胰蛋白酶镜像酶LysargiNase的开发及其在蛋白质组学研究中的应用

蛋白质组学是系统鉴定、定量蛋白质及其翻译后修饰形式,并研究这些蛋白质生物学功能的学科。目前,基于质谱的鸟枪法蛋白质组学技术是蛋白质组学研究的主要手段之一,其技术流程是先将蛋白质组样品经位点特异性蛋白酶消化形成肽组,再进行高效液相色谱分离和质谱检测。而位点特异性蛋白酶对蛋白质样品的消化是质谱检测的前提和基础。随着蛋白质组学研究的深入,多种位点特异性蛋白酶被先后开发利用;而切割发生在相应氨基酸的N端,与传统的C端蛋白酶互为镜像的蛋白酶的鉴定、开发、特性研究和广泛使用更是为蛋白质组学研究提供了新的工具。文中对最近发现的胰蛋白酶的镜像酶——赖氨酸精氨酸N端蛋白酶(LysargiNase)的特点及其应用进行综述,为国内外学者更加广泛的使用创造条件。     

蛋白质组学样品消化条件的优化

随着质谱的飞速发展,基于质谱的"鸟枪法"技术广泛的应用于大规模的蛋白质组学分析。化学反应保护效率过低或者酶切效率过低则会降低鉴定效率,并且在现有的计算方式下会丢失很多肽段信息。因此,蛋白质样品的前处理在现有的蛋白质组学研究中发挥重要作用。本研究对蛋白质的烷基化试剂的反应条件进行优化以提高烷基化效率,同时优化酶解Buffer提高酶切效率以及增加酶量和引入多种酶以提高酶切效率等,最终确定了蛋白质前处理的最优条件,最终使用1μg样品在一次质谱分析鉴定到(2 425±7)个蛋白质,较未优化的方法提高了31%。优化后的蛋白质前处理方法可有效提高现有蛋白质组学的研究效率,可进一步应用于蛋白质的定量及动态分析研究。     

质谱MRM技术在蛋白质组学研究中的应用

质谱多反应监测（multiple reaction monitoring,MRM）技术是一种基于已知信息或假定信息有针对性地获取数据,进行质谱信号采集的技术,具有灵敏、准确和特异等优点。在基于蛋白组学的生物标志物研究、蛋白质翻译后修饰、定量蛋白质组和蛋白质相互作用等研究领域的应用逐渐受到重视。该文概述了该技术在蛋白质组学研究中的应用特点及其最新应用进展。     

质谱MRM技术在蛋白质组学研究中的应用

质谱多反应监测(multiple reaction monitoring,MRM)技术是一种基于已知信息或假定信息有针对性地获取数据,进行质谱信号采集的技术,具有灵敏、准确和特异等优点.在基于蛋白组学的生物标志物研究、蛋白质翻译后修饰.定量蛋白质组和蛋白质相互作用等研究领域的应用逐渐受到重视.该文概述了该技术在蛋白质组学研究中的应用特点及其最新应用进展.     

基于马尔科夫链的胰蛋白酶肽段蛋白酶切位点概率预测

在基于质谱技术的蛋白质鉴定过程中,数据库搜索是主要的方法。漏切位点和酶切规则决定了图谱候选肽段的范围,是数据库搜索算法的重要参数。对于常用的胰蛋白酶切来说,除了局部构象、三维结构、实验条件,以及其它偶然因素会影响赖氨酸K或者精氨酸R后的位点能否被酶切外,该位点附近的其它氨基酸也会影响蛋白水解酶的酶切效果。从质谱图谱中时常会鉴定出包含漏切位点的肽段,因此,预测蛋白质的酶切位点能够为数据库搜索算法提供更为可靠的模型,也能够为了解和分析蛋白质的酶切规律提供依据。本文提出了一种基于马尔科夫(Markov)链的预测方法,能够利用蛋白质的序列信息来预测候选酶切位点的酶切概率,在蛋白酶切过程中,预测肽段的覆盖率可以达到85%以上。     

