基于质谱的蛋白质组学技术在神经退行性疾病生物标志物研究中的应用

蛋白质组学是在整体水平上研究细胞、组织或生物体蛋白质组成及变化规律的科学.与传统的生物学研究相比,蛋白质组学具有快速、灵敏、高通量的优点.神经退行性疾病是一类由神经系统内特定神经细胞的进程性病变或丢失而导致神经功能障碍的疾病,严重危害人类健康.近年来,基于质谱的蛋白质组学技术在神经退行性疾病的研究中得到了广泛应用.本文简要介绍了蛋白质组学在样品分离、多肽定量、质谱检测及生物标志物临床验证等方面的技术发展,并结合实例综述了基于质谱的蛋白质组学在神经退行性疾病生物标志物发现与验证中的研究进展.     

Quantitative mass spectrometry in proteomics: a critical review

The quantification of differences between two or more physiological states of a biological system is among the most important but also most challenging technical tasks in proteomics. In addition to the classical methods of differential protein gel or blot staining by dyes and fluorophores, mass-spectrometry-based quantification methods have gained increasing popularity over the past five years. Most of these methods employ differential stable isotope labeling to create a specific mass tag that can be recognized by a mass spectrometer and at the same time provide the basis for quantification. These mass tags can be introduced into proteins or peptides (i) metabolically, (ii) by chemical means, (iii) enzymatically, or (iv) provided by spiked synthetic peptide standards. In contrast, label-free quantification approaches aim to correlate the mass spectrometric signal of intact proteolytic peptides or the number of peptide sequencing events with the relative or absolute protein quantity directly. In this review, we critically examine the more commonly used quantitative mass spectrometry methods for their individual merits and discuss challenges in arriving at meaningful interpretations of quantitative proteomic data.     

Playing tag with quantitative proteomics

There is steady need for new proteomic strategies on quantitative measurements that provide essential components for detailing dynamic changes in many cellular functions and processes. Stable isotope labeling is a rapidly evolving field, which can be used either after protein extraction with chemical labeling, or in cell culture with metabolic incorporation. In this review, we explore the most frequently utilized quantitation techniques with particular attention paid to chemical labeling using different isotopic tags, including a recent labeling strategy—soluble polymer-based isotopic labeling (SoPIL)—that achieves efficient labeling in homogeneous conditions. Special care should be devoted to the selection of appropriate quantitation approaches according to the needs of the sample and overall experimental design. We evaluate recent advances in quantitative proteomics using stable isotope labeling and their applications to current insightful biological inquiries. Figure Chemical modules of isotopic tags for quantitative proteomics.     

Rapid on-membrane proteolytic cleavage for Edman sequencing and mass spectrometric identification of proteins

A method for the rapid limited enzymatic cleavage of PVDF membrane-immobilized proteins is described. This method allows the fast characterization of PVDF blotted proteins by peptide mass fingerprinting (Henzel, W. J., Billeci, T. M., Stults, J. T., Wong, S. C., Grimley, C., Wantanabe, C., Proc. Natl. Acad. Sci. USA 1993, 90, 5011-5015), LC-MS/MS, or N-terminal sequencing and has been demonstrated on a range of proteins using a full complement of proteolytic enzymes. This technique allows the generation of proteolytic fragments between 5 and 60 min (depending on the enzyme employed), which is significantly faster than previously reported on-membrane digestion methods. To date, this on-membrane rapid digestion protocol has aided in the identification and confirmation of mutation sites in over 200 recombinant proteins.     

基于质谱的蛋白质组学技术在食品真伪鉴别及品质识别方面的应用

蛋白质组学作为后基因组时代的一个新研究方向,近几年发展迅速,目前已应用于多个领域,在食品品质检测和安全控制方面成为有力的研究工具。蛋白质组学为食品科学的相关研究打开了新思路,不仅可以鉴定蛋白质种类,还可进行蛋白质定量,为分析不同物种、产地、成熟阶段的食品蛋白质组分和含量提供了可能。蛋白质组学研究手段多样,质谱是常用技术之一。该文介绍了蛋白质组学的概念、分类、研究技术以及常见蛋白质数据库,综述了基于质谱的蛋白质组学技术在真伪鉴别和品质检测方面的应用,涉及海鲜、肉制品、奶制品、保健食品及高附加值食品等多种食品,并对蛋白质组学的发展进行了展望。     

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.     

