Mass spectrometry based proteomic analysis of human stem cells: a brief review

Stem cells can give rise to various cell types and are capable of regenerating themselves over multiple cell divisions. Pluripotency and self-renewal potential of stem cells have drawn vast interest from different disciplines, with studies on the molecular properties of stem cells being one example. Current investigations on the molecular basis of stem cells pluripotency and self-renewal entail traditional techniques from chemistry and molecular biology. In this mini review, we discuss progress in stem cell research that employs proteomics approaches. Specifically, we focus on studies on human stem cells from proteomics perspective. To our best knowledge, only the following types of human stem cells have been examined via proteomics analysis: human neuronal stem cells, human mesenchymal stem cells, and human embryonic stem cells. Protein expression serves as biomarkers of stem cells and identification and expression level of such biomarkers are usually determined using two-dimensional electrophoresis coupled mass spectrometry or non-gel based mass spectrometry.     

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。     

分离分析技术在蛋白质组学研究中应用的新进展

蛋白质组学研究的核心技术之一是分离分析方法。该综述重点评述了分离分析技术在蛋白质组学研究,即在蛋白质组表达谱构建、翻译后修饰蛋白质组研究、蛋白质复合体和相互作用研究、蛋白质组定量研究中应用的新进展,介绍了各种分离分析方法的优点、应用范围和有待解决的问题。引用文献89篇。     

蛋白质组质谱分析中基于串并联支持向量机的肽段色谱保留时间预测方法

基于质谱的大规模蛋白质鉴定中,在线液相色谱分离发挥了重要作用。色谱保留时间(retention time,RT)是肽段鉴定和定量的重要信息。由于整个色谱分析运行时间中,流动相中的有机相采用了非线性浓度曲线以及样品中肽段之间的相互影响等因素,基于肽段序列的RT预测还存在精度不高、模型推广性能差等问题。本文提出了一种基于串并联支持向量机(serial and parallel support vector machine,SP-SVM)的RT预测方法,能够表征洗脱过程中有机相浓度的非线性变化和肽段之间的相互影响,显著提高了肽段保留时间预测的精度。利用复杂样本数据集验证结果表明,预测RT和实验RT之间的决定系数达到了0.95,超过95%的鉴定肽段的RT预测误差范围小于总运行时间的20%,超过70%的鉴定肽段的RT预测误差范围小于总运行时间的10%。本文提出的模型的性能达到了目前已知的最好水平。     

Mass spectrometry based cellular phosphoinositides profiling and phospholipid analysis: A brief review

Phospholipids are key components of cellular membrane and signaling. Among cellular phospholipids, phosphoinositides, phosphorylated derivatives of phosphatidylinositol are important as a participant in essential metabolic processes in animals. However, due to its low abundance in cells and tissues, it is difficult to identify the composition of phosphoinositides. Recent advances in mass spectrometric techniques, combined with established separation methods, have allowed the rapid and sensitive detection and quantification of a variety of lipid species including phosphoinositides. In this mini review, we briefly introduce progress in profiling of cellular phosphoinositides using mass spectrometry. We also summarize current progress of matrices development for the analysis of cellular phospholipids using matrix-assisted laser desorption/ionization mass spectrometry. The phosphoinositides profiling and phospholipids imaging will help us to understand how they function in a biological system and will provide a powerful tool for elucidating the mechanism of diseases such as diabetes, cancer and neurodegenerative diseases. The investigation of cellular phospholipids including phosphoinositides using electrospray ionization mass spectrometry and matrix-assisted laser desorption/ionization mass spectrometry will suggest new insights on human diseases, and on clinical application through drug development of lipid related diseases.     