蛋白质组学研究中的分离分析技术及其研究进展

在后基因组时代,蛋白质组学成为新的研究热点。蛋白质组学的研究目标是为复杂蛋白质样品建立一个高通量、大规模、自动化的分离分析技术平台,从而实现准确、快速地筛选功能蛋白质。蛋白质的分离分析在蛋白组学研究中起着非常重要的作用。本文主要综述在蛋白质组学研究中二维凝胶电泳、毛细管电泳及其与质谱联用、多维液相分离技术及其与质谱联用和蛋白质芯片等高效分离分析技术的应用研究进展。     

鸟枪法蛋白质鉴定质量控制方法研究进展

鸟枪法串联质谱蛋白质鉴定策略由于其高可靠和高效率而被广泛应用于蛋白质组学研究中,这种方法直接对蛋白质混合物进行酶切,以肽段为鉴定单元,继而推导真实的样品蛋白质．由于利用质谱图推导肽段存在一定的假阳性率,而且直接对蛋白质混合物的酶切也导致了肽段和蛋白质之间关联信息的丢失,所鉴定的蛋白质难免存在部分不可靠结果．因此,蛋白质鉴定的质量控制在蛋白质组学研究中极为重要．蛋白质鉴定的质量控制包含两大类主要方法,其一为利用肽段进行蛋白质组装,当前最常用也被证明最有效的方法是使用简约原则,即用最少的蛋白质解释所有鉴定肽段,现有的方法可以分为布尔型和概率型,其二为鉴定蛋白质的可靠性评估,包括单个蛋白质鉴定置信度和蛋白质鉴定整体水平的假阳性率计算．综合各种可辅助蛋白质鉴定的先验信息,构建普适的概率统计模型,是目前蛋白质鉴定质量控制方法的发展趋势．     

蛋白质胶内酶切技术研究进展

胶内酶切是蛋白质组研究中衔接电泳分离和质谱鉴定的重要环节,对最终的蛋白质定性和定量分析结果有显著的影响。该技术自1992年初步建立以来,一直处于不断完善中,出现了种类繁多的改进方案。为了更有效地利用胶内酶切技术,从凝胶脱色、杂质去除、蛋白酶切、肽段提取4个方面归纳整理了近年来蛋白质胶内酶切技术的主要研究进展。     

测序级胰蛋白酶的制备工艺与质量检测简

目的：制备高纯度、酶解效率高、酶切位点专一的测序级胰蛋白酶,应用于蛋白组学研究的蛋白质鉴定与分析中。方法：取实验室自制的猪胰蛋白酶粗酶,经硼氢化钠、甲醛还原甲基化修饰抑制胰蛋白酶自水解,采用高效液相色谱仪15RPC反相柱纯化,收集对应的甲基化胰蛋白酶峰组分,冷冻干燥;甲基化修饰的胰蛋白酶进一步经甲苯磺酰苯丙氨酰氯甲酮（TPCK）修饰,以抑制糜蛋白酶等非特异性酶切活性,并经反相色谱柱再纯化,获得终产物即质谱测序级胰蛋白酶;自制的测序级胰蛋白酶经SDS-PAGE、HPLC反相色谱分析、酶比活力测定,并应用于胶内蛋白质酶切质谱鉴定氨基酸序列等,检测其纯度、酶水解效率及酶切位点特异性。结果：自制甲基化TPCK修饰的测序级胰蛋白酶纯度大于95％,酶比活力为200U/mgP（TAME）以上,质谱分析酶切特异性好;且酶的制备工艺流程稳定,可应用于测序级胰蛋白酶产品的生产与开发中。结论：制备的测序级胰蛋白酶纯度高、酶解效率优、酶切特异性强,可广泛应用于实验室中蛋白质和肽段测序鉴定、HPLC肽段谱图分析等蛋白组学研究分析中。     

