Evaluation of sample preparation methods for MALDI-TOF MS identification of highly dangerous bacteria

Aims: To propose a universal workflow of sample preparation method for the identification of highly pathogenic bacteria by MALDI‐TOF MS. Methods and Results: Fifteen bacterial species, including highly virulent Gram‐positive (Bacillus anthracis and Clostridium botulinum) and Gram‐negative bacteria (Brucella melitensis, Burkholderia mallei, Francisella tularensis, Shigella dysenteriae, Vibrio cholerae, Yersinia pestis and Legionella pneumophila), were employed in the comparative study of four sample preparation methods compatible with MALDI‐TOF MS. The yield of bacterial proteins was determined by spectrophotometry, and the quality of the mass spectra, recorded in linear mode in the range of 2000–20 000 Da, was evaluated with respect to the information content (number of signals) and quality (S/N ratio). Conclusions: Based on the values of protein concentration and spectral quality, the method using combination of ethanol treatment followed by extraction with formic acid and acetonitrile was the most efficient sample preparation method for the identification of highly pathogenic bacteria using MALDI‐TOF MS. Significance and Impact of the Study: The method using ethanol/formic acid generally shows the highest extraction efficacy and the spectral quality with no detrimental effect caused by storage. Thus, this can be considered as a universal sample preparation method for the identification of highly virulent micro‐organisms by MALDI‐TOF mass spectrometry.     

双向凝胶电泳银染蛋白质点的肽质谱指纹图分析

对双向凝胶电泳后银染显色的蛋白质点经脱色与原位还原和烷基化处理后,用ＴＰＣＫ胰蛋白酶进行酶解,采用带有Ｃ１８反相载体的ＺｉｐＴｉｐ＾ＴＭ吸头进行脱盐处理,再进行ＭＡＬＤＩ－ＴＯＦ肽质谱指纹纹图分析,然后将肽质数据在ＥＭＢＬ数据库中进行搜寻从而对蛋白南点进行鉴定。结果表明用该实验程序可对银染的单一蛋白南点进行快速肽质谱指纹图ｉｐＴｉｐ＾ＴＭ的应用可以明显增加质谱分析的信噪比,提高分析灵敏度。用以上方     

温和过甲酸氧化辅助酶解结合液质联用技术鉴定大鼠大脑皮层质膜蛋白质

由于膜蛋白质尤其是内在膜蛋白的强疏水性,分析和鉴定质膜蛋白质仍然是以质谱为基础的蛋白质组学的方法中的一个难点.过甲酸氧化是一种应用广泛的打开二硫键的方法,温和的过甲酸试剂能完全的将半胱氨酸转化为半胱磺酸,将甲硫氨酸转化为甲硫氨酸砜,从而使目的蛋白更易溶于水介质.采用蔗糖密度梯度离心法纯化得到大鼠大脑皮层质膜,提取的质膜蛋白质经温和过甲酸氧化处理后经胰酶酶解消化得到肽段,利用LC-MS/MS对所得肽段进行质谱分析,采集的原始数据用Mascot软件进行库搜寻鉴定.此方法是研究质膜蛋白质的新方法,温和过甲酸氧化显示出很好的氧化效果却避免其它不利于鉴定的副反应.从大鼠大脑皮层膜提取物共鉴定出220种蛋白质,其中73种为整合膜蛋白,证明对质膜蛋白质直接进行温和过甲酸氧化然后酶解的方法辅助酶解可以有效的鉴定质膜蛋白质.     

纳升电喷雾串联质谱技术对阻断泛素通路诱导凋亡相关蛋白质的鉴定

应用毛细管液相色谱 电喷雾 四极杆 飞行时间串联质谱和纳升电喷雾 四极杆 飞行时间串联质谱技术 ,对阻断白血病细胞泛素通路诱发的凋亡相关蛋白质进行了鉴定。通过双向电泳发现 ,蛋白质斑点H在阻断Mo7e白血病细胞泛素通路 2h之后 ,表达量明显增加 ,6h达到最高。该斑点经MALDI TOF MS肽质量指纹谱分析未获结果 ,但通过上述 2种串联质谱技术获得其胰蛋白酶水解肽段的串联质谱图和肽段的全长序列 ,经检索均确认为RhoGDIβ蛋白。进一步发现阻断泛素通路还诱发了另 2个斑点出现 ,位于H点附近 ,经鉴定为同一蛋白质 ,可能是不同翻译后修饰所造成     

Determination of Biotinylated Proteins as an Index for Purification of Plasma Membrane using Surface Plasmon Resonance-based Optical Biosensor

