蛋白质水解酶和样品预处理优化技术在蛋白质组学研究中的应用

蛋白质组学是全景式鉴定、定量蛋白质,并研究蛋白质功能的学科。基于高分辨质谱的鸟枪法蛋白组学研究技术首先利用不同的位点特异性蛋白酶对复杂蛋白质样品进行酶解,进而利用质谱获得蛋白质相关的定性和定量信息。为了获得高质量的质谱信息,前期的样品处理和质谱数据采集同样重要。本文对蛋白组学中常用的Trypsin、Lys-C、Glu-C等位点特异性蛋白酶的酶切特点进行了总结,并综述了目前常用的几种酶切组合策略和样本预处理技术在提高蛋白质组学研究效率中的作用。     

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。     

生物质谱技术在糖蛋白结构分析中的应用

生物质谱包括基质辅助激光解吸附飞行时间质谱及电喷雾质谱被广泛应用于生物样品如多肽、蛋白质及核酸的分析,由于这种具有软电离方式的质谱具有极高的灵敏度及准确度,目前也被成功地用于糖蛋白的结构分析,与普通的化学方法相比,质谱法快速、简单,结合网上数据库检索、凝集素亲和提取、二维凝胶电泳以及靶上直接酶切等新方法,可以提供糖蛋白的一级结构乃至高级结构的信息。     

液相色谱-质谱法测定纺织品中多种致敏性分散染料的残留量

应用液相色谱-质谱联用技术,建立了纺织品中多种致敏性分散染料残留量同时测定的快速分析方法。纺织品样品经甲醇提取后,过0.22μm滤膜,上样测定。前处理简便快捷,适合大批量样品分析。该方法定量限1 mg.L-1,线性范围1mg.L-1~100 mg.L-1,添加回收率不低于90%,RSD<5%。     

一种适用于质谱分析的简化胶内酶解方法

在常规方法的基础上,设计了一种适于质谱分析的简化胶内酶解方法。改进的步骤包括:(1)通过加大洗涤所用超纯水量、延长涡混时间来强化凝胶洗涤的环节;(2)加入酸化处理来提高胰蛋白酶的活性;(3)在预酶解时不加CaCl_2,减少了酶的自切作用;(4)省略了蛋白质样品脱盐、脱SDS的步骤;(5)直接吸取酶解液进行质谱分析。系统比较该简化酶解法和最新报道的一种酶解方法的质谱鉴定效果,简化法能有效减少酶解后肽段的损失,增加质谱数据库搜索的信息量,得到更可靠的蛋白质鉴定结果。     

高灵敏度电化学发光PCR方法检测基因点突变研究

近年来发展了一种用于定量检测基因点突变的电化学发光PCR方法。该法采用三联吡啶钌标记的上游引物和生物素标记的下游引物对待测基因进行PCR扩增;随后,采用特定的限制性内切酶对扩增产物进行酶切,由于野生型样品和突变型样品间存在酶切位点的变化,其中只有一种基因型样品能被切断;通过生物素与链霉亲和素包被的磁珠连接,将生物素标记的DNA片段收集到样品池中;进行电化学发光检测,通过所得信号的有无可以判断其基因型。我们分别将该法用于Presenilin-1基因和H-ras癌基因的点突变检测,结果均可明显区分突变型样品和野生型样品。该法具有灵敏、快速、简便、安全等优点,是一种实用的基因点突变检测方法。     

红树DNA的十六烷基三甲基溴化铵法提取及其随机扩增多态DNA反应

采用ＣＴＡＢ法提取了耐盐植物红树ＤＮＡ,所得ＤＮＡ样品的紫外吸收Ａ２５８／Ａ２８０比值为１．８９,纯度能符合限制性内切酶酶切、ＰＣＲ反应以及ＤＮＡ重组克隆等分子生物学操作的要求．方法简便,容易掌握,较普通的酚－氯仿法有明显的优点．还探讨了以ＣＴＡＢ法制备的红树ＤＮＡ为模板进行ＲＡＰＤ反应参数的优化组合     