Mass spectrometry studies on meso-substituted corroles and their photochemical decomposition products

Corroles, ring-contracted analogs of porphyrins, are an important class of compounds which have attracted the attention of many researchers in the fields of organic, coordination and physical chemistry. In the present work, the stability and the decomposition pathways of a diverse set of meso-substituted corroles have been studied using mass spectrometry (MS), UV-Vis absorption and preparative methods combined with NMR spectroscopy. Four different ionization methods (electrospray ionization, field desorption, atmospheric pressure photoionization and atmospheric pressure chemical ionization) were utilized to investigate light- and oxygen-induced decomposition in various solvents. It was found that the rate of decomposition in MeCN is significantly higher than in CH(2)Cl(2), hexane, MeOH and ethyl acetate. HR-MS combined with CID-MS/MS enabled us to identify the products of initial decomposition. Surprisingly, numerous smaller open-chain compounds were also detected. Large-scale decomposition of a corrole bearing sterically hindered substituents at positions 5 and 15 allowed us to isolate mg quantities of three decomposition products: isocorrole and isomeric biliverdin-type species. These are formed as a result of oxygen attack on the meso-10 position.     

基于毛细管电泳-质谱联用技术的代谢/蛋白质组学分析

毛细管电泳-质谱(CE-MS)联用技术具有高灵敏度、高分析效率、低样品损耗等优点,在强极性和带电荷的物质分析中具有明显优势,已广泛应用于生命科学、医学、药学等多个领域。在过去的十几年,影响其应用的主要因素包括系统的稳定性、实验的可重复性、数据的准确性等。为解决现有问题,进一步拓展其应用,研究人员在技术设计和改进等方面做了大量工作。医学和分析化学领域的相关研究证明了CE-MS在代谢组学和蛋白质组学中的实用性。这篇文章综述了2015年以来,CE-MS在技术和应用方面的最新进展,为未来的技术发展及应用提供借鉴。为提高CE-MS的分析效率和数据可比性,该文对多个方面的研究进行了讨论,包括涂层与样品的相互作用、接口技术、运行参数和数据处理方法。文中关于复杂样品(组织、细胞、体液等)代谢组学/蛋白质组学的综述研究,使癌症病理分析、药物开发和疾病监测等分析数据更加可视化,为CE-MS临床分析应用提供借鉴。除了对CE-MS的最新发展进行综合评述外,还提出未来应加强3个方面的研究:(i)从样品前处理和分离技术方面优化分析方法;(ii)从毛细管涂层和接口技术方面调整分析技术;(iii)从临床研究和数据分析...     

Mass spectrometry in coupling with affinity capture-release and isotope-coded affinity tags for quantitative protein analysis

Affinity capture-release electrospray ionization mass spectrometry (ACESIMS) and isotope-coded affinity tags (ICAT) are two recently introduced techniques for the quantitation of protein activity and content with applications to clinical enzymology and functional proteomics, respectively. One common feature of these methods is that they use biotinylated tags that function as molecular handles for highly selective and reversible affinity capture of conjugates from complex biological mixtures such as cell homogenates and sub-cellular organelles. ACESIMS uses synthetic substrate conjugates specifically to target cellular enzymes that, when deficient, are the cause of genetic diseases. Multiplex determination of enzyme activities is used for the diagnosis of lysosomal storage diseases. The ICAT method relies on selective conjugation of cysteine thiol groups in proteins, followed by enzymatic digestion and quantitative analysis of peptide conjugates by mass spectrometry. Another common feature of the ACESIMS and ICAT approaches is that both use conjugates labeled with stable heavy isotopes as internal standards for quantitation. Selected applications of the ACESIMS and ICAT techniques are presented that include molecular-level diagnosis of genetic diseases in children and quantitative determination of protein expression in cells.     

Mass spectrometry and planetary exploration: A brief review and future projection

Since the inception of mass spectrometry more than a century ago, the field has matured as analytical capabilities have progressed, instrument configurations multiplied, and applications proliferated. Modern systems are able to characterize volatile and nonvolatile sample materials, quantitatively measure abundances of molecular and elemental species with low limits of detection, and determine isotopic compositions with high degrees of precision and accuracy. Consequently, mass spectrometers have a rich history and promising future in planetary exploration. Here, we provide a short review on the development of mass analyzers and supporting subsystems (eg, ionization sources and detector assemblies) that have significant heritage in spaceflight applications, and we introduce a selection of emerging technologies that may enable new and/or augmented mission concepts in the coming decades.     

