基于不同提取方法的水稻叶片蛋白质组的二维液相色谱分离及高分辨质谱分析

建立了酚法提取-二维液相色谱分离-高分辨质谱分析水稻叶片蛋白质组的方法。水稻叶片蛋白质经过酚法提取,酶解肽段脱盐后用离线反相-反相二维液相色谱分离,然后用线性离子阱/静电场轨道阱组合式高分辨质谱分析,共鉴定到2712种蛋白质。比较了液相色谱分离系统（一维液相色谱与二维液相色谱）和水稻叶片蛋白质提取方法（酚法、十二烷基硫酸钠法（SDS法）和三氯乙酸/丙酮法（TCA/丙酮法））对鉴定蛋白质数量的影响,结果表明：在二维液相色谱条件下,酚法、SDS法和TCA/丙酮法鉴定到的蛋白质数目为2712、2415和1914,分别是一维液相色谱条件下鉴定到的蛋白质数目的2.7、2.5和1.9倍。二维液相色谱条件下,酚法鉴定到的蛋白质数目比SDS法和TCA/丙酮法分别多297和798。与SDS法和TCA/丙酮法相比,酚法不但鉴定到的蛋白质数量多,而且能够鉴定到一些极端蛋白质,如酸性、碱性及高等电点的蛋白质。此外,对二维液相色谱条件下3种蛋白质提取方法提取到的蛋白质进行生物学功能分类,发现3种方法鉴定到的蛋白质的功能存在互补性,但酚法鉴定到的蛋白质功能种类最多。该法为水稻蛋白质组学研究提供了技术支撑,同时也为其他作物的蛋白质组学研究技术提供重要的借鉴。     

反相高效液相色谱双梯度洗脱分离肽混合物及质谱分析

复杂肽段混合物的有效分离是高覆盖率地鉴定蛋白质混合物的前提。“鸟枪法”(Shotgun)蛋白质组学研究策略通常采用蛋白酶切、二维液相色谱-串联质谱分析肽段混合物从而鉴定蛋白质,其中高效率地分离肽段混合物是关键步骤之一。本文通过pH梯度结合有机溶剂梯度的反相高效液相色谱(RP-HPLC)进行一维液相色谱分离,按等时间间隔收集馏分并将一个梯度的前段的一个馏分与后段一个馏分混合,然后进行纳升级液相色谱-质谱联用(nanoRPLC-MS/MS)分析。将该方法应用于酵母蛋白质的分离和鉴定,实验结果为: 与常规的强阳离子色谱-反相液相色谱-质谱分离鉴定方法相比,采用pH梯度结合有机相梯度的RP-HPLC-RPLC-MS分离鉴定方法多鉴定到567个酵母蛋白质(簇,含有3035个唯一肽段);其中鉴定到肽段的pI分布范围为3.42～12.01,相对分子质量范围为587.67～3499.79;蛋白质的pI分布范围为3.82～12.19,相对分子质量范围为3446.55～432905。该结果表明这种方法在复杂体系蛋白质组分离鉴定中具有明显的优势,在蛋白质组学研究中有较好的应用前景。     

微柱高效液相色谱-质谱/质谱快速鉴定混合蛋白质新方法

发展了一种混合蛋白质快速鉴定的新方法.将几种蛋白质的混合物于热变性后直接在溶液中酶解,利用微柱高效液相色谱-离子阱串级质谱进行肽谱/氨基酸序列分析,并结合Mascot数据库搜索处理功能,实现了混合蛋白质快速准确的鉴定.     