对虾白斑综合征病毒基因组快速定性定量方法的建立

对于对虾白斑综合征病毒(white spot syndrome virus,WSSV)全基因组样品,使用限制性内切酶酶切以及荧光绝对定量的方法进行分析,建立了WSSV全基因组快速定性定量的方法。定性实验通过对GenBank中WSSV基因组序列深度分析,选择确定合适的限制性内切酶BamHI对基因组进行酶切,通过比对实际酶切结果和软件模拟酶切结果,以定性待测样品。定量实验使用荧光定量试剂盒,通过建立标准曲线的方法对未知浓度的WSSV基因组样品进行绝对定量。实验结果表明,结合使用酶切分析和荧光定量的方法可以准确、快速、方便、经济地对WSSV基因组样品进行定性定量分析,为进一步深入研究WSSV基因组奠定坚实基础。     

抗CD52单克隆抗体HPLC-肽图分析方法的建立

建立高效液相色谱(HPLC)-肽图分析方法,用于抗人CD52单克隆抗体的专属性鉴别。抗CD52单抗样品经盐酸胍变性、DTT还原,释放出的游离半胱氨酸残基进行烷基化。超滤置换酶切缓冲液后进行胰蛋白酶酶切并终止。色谱条件:采用Eclipse XDB-C18 4.6×250 mm 5μm(Aglient)色谱柱,0.1%TFA水溶液与0.1%TFA乙腈溶液为流动相,梯度洗脱,检测波长为214 nm,柱温为30℃;质谱条件:分析时长135 min;检测方式正离子,TOF;MS+扫描范围350-1 500 Da;Product Ion+扫描范围100-1 500 Da;质谱分辨率40 000;Exceeds,150 Cps。CD52单抗重链CDR1、CDR3、轻链CDR1对应肽段由质谱鉴定出。HPLC-肽图方法专属性验证显示辅料制剂及异种抗体对检测结果无干扰;精密度验证结果显示目标峰的峰面积RSD%均在1.7%-7.6%之间。且目标峰的保留时间RSD%均在0.1%-0.2%之间,小于5%的可接受标准;耐用性结果显示,3μg胰蛋白酶、37℃和18 h的酶切条件是最合适的样品处理条件。基于CDR相关肽段鉴别的HPLC-肽图分析方法可定性鉴定出抗CD52单抗,方法学验证结果显示该方法适用于抗人CD52单抗的专属性鉴别并可用于质量控制及批检验放行。     

固相萃取-超高效液相色谱-串联质谱法测定新鲜块菌子实体中α-雄烷醇

本研究建立了一种固相萃取-超高效液相色谱-串联质谱的检测方法,用于检测新鲜块菌子实体中α-雄烷醇(5α-雄甾-16-烯-3α-醇)的含量。新鲜块菌样品经无水乙醇提取,Qasis HLB柱萃取富集后,采用超高效液相色谱-串联质谱进行分析定量。方法学验证结果表明该方法的回收率为88.49%-92.22%;检出限为0.120 9 ng/mL,定量限为0.398 9 ng/mL。该方法简便、精确,适用于新鲜块菌中α-雄烷醇含量的测定。     

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.     

An in-gel digestion procedure that facilitates the identification of highly hydrophobic proteins by electrospray ionization-mass spectrometry analysis

A procedure is described for in-gel tryptic digestion of proteins that allows the direct analysis of eluted peptides in electrospray ionization (ESI) mass spectrometers without the need of a postdigestion desalting step. It is based on the following principles: (a) a thorough desalting of the protein in-gel before digestion that takes advantage of the excellent properties of acrylamide polymers for size exclusion separations, (b) exploiting the activity of trypsin in water, in the absence of inorganic buffers, and (c) a procedure for peptide extraction using solvents of proven efficacy with highly hydrophobic peptides. Quality of spectra and sequence coverage are equivalent to those obtained after digestion in ammonium bicarbonate for hydrophilic proteins detected with Coomassie blue, mass spectrometry-compatible silver or imidazole-zinc but are significantly superior for highly hydrophobic proteins, such as membrane proteins with several transmembrane domains. ATPase subunit 9 (GRAVY 1.446) is a membrane protein channel, lipid-binding protein for which both the conventional in-gel digestion protocol and in solution digestion failed. It was identified with very high sequence coverage. Sample handling after digestion is notably simplified as peptides are directly loaded into the ESI source without postdigestion processing, increasing the chances for the identification of hydrophobic peptides.     