测序级胰蛋白酶的制备工艺与质量检测

目的:制备高纯度、酶解效率高、酶切位点专一的测序级胰蛋白酶,应用于蛋白组学研究的蛋白质鉴定与分析中。方法:取实验室自制的猪胰蛋白酶粗酶,经硼氢化钠、甲醛还原甲基化修饰抑制胰蛋白酶自水解,采用高效液相色谱仪15RPC反相柱纯化,收集对应的甲基化胰蛋白酶峰组分,冷冻干燥;甲基化修饰的胰蛋白酶进一步经甲苯磺酰苯丙氨酰氯甲酮(TPCK)修饰,以抑制糜蛋白酶等非特异性酶切活性,并经反相色谱柱再纯化,获得终产物即质谱测序级胰蛋白酶;自制的测序级胰蛋白酶经SDS-PAGE、HPLC反相色谱分析、酶比活力测定,并应用于胶内蛋白质酶切质谱鉴定氨基酸序列等,检测其纯度、酶水解效率及酶切位点特异性。结果:自制甲基化TPCK修饰的测序级胰蛋白酶纯度大于95%,酶比活力为200 U/mgP(TAME)以上,质谱分析酶切特异性好;且酶的制备工艺流程稳定,可应用于测序级胰蛋白酶产品的生产与开发中。结论:制备的测序级胰蛋白酶纯度高、酶解效率优、酶切特异性强,可广泛应用于实验室中蛋白质和肽段测序鉴定、HPLC肽段谱图分析等蛋白组学研究分析中。     

Automated in-solution protein digestion using a commonly available high-performance liquid chromatography autosampler

A completely automated peptide mapping liquid chromatography/mass spectrometry (LC/MS) system for characterization of therapeutic proteins in which a common high-performance liquid chromatography (HPLC) autosampler is used for automated sample preparation, including protein denaturation, reduction, alkylation, and enzymatic digestion, is described. The digested protein samples are then automatically subjected to LC/MS analysis using the same HPLC system. The system was used for peptide mapping of monoclonal antibodies (mAbs), known as a challenging group of therapeutic proteins for achieving complete coverage and quantitative representation of all peptides. Detailed sample preparation protocols, using an Agilent HPLC system, are described for Lys-C digestion of mAbs with intact disulfide bonds and tryptic digestion of mAbs after reduction and alkylation. The automated procedure of Lys-C digestion of nonreduced antibody, followed by postdigestion disulfide reduction, produces both the nonreduced and reduced digests that facilitate disulfide linkage analysis. The automated peptide mapping LC/MS system has great utility in preparing and analyzing multiple samples for protein characterization, identification, and quantification of posttranslational modifications during process and formulation development as well as for protein identity and quality control.     

A novel approach and protocol for discovering extremely low-abundance proteins in serum

The proteomic analysis of serum (plasma) has been a major approach to determining biomarkers essential for early disease diagnoses and drug discoveries. The determination of these biomarkers, however, is analytically challenging since the dynamic concentration range of serum proteins/peptides is extremely wide (more than 10 orders of magnitude). Thus, the reduction in sample complexity prior to proteomic analyses is essential, particularly in analyzing low-abundance protein biomarkers. Here, we demonstrate a novel approach to the proteomic analyses of human serum that uses an originally developed serum protein separation device and a sequentially linked 3-D-LC-MS/MS system. Our hollow-fiber-membrane-based serum pretreatment device can efficiently deplete high-molecular weight proteins and concentrate low-molecular weight proteins/peptides automatically within 1 h. Four independent analyses of healthy human sera pretreated using this unique device, followed by the 3-D-LC-MS/MS successfully produced 12 000-13 000 MS/MS spectra and hit around 1800 proteins (>95% reliability) and 2300 proteins (>80% reliability). We believe that the unique serum pretreatment device and proteomic analysis protocol reported here could be a powerful tool for searching physiological biomarkers by its high throughput (3.7 days per one sample analysis) and high performance of finding low abundant proteins from serum or plasma samples.     