比较纳升反相色谱-串联质谱与毛细管区带电泳-串联质谱用于自顶向下蛋白质组学

自顶向下蛋白质组学的一个重要难题是缺乏与质谱可以在线连用并且可以提供高效蛋白质分离的液相分离技术。毛细管区带电泳与纳升反相色谱都可以与质谱在线连用,并且在复杂蛋白质样品分析方面也都有了显著的提升。在这里,我们首次比较了先进的纳升反相色谱-串联质谱与毛细管区带电泳-串联质谱平台用于自顶向下蛋白质组学分析。相对于纳升反相色谱-质谱而言,毛细管区带电泳-质谱可以将标准蛋白质样品的消耗量降低10倍,而且保持与纳升反相色谱-质谱相当的蛋白质信号强度。有意思的是,与毛细管区带电泳-质谱相比,纳升反相色谱-质谱可以获得更高的蛋白质分子的气相价态。这个现象可能是由于反相流动相中的高浓度乙腈使得蛋白质变性的更加充分。从1微克的大肠杆菌蛋白质样品中,毛细管区带电泳-串联质谱可以鉴定到159个蛋白质和513个蛋白质变体,而纳升反相色谱-串联质谱仅鉴定到105个蛋白质和277个蛋白质变体。当将大肠杆菌蛋白质的上样量提高到8微克时,纳升反相色谱-串联质谱可以鉴定到245个蛋白质和1004个蛋白质变体。由于纳升反相色谱-串联质谱具有比毛细管区带电泳-串联质谱更高的上样量与更宽的分离窗口,当蛋白质样品量不受限制时,纳升反相色谱-串联质谱具有明显的优势。但是,在痕量样品分析方面,毛细管区带电泳-串联质谱具有更大的潜力。     

亲水作用-反相二维液相色谱串联质谱法鉴定水稻蛋白质

建立了亲水作用-反相二维液相色谱串联质谱分析水稻叶片蛋白质组学的方法。利用标准肽段系统分析了液相色谱流动相酸碱度对二维亲水作用-反相色谱系统正交性的影响。结果表明,第一维亲水作用色谱在碱性(pH 9.3)和第二维反相色谱在酸性(pH 3.3)的条件下,正交性最佳(R~2=0.34113)。在此基础上,结合馏分收集技术进一步评价了本测试系统在水稻叶片蛋白质分析中的正交性。结果表明,在所有馏分收集组分中,鉴定次数小于2次的水稻叶片肽段占总肽段数目的 50%以上,且一维液相色谱馏分收集的肽段在第二维色谱及质谱分离分析中,可以较好地分布在不同时间段的洗脱窗口,表明本研究建立的亲水作用-反相二维液相色谱串联质谱结合馏分收集技术在复杂水稻叶片蛋白质分离鉴定中可提供良好的的分离正交性。结合水稻蛋白质数据库检索,共鉴定出207345个肽段,归属于2930个蛋白质簇。     

多维色谱串联质谱分析肝癌患者血浆中的低丰度差异蛋白

建立了分析肝癌患者血浆中低丰度差异蛋白的新的多维色谱串联质谱方法.以5例肝细胞癌(HCC)患者血浆为研究对象,选用免疫亲和色谱Agilent MARS Spin Cartridge去除血浆中6种主要高丰度蛋白;离线RP-HPLC预分级血浆低丰度蛋白并收集差异表达蛋白组;液相色谱-芯片(HPLC-CHIP,包含一支纳升级Zorbax 300SB-C18富集柱和一支纳升级Zorbax 300SB-C18分析柱)富集、分离差异表达蛋白组胰酶酶解肽混合物,线性离子阱质谱在线分析,以Spectrum Mill MS Proteomics Workbench自动分析MS和MS/MS数据后在UniProtKB/SWISS-PORT数据库中进行搜索,鉴定得到27种差异蛋白,其中23种蛋白与各种疾病或肿瘤相关,IMP3、ARNT2和GRIP1可能成为诊断HCC的潜在生物标志物.     

气相色谱-离子阱质谱联用测定玩具中8种酯类致敏性芳香剂

建立了气相色谱-离子阱质谱联用测定玩具中丙烯酸乙酯等8种酯类致敏性芳香剂的方法。对于布绒和贴纸玩具样品,采用丙酮超声提取20 min后过0.45μm滤膜直接进样,对于塑料玩具样品,经相应溶剂提取,离心后,澄清溶液经Envi-carb石墨化碳固相萃取小柱净化,丙酮定容,过0.45μm滤膜后通过30 m HP-5 MS色谱柱分离,质谱进行检测,外标法定量。方法对于不同物质的定量限(LOQ)在0.05～8.0 mg/kg之间,线性范围为0.005～50 mg/L,在3个添加水平的平均回收率在80.2%～105.9%之间,RSD在0.7%～8.9%之间。该方法可用于玩具中丙烯酸乙酯等8种酯类致敏性芳香剂的检测。     

液相色谱-离子阱-飞行时间串联质谱同时检测肉制品中14种杂环胺

建立了液相色谱-离子阱-飞行时间串联质谱(LCMS - IT - TOF)同时测定肉制品中14种杂环胺( HAAs)残留量的分析方法.样品以乙腈提取,经Phenomenex strata-X-C小柱净化,甲醇-氨水(9:1)洗脱,氮吹至干,经1 mL乙腈-水(1:9)定容离心后上机测定,基质曲线内标法定量.结果表明,...     