A magnetic bead‐based serum proteomic fingerprinting method for parallel analytical analysis and micropreparative purification

ProteinChip surface‐enhanced laser desorption/ionization technology and magnetic beads‐based ClinProt system are commonly used for semi‐quantitative profiling of plasma proteome in biomarker discovery. Unfortunately, the proteins/peptides detected by MS are non‐recoverable. To obtain the protein identity of a MS peak, additional time‐consuming and material‐consuming purification steps have to be done. In this study, we developed a magnetic beads‐based proteomic fingerprinting method that allowed semi‐quantitative proteomic profiling and micropreparative purification of the profiled proteins in parallel. The use of different chromatographic magnetic beads allowed us to obtain different proteomic profiles, which were comparable to those obtained by the ProteinChip surface‐enhanced laser desorption/ionization technology. Our assays were semi‐quantitative. The normalized peak intensity was proportional to concentration measured by immunoassay. Both intra‐assay and inter‐assay coefficients of variation of the normalized peak intensities were in the range of 4–30%. Our method only required 2 μL of serum or plasma for generating enough proteins for semi‐quantitative profiling by MALDI‐TOF‐MS as well as for gel electrophoresis and subsequent protein identification. The protein peaks and corresponding gel spots could be easily matched by comparing their intensities and masses. Because of its high efficiency and reproducibility, our method has great potentials in clinical research, especially in biomarker discovery.     

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。     

基于超高效液相色谱-质谱法的肽段分析中非特异性吸附评估及通用型最小化策略北大核心CSCD

蛋白质组学技术在多肽和蛋白质类新型治疗药物的开发、临床诊断生物标志物的深入发掘中应用广泛。然而,多肽和蛋白质类大分子的非特异性吸附性质给分析方法的开发带来极大挑战,亟须一种通用型的策略去评估和降低非特异吸附对超高效液相色谱-质谱(UPLC-MS)大分子检测造成的负面影响。研究以牛血清白蛋白(BSA)为模型,探讨其酶解后多肽组理化性质与吸附程度之间的相关性;根据肽段的响应和吸附程度设计分级策略;针对高响应、强吸附的ClassⅡ类肽段,从样品制备中离心管、进样瓶的选择,乃至液相色谱系统中色谱柱固定相、流速、梯度、柱温、洗针液的选择全过程设计试验,探讨非特异吸附的影响因素及其通用型最小化策略。结果显示,肽段的被吸附程度与其理化参数HPLC指数(HPLC Index)、肽段长度等显著相关(p<0.05),但仅凭上述参数仅能解释30%肽段的被吸附程度。改性的聚丙烯材料可使肽段溶液在储存或前处理过程中获得较高的回收率(24 h内回收率大于80%)。在对液相色谱条件的考察和优化过程中发现,C_(8)填料的色谱柱、高流速、缓梯度以及强洗针液,可使残留量降至最低(降低为原来的1/150)。柱温对残留的影响在肽段间存在较大个体差异,需要对不同的肽段具体分析以得到较少量的残留。研究以详实的数据考察并最小化模型肽段组在分析过程中的非特异吸附,提示了蛋白质类大分子药物分析方法建立中应重点关注的影响因素及其有效的解决方案。     

“Polymeromics”: Mass spectrometry based strategies in polymer science toward complete sequencing approaches: A review

Mass spectrometry (MS) is the most versatile and comprehensive method in “OMICS” sciences (i.e. in proteomics, genomics, metabolomics and lipidomics). The applications of MS and tandem MS (MS/MS or MSⁿ) provide sequence information of the full complement of biological samples in order to understand the importance of the sequences on their precise and specific functions. Nowadays, the control of polymer sequences and their accurate characterization is one of the significant challenges of current polymer science. Therefore, a similar approach can be very beneficial for characterizing and understanding the complex structures of synthetic macromolecules. MS-based strategies allow a relatively precise examination of polymeric structures (e.g. their molar mass distributions, monomer units, side chain substituents, end-group functionalities, and copolymer compositions). Moreover, tandem MS offer accurate structural information from intricate macromolecular structures; however, it produces vast amount of data to interpret. In “OMICS” sciences, the software application to interpret the obtained data has developed satisfyingly (e.g. in proteomics), because it is not possible to handle the amount of data acquired via (tandem) MS studies on the biological samples manually. It can be expected that special software tools will improve the interpretation of (tandem) MS output from the investigations of synthetic polymers as well. Eventually, the MS/MS field will also open up for polymer scientists who are not MS-specialists. In this review, we dissect the overall framework of the MS and MS/MS analysis of synthetic polymers into its key components. We discuss the fundamentals of polymer analyses as well as recent advances in the areas of tandem mass spectrometry, software developments, and the overall future perspectives on the way to polymer sequencing, one of the last Holy Grail in polymer science.     