Usage of electrostatic eliminator reduces human keratin contamination significantly in gel-based proteomics analysis

In the field of bottom-up proteomics, heavy contamination of human keratins could hinder the comprehensive protein identification, especially for the detection of low abundance proteins. In this study, we examined the keratin contamination in the four major experimental procedures in gel-based proteomic analysis including gel preparation, gel electrophoresis, gel staining, and in-gel digestion. We found that in-gel digestion procedure might be of importance corresponding to keratin contaminants compared to the other three ones. The human keratin contamination was reduced significantly by using an electrostatic eliminator during in-gel digestion, suggesting that static electricity built up on insulated experimental materials might be one of the essential causes of keratin contamination. We herein proposed a series of methods for improving experimental conditions and sample treatment in order to minimize the keratin contamination in an economical and practical way.     

Label-free mass spectrometry-based relative quantification of proteins separated by one-dimensional gel electrophoresis

Here we present a matrix-assisted laser desorption/ionization tandem time-of-flight (MALDI–TOF/TOF)-based label-free relative protein quantification strategy that involves sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) separation of proteins followed by in-gel trypsin digestion. The main problem encountered in gel-based protein quantification is the difficulty in achieving complete and consistent proteolytic digestion. To solve this problem, we developed a high-pressure-assisted in-gel trypsin digestion method that is based on pressure cycling technology (PCT). The PCT approach performed at least as well as the conventional overnight in-gel trypsin digestion approach in parameters such as number of peaks detected, number of peptides identified, and sequence coverage, and the digestion time was reduced to 45 min. The gel/mass spectrometry (MS)-based label-free protein quantification method presented in this work proved the applicability of the signal response factor concept for relative protein quantification previously demonstrated by other groups using the liquid chromatography (LC)/MS platform. By normalizing the average signal intensities of the three most intense peptides of each protein with the average intensities of spiked synthetic catalase tryptic peptides, which we used as an internal standard, we observed spot-to-spot and lane-to-lane coefficients of variation of less than 10 and 20%, respectively. We also demonstrated that the method can be used for determining the relative quantities of proteins comigrating during electrophoretic separation.     

Comparative study of workflows optimized for in-gel, in-solution, and on-filter proteolysis in the analysis of plasma membrane proteins

Proteomic studies of plasma membrane proteins are challenged by the limited solubility of these proteins and the limited activity of proteolytic enzymes in solubilizing agents such as SDS. In this work, we have evaluated three bottom-up workflows to obtain tryptic peptides from plasma membrane proteins solubilized with 2% SDS. The workflows are in-gel digestion, in-solution digestion, and on-filter digestion. The efficiencies of these strategies, optimized to employ different matrices for trypsin cleavage, were compared using a plasma membrane sample enriched from multiple myeloma cells using a nanoparticle pellicle. On the basis of the number of proteins identified, number of transmembrane proteins identified, hydrophobicity, and spectral count per protein, the workflow that uses in-gel digestion is the most advantageous approach for analysis of plasma membrane proteins.     

A new application of microwave technology to proteomics

Two-dimensional electrophoresis (2-DE) combined with mass spectrometry has significantly improved the possibilities of large-scale identification of proteins. However, 2-DE is limited by its inability to speed up the in-gel digestion process. We have developed a new approach to speed up the protein identification process utilizing microwave technology. Proteins excised from gels are subjected to in-gel digestion with endoprotease trypsin by microwave irradiation, which rapidly produces peptide fragments. The peptide fragments were further analyzed by matrix-assisted laser desorption/ionization technique for protein identification. The efficacy of this technique for protein mapping was demonstrated by the mass spectral analyses of the peptide fragmentation of several proteins, including lysozyme, albumin, conalbumin, and ribonuclease A. The method reduced the required time for in-gel digestion of proteins from 16 hours to as little as five minutes. This new application of microwave technology to protein identification will be an important advancement in biotechnology and proteome research.     