气相色谱-离子阱质谱联用测定玩具中21种致敏性芳香剂

建立了气相色谱-离子阱质谱联用测定布绒、贴纸和塑料玩具中苯甲醇等21种致敏性芳香剂的方法。对于布绒和贴纸玩具样品,采用丙酮超声振荡提取20 min后过0.45 μm滤膜,经HP-1MS色谱柱(50 m×0.2 mm×0.5 μm)分离,离子阱质谱检测。对于塑料玩具样品,采取溶解-沉淀方式提取,经Envi-carb石墨化碳固相萃取小柱净化,旋蒸、氮吹浓缩,过0.45 μm滤膜后进行测定,外标法定量。方法对不同物质的定量限(LOQ)为0.02～40 mg/kg,线性范围为0.002～50 mg/L,低、中、高3个添加水平的平均回收率为82.2%～110.8%,相对标准偏差(RSD)为0.6%～10.5%。该方法准确、灵敏,可用于玩具中苯甲醇等21种致敏性芳香剂含量的检测。     

高效液相色谱-串联质谱法结合稳定同位素标记肽段同时测定稻米及其制品中3种过敏蛋白质

基于稳定同位素标记特征肽段和液相色谱-质谱联用仪建立稻米及制品中3种过敏蛋白质的同时定量方法。稻米及制品样品经盐溶液提取,赖氨酰基内切酶(Lys-C)和胰蛋白酶依次水解,C18-SD柱净化后,采用纳升高效液相色谱-线性离子阱-静电场轨道阱(NanoLC-LTQ-Orbitrap)采集和Protein Discovery软件鉴定,NCBI和Uniprot数据库的基本局部搜索比对工具(BLAST)筛选验证,最终获得表征稻米及制品中α-淀粉酶/胰蛋白酶抑制剂类蛋白质(seed allergenic protein RAG2, RAG2)、乙二醛酶Ⅰ活性蛋白(glyoxalase Ⅰ)和α-球蛋白(19 kDa globulin)3种过敏蛋白质的特异性肽段。3个特异性肽段经液相色谱梯度洗脱,在Poroshell色谱柱上实现完全分离,由三重四极杆质谱仪分析。实验通过优化多反应监测(MRM)质谱参数,比较不同溶剂体系、水解酶种类和酶量等酶解条件,结合内标法定量,实现对稻米及制品中3种蛋白质的绝对定量。实验结果表明,当酶解溶剂中含1 g/L十二烷基硫酸钠,采用Lys-C和胰蛋白酶组合消化策略,可有效提高3种蛋白质的酶切效率至65.7%~97.3%。该方法在1~200 nmol/L范围内线性关系良好,相关系数均大于0.9972, 3种蛋白质的检出限和定量限分别为3 mg/kg和10 mg/kg。3种蛋白质在空白稻米制品基质中3个水平下的加标回收率为80.6%~103.7%,日间和日内精密度均小于11.5%。该方法稳定性好,检测灵敏度高,操作简便,在分析各类稻米及制品中3种过敏蛋白质含量具有广泛的应用前景。     

Evaluation of the combinative application of SDS and sodium deoxycholate to the LC–MS‐based shotgun analysis of membrane proteomes

SDS and sodium deoxycholate (SDC) as two representative detergents have been widely used in LC–MS/MS‐based shotgun analysis of membrane proteomes. However, some inherent disadvantages limit their applications such as interference with MS analysis or their weak ability to disrupt membranes. To address this, the combinative application of SDS and SDC was developed and evaluated in our study, which comprehensively used the strong ability of SDS to lyse membranes and solubilize hydrophobic membrane proteins, and the high efficiencies of an optimized acetone precipitation method and SDC in sample clean‐up, protein recovery, and redissolution and digestion of precipitated proteins. The comparative study using a rat‐liver‐membrane‐enriched sample showed that, compared with other three commonly used methods including the filter‐aided sample preparation strategy, the combinative method not only increased the identified number of total proteins, membrane proteins, and integral membrane proteins by an average of 19.8, 23.9, and 24.8%, respectively, but also led to the identification of the highest number of matching peptides. All these results demonstrate that the method yielded better recovery and reliability in the identification of the proteins especially highly hydrophobic integral membrane proteins than the other three methods, and thereby has more potential in shotgun membrane proteomics.     