Proteins of plasma membrane could be an index of purification of the plasma membrane of animal cells. A convenient method is proposed for determining the plasma membrane proteins by a surface plasmon resonance (SPR) biosensor. Biotinylated proteins were observed only in the peripheral areas of MOLT-4 cells which were treated by 5-[5-(N-succinimidyloxycarbonyl) pentylamido] hexyl-d-biotinamide. The proteins on HeLa cells were also biotinylated. And then the membrane samples of the HeLa cells were injected onto the avidin-immobilized SPR-surface, and components bound non-specifically on the surface were removed by a washout solution. The amount of biotinylated protein (BP) was determined directly from the absolute resonance unit (RU) after injection of the washout solution. In the method a reference surface was not needed. The amount of BP bound to the surface was gradually attenuated with the repeated injection, and a method for calibrating the RU value was introduced by considering the ratio of attenuation by every injection. The correlation between the BP titer calculated by the calibration and the theoretically-estimated one was greatly improved. Three cycles of the BP determination on a sensor surface was performed successfully. During the purification process of membrane fractions, the degree of purification as judged by the BP titer was in good agreement with the degree of increase in aminopeptidase N activity in the membrane fraction. Thus, the BP titer could be used as an index for purification of plasma membrane.     

不同失水状态发菜类囊体膜蛋白的分离及鉴定

采用液氮研磨与超声波破碎相结合的方法,破碎不同失水状态的发菜藻体和细胞,用差速离心法制备发菜类囊体膜粗制品,蔗糖密度梯度高速离心纯化类囊体膜,SDS-PAGE电泳分离类囊体膜蛋白,并对膜蛋白进行了MALDI-TOF-TOF/MS质谱分析和鉴定。结果表明:(1)充分吸胀4h后失水6h的发菜(含水量51.2%)和失水24h的发菜(含水量14.9%),经过多步差速离心后再进行蔗糖密度梯度高速离心,可得到纯化的类囊体膜。(2)发菜类囊体膜蛋白SDS-PAGE电泳分离到14个条带,共鉴定出8种蛋白,根据其功能可分为4类——光合作用相关蛋白(光系统Ⅱ锰稳定蛋白PsbO,F1F0 ATP合成酶α亚基和β亚基)、结构域蛋白、选择性通道蛋白OprB、未知蛋白(hypothetical protein Npun_R1321、Npun_R3785、N9414_02186),它们在发菜的光合作用中具有重要作用。     

Proteome Analysis of Light-induced Proteins in Synechocystis sp. PCC 6803: Identification of Proteins Separated by 2D-PAGE Using N-terminal Sequencing and MALDI-TOF MS

The cyanobacterium Synechocystis sp. PCC 6803 is an ideal model organism for the proteome study of light-induced gene expression because the whole genomic sequence has been determined. The soluble proteins extracted from light- and dark-cultured cells were separated by two-dimensional polyacrylamide gel electrophoresis. Light-induced protein spots electroblotted on a polyvinyldiene difluoride membrane were analyzed by N-terminal Edman sequence determination and followed by CyanoBase. The tryptic digests of some proteins were also confirmed by matrix-assisted laser desorption ionization/time-of-flight (MALDI-TOF) and MS-Fit search. Interestingly, eight proteins were related to photosynthesis and respiration (RbcS/L, CbbA, Gap2, AtpB, CpcB, PsbO, and PsbU). Four proteins (SodB, DnaK, GroEL2, and Tig) were involved in cellular processes and the functions of another two proteins (rehydrin and membrane protein) were unknown. The proteome analysis by N-terminal Edman sequencing and MALDI-TOF enabled us to characterize one-shot protein profiles expressed under different physiological conditions.     

An update on the routine application of MALDI-TOF MS in clinical microbiology

Introduction: Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) has entered clinical diagnostics and is today a generally accepted and integral part of the workflow for microbial identification. MALDI-TOF MS identification systems received approval from national and international institutions, such as the USA-FDA, and are continuously improved and adopted to other fields like veterinary and industrial microbiology. The question is whether MALDI-TOF MS also has the potential to replace other conventional and molecular techniques operated in routine diagnostic laboratories.

Areas covered: We give an overview of new advancements of mass spectral analysis in the context of microbial diagnostics. In particular, the expansion of databases to increase the range of readily identifiable bacteria and fungi, the refined discrimination of species complexes, subspecies, and types, the testing for antibiotic resistance or susceptibility, progress in sample preparation including automation, and applications of other mass spectrometry techniques are discussed.

Expert opinion: Although many new approaches of MALDI-TOF MS are still in the stage of proof of principle, it is expectable that MALDI-TOF MS will expand its role in the clinical microbiology laboratory of the future. New databases, instruments and analytical software modules will continue to be developed to further improve diagnostic efficacy.     