Sequencing of rat liver cytosolic proteins by matrix-assisted laser desorption ionization-quadrupole time-of-flight mass spectrometry following electrophoretic separation and extraction

A new technique is described that enables the direct determination of the complete or partial amino acid sequence of cytosolic proteins separated by gel electrophoresis and allows for the further observation of disease- or drug-induced posttranslational modifications. The procedure uses a two-phase extraction strategy (ethyl acetate/ammonium bicarbonate) for the efficient separation of proteins/peptides from an electrophoretic matrix and subsequent sequence analysis by matrix-assisted laser desorption ionization-quadrupole time-of-flight mass spectrometry. The method was tested using hepatocyte cytosolic proteins and compared to a complementary approach using direct solvent extraction from in-gel digests. Although the latter procedure identified the proteins, it did not enable complete amino acid sequence determination. In contrast, high sequence coverage was obtained using the peptide extraction procedure, without any apparent dependence on protein size. The technique minimized the chemically inconsistent modifications generated from in-gel digestion, thus aiding mass spectrometric interpretation and valid protein sequence identification.     

Ultra fast trypsin digestion of proteins by high intensity focused ultrasound

Proteolytic digestion of proteins in seconds under an ultrasonic field provided by high-intensity focused ultrasound (HIFU) has been achieved. Successful in-solution and in-gel tryptic digestion of proteins in 60 s or less was demonstrated by either MALDI-TOF mass spectrometry or liquid chromatography-electrospray ion trap mass spectrometry (RP-HPLC-ESI-IT-MS/MS). The efficiency of this new procedure for protein digestion compared favorably with those attained using conventional overnight incubation methods. The performance of the method was also demonstrated by the specific identification of three proteins in a whole proteome in less than 1 h. The method greatly reduces the time needed for protein digestion, is of easy implementation, environmental friendly, and economic. Adaptation of this method to on-line procedures and robotic platforms could have promising applications in the proteomics field.     

Analysis of modified apolipoprotein B-100 structures formed in oxidized low-density lipoprotein using LC-MS/MS

Oxidatively modified low-density lipoprotein (oxLDL) is one of the major factors involved in the development of atherosclerosis. Because of the insolubility of apolipoprotein B-100 (apoB-100) and the heterogeneous nature of oxidative modification, modified structures of apoB-100 in oxLDL are poorly understood. We applied an on-Membrane sample preparation procedure for LC-MS/MS analysis of apoB-100 proteins in native and modified low-density lipoprotein (LDL) samples to eliminate lipid components in the LDLs followed by collection of tryptic digests of apoB-100. Compared with a commonly used in-gel digestion protocol, the sample preparation procedure using PVDF membrane greatly increased the recovery of tryptic peptides and resulted in improved sequence coverage in the final analysis, which lead to the identification of modified amino acid residues in copper-induced oxLDL. A histidine residue modified by 4-hydroxynonenal, a major lipid peroxidation product, as well as oxidized histidine and tryptophan residues were detected. LC-MS/MS in combination with the on-Membrane sample preparation procedure is a useful method to analyze highly hydrophobic proteins such as apoB-100.     

In-gel digestion of proteins for internal sequence analysis after one- or two-dimensional gel electrophoresis.

We examined the different steps necessary for the enzymatic digestion of proteins in the polyacrylamide matrix after gel electrophoresis. As a result, we developed an improved method for obtaining peptides for internal sequence analysis from 1-2 micrograms of in-gel-digested proteins. The long washing-lyophilization-equilibration steps necessary to eliminate the dye, sodium dodecyl sulfate, and other gel-associated contaminants that perturb protein digestion in Coomassie blue-stained gels have been replaced by washing for 40 min with 50% acetonitrile, drying for 10 min at room temperature, and then rehydrating with a protease solution. The washing and drying steps result in a substantial reduction of the gel slice volume that, when next swollen in the protease solution, readily absorbs the enzyme, facilitating digestion. The Coomassie blue staining procedure has also been modified by reducing acetic acid and methanol concentrations in the staining solution and by eliminating acetic acid in the destaining solution. The peptides resulting from the in-gel digestion are easily recovered by passive elution, in excellent yields for structural characterization. This simple and rapid method has been successfully applied for the internal sequence analysis of membrane proteins from the rat mitochondria resolved in preparative two-dimensional gel electrophoresis.