用液相等电聚焦电泳纯化藻蓝蛋白亚基

以纯藻蓝蛋白(C-phycocyanin, C-PC)为材料, 采用Rotofor系统进行液相等电聚焦(Liquid-phase isoeletric focusing, LP-IEF)电泳纯化C-PC的α, β亚基, 探讨蛋白质亚基纯化的制备电泳(Preparative eletrophoresis)技术. 结果显示, 样品经2次等电聚焦电泳后, C-PC 的α, β亚基分别浓集在pH=4.9和pH=4.1附近, 平板超薄等电聚焦(Slab ultra thin IEF)和SDS-PAGE电泳鉴定表明分别为高纯度的C-PC α, β亚基. 提示LP-IEF是分离纯化等电点差异蛋白质活性亚基的简便有效的方法.     

Use of chromatofocusing for separation of beta-lactamases. IX. Analytical chromatofocusing for the separation of a chromosomal cephalosporinase from Proteus vulgaris 1028

Simultaneous purification and isoelectric point (pI) determination was carried out at analytical scale of the chromosomal cephalosporinase from the Proteus vulgaris 1028 strain. Comparison of the enzyme to the purification results with m-aminophenylboronic acid-agarose affinity chromatography with sodium dodecyl sulphate-polyacrylamide gel electrophoresis revealed that minute amounts of accompanying proteins having identical pI values but different molecular masses were found in the chromatofocused preparation. The molecular mass of the enzyme was 24,000 dalton. The pI was found to be 8.3.     

Efficient and non-denaturing membrane solubilization combined with enrichment of membrane protein complexes by detergent/polymer aqueous two-phase partitioning for proteome analysis

It is of central interest in membrane proteomics to establish methods that combine efficient solubilization with enrichment of proteins and intact protein complexes. We have investigated the quantitative and qualitative solubilization efficiency of five commercially available detergents using mitochondria from the yeast Saccharomyces cerevisiae as model system. Combining the zwitterionic detergent Zwittergent 3-10 and the non-ionic detergent Triton X-114 resulted in a complementary solubilization of proteins, which was similar to that of the anionic detergent sodium dodecyl sulfate (SDS). The subsequent removal of soluble proteins by detergent/polymer two-phase system partitioning was further enhanced by addition of SDS and increasing pH. A large number of both integral and peripheral membrane protein subunits from mitochondrial membrane protein complexes were identified in the detergent phase. We suggest that the optimized solubilization protocol in combination with detergent/polymer two-phase partitioning is a mild and efficient method for initial enrichment of membrane proteins and membrane protein complexes in proteomic studies.     

Substructure of human erythrocyte spectrin.

The human erythrocyte structural protein spectrin and its subunits I, II were isolated in the presence of Na-dodecyl-sulfate by gel filtration and preparative gel electrophoresis. After removal of the detergent, spectrin alpha-helical content is comparable to spectrin isolated without detergent. Subunits I and II formed single bands in isoelectric focusing (pI = 5.6) and in Ornstein-Davis disc gel electrophoresis systems, indicating the individual subunits are homogenous in nature. The molecular weights of the subunits I and II, determined by Ferguson plot, are 237,500 and 238,600, respectively, which is in good agreement with values obtained by the standard SDS gel relative mobility method. Limited tryptic digestion of spectrin and two-dimensional peptide maps of the individual subunits cleaved by S-cyanylation reaction showed dissimilar patterns, suggesting differences in primary structure between the two subunits.     