Proteolysis and mass spectrometric analysis of an integral membrane: aquaporin 0

Due to hydrophobicity, structural analysis of integral membrane proteins poses a formidable challenge for current mass spectrometry-based proteomics approaches. Herein, we demonstrate results from optimized sample preparation and enzymatic proteolysis procedures for the complete primary structure determination of a targeted integral membrane protein, lens aquaporin 0 (AQP0). Plasma membrane from bovine lens tissue was alkali treated and tryptic digestion was performed in optimized acetonitrile-ammonium bicarbonate solution. Full sequence coverage of AQP0 was observed as tryptic peptides using both matrix-assisted laser desorption/ionization mass spectrometry (MALDI MS) and capillary liquid chromatography tandem mass spectrometry (cLC/MS/MS). An amino acid mutation of Thr to Ile/Leu at residue 199 was deduced based on MS/MS results. In a complementary effort to fully sequence the protein, peptic digestion was developed to take advantage of hydrophobic protein solubility in organic acid as well as the decreased activity of pepsin at low pH. Peptic digestion in 10% formic acid (pH 1.2) generated peptides of 500 to 3000 Da and gave 100% sequence coverage by cLC/MS/MS. In addition to post-translational modifications reported previously, a new phosphorylation site at serine 229 and two oxidation sites at tryptophan 202 and 205 were detected on the protein. These methodologies provide complementary detergent- and CNBr-free procedures for detailed analysis of this important membrane channel protein and offer promise for analysis of the integral membrane proteome.     

Improvement of an in-gel tryptic digestion method for matrix-assisted laser desorption/ionization-time of flight mass spectrometry peptide mapping by use of volatile solubilizing agents

The combination of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS), in-gel enzymatic digestion of proteins separated by two-dimensional gel electrophoresis and searches of molecular weight in peptide-mass databases is a powerful and well established method for protein identification in proteomics analysis. For successful protein identification by MALDI-TOF mass spectrometry of peptide mixtures, critical parameters include highly specific enzymatic cleavage, high mass accuracy and sufficient numbers and sequence coverage of the peptides which can be analyzed. For in-gel digestion with trypsin, the method employed should be compatible both with enzymatic cleavage and subsequent MALDI-TOF MS analysis. We report here an improved method for preparation of peptides for MALDI-TOF MS mass fingerprinting by using volatile solubilizing agents during the in-gel digestion procedure. Our study clearly demonstrates that modification of the in-gel digestion protocols by addition of dimethyl formamide (DMF) or a mixture of DMF/N,N-dimethyl acetamide at various concentrations can significantly increase the recovery of peptides. These higher yields of peptides resulted in more effective protein identification.     

Isolation of N-terminal protein sequence tags from cyanogen bromide cleaved proteins as a novel approach to investigate hydrophobic proteins

A novel method for the isolation of protein sequence tags to identify proteins in a complex mixture of hydrophobic proteins is described. The PST (Protein Sequence Tag) technology deals with the isolation and MS/MS based identification of one N-terminal peptide from each polypeptide fragment generated by cyanogen bromide cleavage of a mixture of proteins. PST sampling takes place after sub-cellular fractionation of a complex protein mixture to give enrichment of mitochondrial proteins. The method presented here combines effective sample preparation with a novel peptide isolation protocol involving chemical and enzymatic cleavage of proteins coupled to chemical labeling and selective capture procedures. The overall process has been very successful for the analysis of complex mixtures of hydrophobic proteins, particularly membrane proteins. This method substantially reduces the complexity of a protein digest by "sampling" the peptides present in the digest. The sampled digest is amenable to analysis by liquid chromatography tandem mass spectrometry (LC-MS/MS). Methods of "sampling" protein digests have great value' if they can provide sufficient information to identify substantially all of the proteins in the sample while reducing the complexity of the sample to maximize the efficient usage of LC-MS/MS capacity. The validity of the process is demonstrated for mitochondrial samples from S. cerevisiae. The proteins identified by the PST technology are compared to the proteins identified by the conventional technology 2-D gel electrophoresis as a control.     

Comparison of some new pretreatment methods for second generation bioethanol production from wheat straw and water hyacinth

Pretreatment of lignocellulosic residues like water hyacinth (WH) and wheat straw (WS) using crude glycerol (CG) and ionic liquids (IL) pretreatment was evaluated and compared with conventional dilute acid pretreatment (DAT) in terms of enzymatic hydrolysis yield and fermentation yield of pretreated samples. In the case of WS, 1-butyl-3-methylimidazolium acetate pretreatment was found to be the best method. The hydrolysis yields of glucose and total reducing sugars were 2.1 and 3.3 times respectively higher by IL pretreatment than DAT, while it was 1.4 and 1.9 times respectively higher with CG pretreatment. For WH sample, CG pretreatment was as effective as DAT and more effective than IL pretreatment regarding hydrolysis yield. The fermentation inhibition was not noticeable with both types of pretreatment methods and feedstocks. Besides, CG pretreatment was found as effective as pure glycerol pretreatment for both feedstocks. This opens up an attractive economic route for the utilization of CG.     