Identification of human hepatocellular carcinoma-related proteins by proteomic approaches

Hepatocellular carcinoma (HCC) is the most common malignant liver tumor. Analysis of human serum from HCC patients using two-dimensional gel electrophoresis (2DE) combined with nano-high-performance liquid chromatography electrospray ionization tandem mass spectrometry (nano-HPLC–ESI-MS/MS) identified fourteen different proteins differentially expressed between HCC patients and the control group. Twelve proteins were up-regulated and two down-regulated. By using nano-HPLC–MS/MS system to analyze proteome in human serum, 317 proteins were identified, twenty-nine of which to high confidence levels (protein matched at last two unique peptide sequences). Of these twenty-nine proteins, six were present only in HCC patients and may serve as biomarkers for HCC. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Sample preparation by in-gel digestion for mass spectrometry-based proteomics

The proteomic characterization of proteins and protein complexes from cells and cell organelles is the next challenge for investigation of the cell. After isolation of the cell compartment, three steps have to be performed in the laboratory to yield information about the proteins present. The protein mixtures must be separated into single species, broken down into peptides, and, finally, identified by mass spectrometry. Most scientists engaged in proteomics separate proteins by electrophoresis. For characterization and identification of proteomes, mass spectrometry of peptides is the method of choice. To combine electrophoresis and mass spectrometry, sample preparation by “in-gel digestion” has been developed. Many procedures are available for in-gel digestion, which inspired us to review in-gel digestion approaches. Figure Classical in-gel digestion process for a protein band stained with CBB. Protein bands are cut from the polyacrylamide gel (1). CBB molecules (blue circles) bound to the protein are released by iterative incubation in a buffered organic solvent system (2). To increase digestion efficiency and sequence coverage proteins are reduced (3) and alkylated (4). Proteins are subsequently digested with proteolytic enzymes (scissors symbols), typically trypsin (5). Trypsin cleaves at the amino acid residues arginine (R) and lysine (K). The resulting peptides (A, B, and C) are extracted from the polyacrylamide matrix (6). The peptide solution can be further purified for analysis by mass spectrometry (Section “Concentration and desalting of peptides”)     

An integrated workflow for extraction and solubilization of intermediate filaments from colorectal biopsies for proteomic analysis

We report a technique for isolation and solubilization of intermediate filament (IF) proteins from colonic biopsies compatible with both gel electrophoresis and liquid chromatography "shotgun" proteomics using mass spectrometry (MS). This is important because changes in the IF proteome, particularly in keratin expression and modification, are noted in colonic mucosa of patients with colorectal cancer. Though keratins have traditionally been dissolved in high concentration of urea, the latter solvent precludes efficient proteolytic digestion by trypsin prior to gel-free LC-MS/MS approaches. The extraction of cytoskeletal proteins was initially evaluated using MCF-7 cancer cell lines using a published, differential detergent solubilization protocol. IF proteins were extracted from colonic biopsies using a combination of homogenization and sonication. Since comparable efficiency of solubilization was noted on the extracted IF from cell lines between urea and guanidine hydrochloride (GuHCl) in triethylammonium bicarbonate buffer, isolated proteins from endoscopic biopsies were solubilized in GuHCl. Using immunoblotting techniques, we successfully demonstrated isolation of keratins and preservation of posttranslational modifications (phosphorylation, acetylation). Dissolved proteins were tryptically digested and peptides analyzed by MS, showing the functionality of the workflow in shotgun proteomic applications, specifically compatibility of the workflow for isobaric tagging relative and absolute quantification based quantitation approaches.     

Elemental mass spectrometry for quantitative proteomics

In the last decade mass-spectrometry-based proteomics has become an indispensable analytical tool for molecular biology, cellular biology and, lately, for the emerging systems biology. This review summarises the evolution and great potential of analytical methods based on elemental mass-spectrometric detection for quantitative proteomic analysis.     