Capillary sodium dodecyl sulfate-DALT electrophoresis with laser-induced fluorescence detection for size-based analysis of proteins in human colon cancer cells

Capillary sodium dodecyl sulfate (SDS)-DALT electrophoresis (SDS-DALT-CE) refers to CE separation of proteins based on their size; DALT is the abbreviation for Dalton, the unit used to describe molecular weight. In this work, seven proteins from 18 to 116 kDa were denatured by SDS, labeled by 3-(2-furoyl) quinoline-2-carboxaldehyde, separated by SDS-DALT-CE in polyethylene oxide sieving matrix, and detected by laser-induced fluorescence (LIF) in a sheath flow cuvette. This method was combined with detergent differential fractionation, which is a protein fractionation method using a series of detergent-containing buffers to sequentially extract protein fractions from cells, to analyze the proteins in HT29 human colon adenocarcinoma cells. In addition, on-column labeling was demonstrated for protein analysis by SDS-DALT-CE with LIF, and applied to analysis of proteins in a single HT29 cancer cell. Most proteins had molecular masses from 10 to 120 kDa. Similar protein profiles were obtained for single cells and protein extract of a large cell population.     

Identification of proteins from membrane preparations by a combination of MALDI TOF-TOF and LC-coupled linear ion trap MS analysis of an Antarctic bacterium Pseudomonas syringae Lz4W, a strain with unsequenced genome

Multidimensional protein identification technology helps in identifying a large number of proteins with ESI by sequencing several peptides with MS/MS methods. When ionization and separation of different hydrophobic and hydrophilic peptides in a single process are difficult, a combination of LC-coupled linear ion trap MS and MALDI TOF/TOF can be used for identification of proteins as shown in the present study. We have used this combinational approach to identify membrane proteins of the Antarctic bacterium Pseudomonas syringae Lz4W, which are separated by SDS gel electrophoresis. Although the genome of P. syringae Lz4W has not been sequenced, the known genome sequences of mesophilic Pseudomonas species have been used for the identification of the proteins. Broadly, many membrane proteins, proteins with a wide range of molecular weight and pI including some integral membrane proteins could be identified using this procedure. Some of the identified proteins are involved in low temperature adaptation.     

Assessing matrix assisted laser desorption/ ionization-time of flight-mass spectrometry as a means of rapid embryo protein identification in rice.

Rice embryo proteins were separated by two-dimensional gel electrophoresis (2-DE). A total of 105 spots were digested with trypsin and the resultant peptides were analyzed by matrix assisted laser desorption/ionization-time of flight-mass spectrometry (MALDI-TOF-MS). Raw mass spectra were fully-automatically processed and searched with selected monoisotopic masses against SWISS-PROT/TrEMBL and NCBInr databases. High quality mass spectra were obtained from 53 spots, of which 36 spots were identified including 29 not registered in databases. Fifty percent of the rice embryo proteins resolved in 2-DE could not be identified, indicating more efficient sample preparation techniques need to be developed in the future. At least four to five matching peptides were found to be essential for unambiguous identification of rice embryo proteins; peptide matching of less than four lead to ambiguous results. The suitability of peptide mass fingerprinting method as a means of rapid embryo protein identification in rice was discussed.     

Novel staining-free proteomic method for simultaneous identification of proteins and determination of their pI values by using low-molecular-mass pI markers

A new proteomic staining-free method for simultaneous identification of proteins and determination of their pI values by using low-molecular-mass pI markers is described. It is based on separation of proteins in gels by IEF in combination with mass spectrometric analysis of both peptides derived by in-gel digestion and low-molecular-mass pI markers extracted form the same piece excised from the gel. In this method, the pI markers are mixed with a protein mixture (a commercial malted barley protein extract) deposited on a gel and separated in a pH gradient. Color pI markers enable supervision of progress of focusing process. Several separated bands of the pI markers (including separated proteins) were excised and the pI markers were eluted from each gel piece by water/ethanol and identified by MALDI-TOF/TOF MS. The remaining carrier ampholytes were then washed out from gel pieces and proteins were in-gel digested with trypsin or chymotrypsin. Obtained peptides were measured by MALDI-TOF/TOF MS and proteins were identified via protein database search. This procedure allows omitting time-consuming protein staining and destaining procedures, which shortens the analysis time. For comparison, other IEF gels were stained with CBB R 250 and proteins in the gel bands were identified. Similarity of the results confirmed that our approach can give information about the correct pI values of particular proteins in complex samples at significantly shorter analysis times. This method can be very useful for identification of proteins and their post-translational modifications in prefractioned samples, where post-translational modifications (e.g., glycation) are frequent.