. By using the exponentially modified protein abundance index (emPAI) to determine the abundance of the individual component proteins for the spots containing multiple proteins, we found that the top-hit proteins from 40 out of 43 spots identified by MALDI matched the most abundant proteins determined by LC-MS/MS. Furthermore, our 2-D-GeLC-MS/MS results show that the top-hit proteins in 44 identified spots contributed on average 81% of the spots' staining intensity. This is the first quantitative measurement of the average rate of false assignment for direct MALDI analysis of 2-D gel spots using a new integrated workflow (2-D gel imaging, "2-D GeLC-MS/MS", and emPAI analysis). Here, the new approach is proposed as an alternative to traditional gel-based quantitative proteomics studies.     

Overview of software options for processing,analysis and interpretation of mass spectrometric proteomic data

Recently, the interests in proteomics have been intensively increased, and the proteomic methods have been widely applied to many problems in cell biology. If the age of 1990s is considered to be a decade of genomics, we can claim that the following years of the new century is a decade of proteomics. The rapid evolution of proteomics has continued through these years, with a series of innovations in separation techniques and the core technologies of two‐dimensional gel electrophoresis and MS. Both technologies are fueled by automation and high throughput computation for profiling of proteins from biological systems. As Patterson ever mentioned, ‘data analysis is the Achilles heel of proteomics and our ability to generate data now outstrips our ability to analyze it’. The development of automatic and high throughput technologies for rapid identification of proteins is essential for large‐scale proteome projects and automatic protein identification and characterization is essential for high throughput proteomics. This review provides a snap shot of the tools and applications that are available for mass spectrometric high throughput biocomputation. The review starts with a brief introduction of proteomics and MS. Computational tools that can be employed at various stages of analysis are presented, including that for data processing, identification, quantification, and the understanding of the biological functions of individual proteins and their dynamic interactions. The challenges of computation software development and its future trends in MS‐based proteomics have also been speculated. Copyright © 2014 John Wiley & Sons, Ltd.     

基于毛细管电泳技术的高灵敏度蛋白质组学技术发展

近年来,蛋白质组学技术在样品前处理、分离技术和质谱检测技术方面获得了快速发展,已经可以实现在几小时内对上万种蛋白的同时定性和定量分析。然而,目前的主流蛋白质组学技术仍无法满足极微量生物样品,尤其是单细胞样品的组学分析需求。毛细管电泳分离技术具有峰宽窄、柱效高、样品用量少等优势,是与高分辨质谱在线联用的理想选择之一。该文评述了集成化和在线样品前处理以及主流的纳升液相色谱-质谱联用系统在高灵敏度蛋白质组学分析领域的发展现状和挑战,认为该领域的重要技术挑战之一在于目前的纳升液相色谱分离已经无法完全匹配现代高分辨质谱超过40 Hz的超高扫描速度,从而导致质谱使用效率的降低。针对上述技术挑战,该文重点探讨了毛细管电泳-质谱联用技术的独特技术优势和潜在发展机遇,主要包括：（1）面向微量酶解多肽样品的高柱效毛细管电泳分离。通过采用毛细管电色谱可以进一步改善毛细管电泳柱容量不足的局限;（2）面向高灵敏度分析的无鞘液/鞘液接口开发;（3）高效毛细管电泳分离与高扫描速度质谱检测的协同化使用。总之,我们预期毛细管电泳-质谱联用技术的进一步发展有望在针对单细胞等超微量生物学样品的蛋白质组学分析中获得更广泛的应用。     

A protein extraction method for low protein concentration solutions compatible with the proteomic analysis of rubber particles

The extraction of high‐purity proteins from the washing solution (WS) of rubber particles (also termed latex‐producing organelles) from laticifer cells in rubber tree for proteomic analysis is challenging due to the low concentration of proteins in the WS. Recent studies have revealed that proteins in the WS might play crucial roles in natural rubber biosynthesis. To further examine the involvement of these proteins in natural rubber biosynthesis, we designed an efficiency method to extract high‐purity WS proteins. We improved our current borax and phenol‐based method by adding reextraction steps with phenol (REP) to improve the yield from low protein concentration samples. With this new method, we extracted WS proteins that were suitable for proteomics. Indeed, compared to the original borax and phenol‐based method, the REP method improved both the quality and quantity of isolated proteins. By repeatedly extracting from low protein concentration solutions using the same small amount of phenol, the REP method yielded enough protein of sufficiently high‐quality from starting samples containing less than 0.02 mg of proteins per milliliter. This method was successfully applied to extract the rubber particle proteins from the WS of natural rubber latex samples. The REP‐extracted WS proteins were resolved by 2DE, and 28 proteins were positively identified by MS. This method has the potential to become widely used for the extraction of proteins from low protein concentration solutions for proteomic analysis.     