含硫醚、硒醚及二硫键链接侧链的小檗碱衍生物的合成及体外抗肿瘤活性

为开发高效低毒的抗肿瘤天然产物衍生物,依据药物拼合原理设计并合成了10个新型小檗碱衍生物。将盐酸小檗碱与含单硫醚、单硒醚和二硫键脂肪链片段通过缩合反应得到目标化合物,其化学结构均经过核磁共振氢谱、碳谱以及高分辨质谱确证。采用噻唑蓝（MTT）法考察其体外抗肿瘤活性,结果表明,7个化合物针对不同的癌细胞株的抑制活性优于盐酸小檗碱,同时对人正常肝细胞L02的毒性较低。化合物3e的体外抗肿瘤活性最佳(针对BEL-7402的IC₅₀值为8.70μmol/L,选择性指数为3.61),可对其深入研究用以开发高效低毒的抗肿瘤药物。     

蛋白质组学在人乳蛋白质研究中的应用

人乳是新生儿最理想的天然食物，蛋白质是人乳中最主要的营养成分之一。随着蛋白质组学技术的发展，利用蛋白质组学的方法研究人乳蛋白质也取得了一些研究成果。本文综述了近年来蛋白质组学技术在人乳蛋白质研究中的应用，分别从人乳蛋白质的组成研究、动态变化、人乳与其他来源乳汁的蛋白质差异比较、人乳磷酸化蛋白和糖基化蛋白研究、人乳内源肽的研究及人乳蛋白与疾病等几个方面进行了阐述。蛋白质组学技术使人乳蛋白质的研究进入了微量营养研究的时代，人乳蛋白质组学的研究成果将为母婴健康提供更好的保障。     

Characterization of the human serum depletome by label-free shotgun proteomics

While it is known that immunoaffinity depletion of abundant proteins in serum removes additional proteins beyond those targeted, there has been little characterization of the co-depleted proteins in the high abundant fraction, which we refer to here as the "depletome". We present evidence of co-depletion of non-targeted proteins in human serum using a top-20 immunodepletion column, as shown by label-free liquid chromatography mass spectrometry (LC-MS(E)) profiling. This led to identification of 147 proteins which were specific for this fraction and comprised proteins with functions predominantly in binding and transport of nucleotides, metal ions, carbohydrates and lipids. These results suggest that further studies on this commonly ignored serum fraction may provide new insights into clinical proteomics.     

Fractionation of the human plasma proteome for monoclonal antibody proteomics‐based biomarker discovery 2: Antigen identification by dot‐blot array screening

Immunization with complex mixtures, like the human plasma resulted in the generation of cloned mAb libraries (PlasmaScan? and QuantiPlasma? libraries, with >1000 individual mAbs) reacting with a nonredundant set of antigenic epitopes. mAb proteomics refers to quasi‐hypothesis‐free profiling of plasma samples with nascent or cloned mAb libraries for the discovery of disease‐specific biomarkers. Once mAbs with biomarker potential have been identified, the next task is the determination of cognate antigens recognized by the respective mAbs. To determine the cognate protein antigen corresponding to each individual mAbs in the cloned mAb libraries, we have separated human plasma by consecutive steps of desalting and various chromatography procedures. The process resulted in 783 fractions, which we termed “Analyte Library” (AL). The AL represents the human plasma proteome in relatively low‐protein complexity fractions. Here, to determine the utility of the AL, we selected ten plasma proteins and checked for their presence in the fractions. Among the ten cases, the distribution of four selected plasma proteins matched expectations, as these proteins were present only in a few fractions corresponding to their physical, chemical, and biochemical properties. However, in six cases, we observed “smear” ‐like distribution or complete absence of the proteins, suggesting that protein–protein interactions or protein variants may alter the observed plasma distribution profiles. Nevertheless, we conclude that the AL is an efficient, high throughput tool to complement the mAb biomarker discovery process with cognate protein antigen identification for each mAbs.     

Ion mobility–mass spectrometry: a new paradigm for proteomics

Matrix-assisted laser desorption/ionization (MALDI) coupled with ion mobility–mass spectrometry (IM–MS) provides a rapid (μs–ms) means for the two-dimensional (2D) separation of complex biological samples (e.g., peptides, oligonucleotides, glycoconjugates, lipids, etc.), elucidation of solvent-free secondary structural elements (e.g., helices, β-hairpins, random coils, etc.), rapid identification of post-translational modifications (e.g., phosphorylation, glycosylation, etc.) or ligation of small molecules, and simultaneous and comprehensive sequencing information of biopolymers. In IM–MS, protein-identification information is complemented by structural characterization data, which is difficult to obtain using conventional proteomic techniques. New avenues for enhancing the figures of merit (e.g., sensitivity, limits of detection, dynamic range, and analyte selectivity) and optimizing IM–MS experimental parameters are described in the context of deriving new information at the forefront of proteomics research.     