Mass spectrometry and NMR analysis of ligand binding by human liver fatty acid binding protein

Human liver fatty acid binding protein (hL‐FABP) is the most abundant cytosolic protein in the liver. This protein plays important roles associated to partitioning of fatty acids (FAs) to specific metabolic pathways, nuclear signaling and protection against oxidative damage. The protein displays promiscuous binding properties and can bind two internal ligands, unlike FABPs from other tissues. Different topologies for the ligand located in the more accessible site have been reported, with either a ‘head‐in’ or ‘head‐out’ orientation of the carboxylate end. Electrospray‐ionization mass spectrometry and nuclear magnetic resonance titrations are employed here in order to investigate in further detail the binding properties of this system, the equilibria established in solution and the pH dependence of the complexes. The results are consistent with two binding sites with different affinity and a unique head‐out topology for the second molecule of either ligand. Competition experiments indicate a higher affinity for oleic acid relative to palmitic acid at each binding site. Copyright © 2013 John Wiley & Sons, Ltd.     

Trypsin and MALDI matrix pre‐coated targets simplify sample preparation for mapping proteomic distributions within biological tissues by imaging mass spectrometry

Prefabricated surfaces containing α‐cyano‐4‐hydroxycinnamic acid and trypsin have been developed to facilitate enzymatic digestion of endogenous tissue proteins prior to matrix‐assisted laser desorption/ionization (MALDI) imaging mass spectrometry (IMS). Tissue sections are placed onto slides that were previously coated with α‐cyano‐4‐hydroxycinnamic acid and trypsin. After incubation to promote enzymatic digestion, the tissue is analyzed by MALDI IMS to determine the spatial distribution of the tryptic fragments. The peptides detected in the MALDI IMS dataset were identified by Liquid chromatography‐tandem mass spectrometry/mass spectrometry. Protein identification was further confirmed by correlating the localization of unique tryptic fragments originating from common parent proteins. Using this procedure, proteins with molecular weights as large as 300 kDa were identified and their distributions were imaged in sections of rat brain. In particular, large proteins such as myristoylated alanine‐rich C‐kinase substrate (29.8 kDa) and spectrin alpha chain, non‐erythrocytic 1 (284 kDa) were detected that are not observed without trypsin. The pre‐coated targets simplify workflow and increase sample throughput by decreasing the sample preparation time. Further, the approach allows imaging at higher spatial resolution compared with robotic spotters that apply one drop at a time. Copyright © 2016 John Wiley & Sons, Ltd.     

Profiling and comprehensive expression analysis of ABC transporter solute-binding proteins of Bacillus subtilis membrane based on a proteomic approach