Isoforms of a cuticular protein from larvae of the meal beetle, Tenebrio molitor, studied by mass spectrometry in combination with Edman degradation and two-dimensional polyacrylamide gel electrophoresis.

Simultaneous sequencing, using a combination of mass spectrometry and Edman degradation, of three approximately 15-kDa variants of a cuticular protein extracted from the meal beetle Tenebrio molitor larva is demonstrated. The information obtained by matrix-assisted laser desorption ionization mass spectrometry (MALDI MS) time-course monitoring of enzymatic digests was found essential to identify the differences among the three variants and for alignment of the peptides in the sequence. To determine whether each individual insect larva contains all three protein variants, proteins extracted from single animals were separated by two-dimensional gel electrophoresis, electroeluted from the gel spots, and analyzed by MALDI MS. Molecular weights of the proteins present in each sample could be obtained, and mass spectrometric mapping of the peptides after digestion with trypsin gave additional information. The protein isoforms were found to be allelic variants.     

The proteomic reactor facilitates the analysis of affinity-purified proteins by mass spectrometry: application for identifying ubiquitinated proteins in human cells

Mass spectrometry (MS) coupled to affinity purification is a powerful approach for identifying protein-protein interactions and for mapping post-translational modifications. Prior to MS analysis, affinity-purified proteins are typically separated by gel electrophoresis, visualized with a protein stain, excised, and subjected to in-gel digestion. An inherent limitation of this series of steps is the loss of protein sample that occurs during gel processing. Although methods employing in-solution digestion have been reported, they generally suffer from poor reaction kinetics. In the present study, we demonstrate an application of a microfluidic processing device, termed the Proteomic Reactor, for enzymatic digestion of affinity-purified proteins for liquid chromatography tandem mass spectrometry (LC-MS/MS) analysis. Use of the Proteomic Reactor enabled the identification of numerous ubiquitinated proteins in a human cell line expressing reduced amounts of the ubiquitin-dependent chaperone, valosin-containing protein (VCP). The Proteomic Reactor is a novel technology that facilitates the analysis of affinity-purified proteins and has the potential to aid future biological studies.     

pH catalyzed pretreatment of corn bran for enhanced enzymatic arabinoxylan degradation

Corn bran is mainly made up of the pericarp of corn kernels and is a byproduct stream resulting from the wet milling step in corn starch processing. Through statistic modeling this study examined the optimization of pretreatment of corn bran for enzymatic hydrolysis. A low pH pretreatment (pH 2, 150 °C, 65 min) boosted the enzymatic release of xylose and glucose and maximized biomass solubilization. With more acidic pretreatment followed by enzymatic hydrolysis the total xylose release was maximized (at pH 1.3) reaching ～ 50% by weight of the original amount present in destarched corn bran, but the enzyme catalyzed xylose release was maximal after pretreatment at approx. pH 2. The total glucose release peaked after pretreatment of approx. pH 1.5 with an enzymatic release of approx. 68% by weight of the original amounts present in destarched corn bran. For arabinose the enzymatic release was negatively affected by the acidic pretreatment as labile arabinosyl-linkages were presumably hydrolysed directly during the pretreatment. A maximum of 60% arabinose release was achieved directly from the optimal (acidic) pretreatment. The total content of diferulic acids, supposedly involved in the cross-linking of the arabinoxylan polymers, decreased by both alkaline and acidic pretreatment pH, with the loss by alkaline pretreatments being highest. No direct correlation between the enzymatic release of xylose and the content of diferulic acids in the substrate could be verified. On the contrary the enzymatic release of xylose was significantly correlated to the total release of arabinose, indicating that the degree of arabinosyl-substitutions on the xylan backbone is an essential parameter for enzymatic hydrolysis of corn bran arabinoxylan.