Rapid 3-dimensional shape determination of globular proteins by mobility capillary electrophoresis and native mass spectrometry

Established high-throughput proteomics methods provide limited information on the stereostructures of proteins. Traditional technologies for protein structure determination typically require laborious steps and cannot be performed in a high-throughput fashion. Here, we report a new medium throughput method by combining mobility capillary electrophoresis (MCE) and native mass spectrometry (MS) for the 3-dimensional (3D) shape determination of globular proteins in the liquid phase, which provides both the geometric structure and molecular mass information of proteins. A theory was established to correlate the ion hydrodynamic radius and charge state distribution in the native mass spectrum with protein geometrical parameters, through which a low-resolution structure (shape) of the protein could be determined. Our test data of 11 different globular proteins showed that this approach allows us to determine the shapes of individual proteins, protein complexes and proteins in a mixture, and to monitor protein conformational changes. Besides providing complementary protein structure information and having mixture analysis capability, this MCE and native MS based method is fast in speed and low in sample consumption, making it potentially applicable in top–down proteomics and structural biology for intact globular protein or protein complex analysis.

Using native mass spectrometry and mobility capillary electrophoresis, the ellipsoid dimensions of globular proteins or protein complexes could be measured efficiently.     

Edman降解中异硫氰酸苯酯新修饰位点的发现和验证

Edman降解是最早建立的一种用于多肽和蛋白质氨基端测序的方法,该方法现在仍被广泛用于生物化学领域。随着高通量蛋白质组学技术的发展和应用,该方法中的异硫氰酸苯酯反应被用于修饰蛋白质氨基端,并用于检测蛋白质水解位点。但还没有异硫氰酸苯酯是否可以修饰其他氨基酸侧链并影响多肽序列分析的研究。为了探究其修饰其他氨基酸的可能性,本文利用基质辅助激光解吸电离飞行时间质谱（MALDI-TOF-MS）和液相色谱-串联质谱（LC-MS/MS）研究了异硫氰酸苯酯对一个模型肽的化学修饰。质谱数据解析后发现在高浓度异硫氰酸苯酯的反应条件下,组氨酸上可以引入一个新的异硫氰酸苯酯修饰位点。这一修饰位点的发现预示着通过改变实验条件或分析方法,可以更准确地利用Edman降解和蛋白质组学技术分析多肽和蛋白质。     

Proteome profile of salt gland‐rich epidermis extracted from a salt‐tolerant tree species

Preparation of proteins from salt‐gland‐rich tissues of mangrove plant is necessary for a systematic study of proteins involved in the plant's unique desalination mechanism. Extraction of high‐quality proteins from the leaves of mangrove tree species, however, is difficult due to the presence of high levels of endogenous phenolic compounds. In our study, preparation of proteins from only a part of the leaf tissues (i.e. salt gland‐rich epidermal layers) was required, rendering extraction even more challenging. By comparing several extraction methods, we developed a reliable procedure for obtaining proteins from salt gland‐rich tissues of the mangrove species Avicennia officinalis. Protein extraction was markedly improved using a phenol‐based extraction method. Greater resolution 1D protein gel profiles could be obtained. More promising proteome profiles could be obtained through 1D‐LC‐MS/MS. The number of proteins detected was twice as much as compared to TUTS extraction method. Focusing on proteins that were solely present in each extraction method, phenol‐based extracts contained nearly ten times more proteins than those in the extracts without using phenol. The approach could thus be applied for downstream high‐throughput proteomic analyses involving LC‐MS/MS or equivalent. The proteomics data presented herein are available via ProteomeXchange with identifier PXD001691.