We analyzed ABC transporter solute-binding proteins (SBPs) of the Bacillus subtilis membrane using a proteomic approach. We prepared a washed cell membrane fraction that was insoluble in 134 mM nondetergent sulfobetaine and then extracted proteins using mixtures of detergents in a stepwise manner. The membrane proteins were resolved by three two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) or two one-dimensional (1-D) PAGE procedures, electroblotted, and digested in the presence of 5% or 80% acetonitrile. Thereafter, matrix-assisted laser desorption/ionization-time of flight-mass spectrometry (MALDI-TOF MS) identified 637 proteins corresponding to 15.9% of the total cellular proteins. We predicted that among these, 256 were membrane proteins, 101 were lipoproteins or secretory proteins and 280 were soluble proteins containing peripheral proteins that function in both the cytoplasm and the cell membrane such as SecA and FtsY. Among the 637 proteins, we identified 30 SBPs among 38 importers predicted by a bioinformatic search of the genome. We confirmed expression of the genes for the 30 SBPs using DNA microarray analysis. We compared the 2-D gel separation profiles of submembrane fractions solubilized by 1% n-dodecyl-beta-D-maltoside from cells cultured on Luria Bertani (LB), S7, and S7 medium without glutamate as well as DNA microarray data on LB and S7. The results suggested that YcdH, YtmK and YurO are binding proteins for Mn(++), glutamate and glucose, respectively, and that YqiX and YxeM are binding proteins for amino acids (tryptophan in S7 medium).     

基于生物质谱技术的磷酰化修饰策略在多肽测序中的应用

该文建立了一种利用磷酰化修饰结合电喷雾质谱(ESI-Q-TOF)测定多肽氨基酸序列的有效方法。利用Atherton-Todd反应,以二丙基亚磷酰酯(DPP)为磷酰化试剂,应用生物质谱技术,对磷酰化修饰后的5种模型肽的磷酰化反应情况进行了系统研究,考察了磷酰化肽的二级质谱特征,并与未经磷酰化反应的肽的二级质谱特征对比。结果表明,经过磷酰化修饰后,肽的二级质谱中的a1离子信号强度明显增加,可以准确鉴定其N端氨基酸;b系列离子信息完整,信号强度增强,使得多肽C ID测序的谱图简单、清晰,有利于肽的氨基酸序列的测定;赖氨酸(K,128.10 u)和谷氨酰胺(Q,128.13 u)两种氨基酸质荷比相近,由于二者磷酰化修饰后的差异性,使其得到准确区分。经过5种已知氨基酸序列的模型肽的磷酰化后结合质谱技术进行氨基酸序列测定验证,结果表明该方法简单、快速、准确,提高了利用质谱技术进行多肽测序的准确度和灵敏度,可为蛋白质组学研究提供有效的技术手段。     

Two terphenyl‐based diol hosts and corresponding solvent inclusions with dimethylformamide and acetonitrile

Having reference to an elongated structural modification of 2,2′‐bis(hydroxydiphenylmethyl)biphenyl, (I), the two 1,1′:4′,1′′‐terphenyl‐based diol hosts 2,2′′‐bis(hydroxydiphenylmethyl)‐1,1′:4′,1′′‐terphenyl, C₄₄H₃₄O₂, (II), and 2,2′′‐bis[hydroxybis(4‐methylphenyl)methyl]‐1,1′:4′,1′′‐terphenyl, C₄₈H₄₂O₂, (III), have been synthesized and studied with regard to their crystal structures involving different inclusions, i.e. (II) with dimethylformamide (DMF), C₄₄H₃₄O₂·C₂H₆NO, denoted (IIa), (III) with DMF, C₄₈H₄₂O₂·C₂H₆NO, denoted (IIIa), and (III) with acetonitrile, C₄₈H₄₂O₂·CH₃CN, denoted (IIIb). In the solvent‐free crystals of (II) and (III), the hydroxy H atoms are involved in intramolecular O—H...π hydrogen bonding, with the central arene ring of the terphenyl unit acting as an acceptor. The corresponding crystal structures are stabilized by intermolecular C—H...π contacts. Due to the distinctive acceptor character of the included DMF solvent species in the crystal structures of (IIa) and (IIIa), the guest molecule is coordinated to the host via O—H...O=C hydrogen bonding. In both crystal structures, infinite strands composed of alternating host and guest molecules represent the basic supramolecular aggregates. Within a given strand, the O atom of the solvent molecule acts as a bifurcated acceptor. Similar to the solvent‐free cases, the hydroxy H atoms in inclusion structure (IIIb) are involved in intramolecular hydrogen bonding, and there is thus a lack of host–guest interaction. As a result, the solvent molecules are accommodated as C—H...N hydrogen‐bonded inversion‐symmetric dimers in the channel‐like voids of the host lattice.