Studies on the Basic Conformation of Lysozyme by Electrospray Ionization-Mass Spectrometry (ESI- MS)

In the present work, 2-mercaptoethanol and dithiothreitol were used to hydrogenise the internal disulfide bond in lysozyme. The experimental results indicate that the charge distribution of the proteins are different in the reaction process. From the calculated molecular weight, the reduction process of the disulfide bond in the molecules can be described, and the number of the disulfide bonds in the molecule can also be determined.     

Interpretation of mass spectrometry data for high-throughput proteomics

Recent developments in proteomics have revealed a bottleneck in bioinformatics: high-quality interpretation of acquired MS data. The ability to generate thousands of MS spectra per day, and the demand for this, makes manual methods inadequate for analysis and underlines the need to transfer the advanced capabilities of an expert human user into sophisticated MS interpretation algorithms. The identification rate in current high-throughput proteomics studies is not only a matter of instrumentation. We present software for high-throughput PMF identification, which enables robust and confident protein identification at higher rates. This has been achieved by automated calibration, peak rejection, and use of a meta search approach which employs various PMF search engines. The automatic calibration consists of a dynamic, spectral information-dependent algorithm, which combines various known calibration methods and iteratively establishes an optimised calibration. The peak rejection algorithm filters signals that are unrelated to the analysed protein by use of automatically generated and dataset-dependent exclusion lists. In the "meta search" several known PMF search engines are triggered and their results are merged by use of a meta score. The significance of the meta score was assessed by simulation of PMF identification with 10,000 artificial spectra resembling a data situation close to the measured dataset. By means of this simulation the meta score is linked to expectation values as a statistical measure. The presented software is part of the proteome database ProteinScape which links the information derived from MS data to other relevant proteomics data. We demonstrate the performance of the presented system with MS data from 1891 PMF spectra. As a result of automatic calibration and peak rejection the identification rate increased from 6% to 44%.Abbreviations 2-DE Two-dimensional gel electrophoresis - MALDI Matrix-assisted laser desorption ionisation - PMF Peptide mass fingerprinting - MS Mass spectrometry - TOF Time of flight     

A mass spectrometry approach for the study of deglycosylated proteins

Mass spectrometry (MS) analysis, after enzymatic or chemical deglycosylation, requires preparatory steps to remove salts and buffers. In this work, the glycosylated protein fetuin and a lectin protein isolated from the serum of Alligator mississippiensis were used to evaluate methods for desalting samples after an enzymatic or chemical deglycosylation. Precipitation and dialysis were used to prepare the deglycosylated samples for MS analysis. Both the precipitation and dialysis methods were suitable for sample preparation prior to analysis by matrix assisted laser desorption ionization (MALDI) MS.     

Limitations of current proteomics technologies

Application of proteomics technologies in the investigation of biological systems creates new possibilities in the elucidation of biopathomechanisms and the discovery of novel drug targets and early disease markers. A proteomic analysis involves protein separation and protein identification as well as characterization of the post-translational modifications. Proteomics has been applied in the investigation of various disorders, like neurological diseases, and the application has resulted in the detection of a large number of differences in the levels and the modifications of proteins between healthy and diseased states. However, the current proteomics technologies are still under development and show certain limitations. In this article, we discuss the major drawbacks and pitfalls of proteomics we have observed in our laboratory and in particular during the application of proteomics technologies in the investigation of the brain.     

Combination of mathematics and label-free proteomics for discovering keratin-derived specific peptide biomarkers to distinguish animal horn-derived traditional Chinese medicines

Saiga antelope horn and Rhinoceros horn have been used in traditional Chinese medicine for thousands of years. However, due to the protection of wildlife, the application of these rare animal horns has been restricted or prohibited. Therefore, water buffalo horn, goat horn, and yak horn have been applied as alternatives to Rhinoceros horn or Saiga antelope horn in a clinic. It is extremely difficult to distinguish normal animal horns in powdered or decocted form, especially identifying related species such as water buffalo horn, yak horn, and cattle horn. In this work, mathematics set and label-free proteomics analysis were combined for discovering keratin-derived specific peptide biomarkers. By using mathematics set analysis after nano liquid chromatography-tandem mass spectrometry-based proteomics, the selected species-specific peptides could be used to identify the authenticity of the Saiga antelope horn and goat horn. Furthermore, peptide biomarkers were selected to distinguish related species-derived horns, water buffalo horn, yak horn, and cattle horn. In total, eight peptide biomarkers were selected and applied for simultaneously distinguishing different horn samples. The present strategy provides a method for peptide biomarkers discovery and also has positive significance for ensuring the quality and efficacy of animal horn-derived traditional Chinese medicines and their products.