Proteome Analysis of Light-induced Proteins in Synechocystis sp. PCC 6803: Identification of Proteins Separated by 2D-PAGE Using N-terminal Sequencing and MALDI-TOF MS

The cyanobacterium Synechocystis sp. PCC 6803 is an ideal model organism for the proteome study of light-induced gene expression because the whole genomic sequence has been determined. The soluble proteins extracted from light- and dark-cultured cells were separated by two-dimensional polyacrylamide gel electrophoresis. Light-induced protein spots electroblotted on a polyvinyldiene difluoride membrane were analyzed by N-terminal Edman sequence determination and followed by CyanoBase. The tryptic digests of some proteins were also confirmed by matrix-assisted laser desorption ionization/time-of-flight (MALDI-TOF) and MS-Fit search. Interestingly, eight proteins were related to photosynthesis and respiration (RbcS/L, CbbA, Gap2, AtpB, CpcB, PsbO, and PsbU). Four proteins (SodB, DnaK, GroEL2, and Tig) were involved in cellular processes and the functions of another two proteins (rehydrin and membrane protein) were unknown. The proteome analysis by N-terminal Edman sequencing and MALDI-TOF enabled us to characterize one-shot protein profiles expressed under different physiological conditions.     

Labeling elastase digests with TMT: informational gain by identification of poorly detectable peptides with MALDI-TOF/TOF mass spectrometry

The applicability of the less specific protease elastase for the identification of membrane and cytosolic proteins has already been demonstrated. MALDI as ionization technique particularly favors the detection of basic and to a lesser extent of weakly acidic peptides, whereas neutral peptides often remain undetected. Moreover, peptides below 700 Da are routinely excluded. In the following study, the advantage of additional information gained from tandem mass tag zero labeled peptides and the resultant increase in sequence coverage was evaluated. Through derivatization with tandem mass tag reagents, peptide measurement within the standard mass range of the MALDI reflector mode is achievable due to the mass increase. Compared to the unlabeled sample, peptides exhibiting relatively low molecular masses, pI values or higher hydrophobicity could be identified.     

Expression of proteins containing disulfide bonds in an insect cell-free system and confirmation of their arrangements by MALDI-TOF MS

Escherichia coli alkaline phosphatase (AP) and human lysozyme (h-LYZ), which contain two and four disulfide bonds, respectively, were expressed in a cell-free protein synthesis system constructed from Spodoptera frugiperda 21 (Sf21) cells. AP was expressed in a soluble and active form using the insect cell-free system under non-reducing conditions, and h-LYZ was expressed in a soluble and active form under non-reducing conditions after addition of reduced glutathione (GSH), oxidized glutathione (GSSG), and protein disulfide isomerase (PDI). The in vitro synthesized proteins were purified by means of a Strep-tag attached to their C termini. Approximately 41 microg AP and 30 microg h-LYZ were obtained from 1 mL each of the reaction mixture. The efficiency of protein synthesis approached that measured under reducing conditions. Analysis of the disulfide bond arrangements by MALDI-TOF MS showed that disulfide linkages identical to those observed in the wild-type proteins were formed.     

固相化学辅助MALDI-TOF质谱源后衰变技术对2D-胶银染蛋白质点的鉴定

利用O 甲基异脲氢硫酸 (O methylisoureahydrogensulfate)修饰 2D 胶上胰蛋白酶原位酶切后产生的Lys 肽 ,使其具有Arg 肽的特性 ,提高质谱测定的灵敏度 ,然后用化学试剂 3 磺酸丙酸N 羟基琥珀酰胺酯 ( 3 sulfopropionicacidNHS ester)修饰Lys 肽及Arg 肽 ,修饰的产物在进行PSD测序时能得到单一的y 离子系列 ,有利于蛋白质酶解片段序列的从头解析 ,为 2D 胶上蛋白质点的准确鉴定提供有力的依据 ;该方法应用于鼻咽癌细胞 2D胶银染蛋白质点的鉴定取得可靠结果     

雪莲愈伤组织蛋白质提取及双向电泳分析

以产自西藏绵头雪莲为材料,分别采用TCA/丙酮法、尿素法和酚法,提取雪莲愈伤组织蛋白质并进行双向电泳,对蛋白产量和纯度以及电泳图谱进行比较。结果表明:(1)酚法较TCA/丙酮法和尿素法获得的蛋白质更纯,杂质少,蛋白点多且分辨率较高,在二维电泳图谱中背景清晰,横纹和纵纹较少。(2)对酚法提取的蛋白质样品进行上样量的比较结果显示,17cm胶条500μg上样量二维图谱的背景和蛋白点分布较好。(3)用0℃处理雪莲愈伤组织12h,利用建立的双向电泳体系分析结果发现,有33个蛋白点比常温(23℃)上调1.5倍表达,有5个蛋白点的表达下调2倍。(4)质谱鉴定结果表明,低温诱导的蛋白包括NADP-依赖型异柠檬酸脱氢酶、腺苷高半胱氨酸酶、抗性RPP8类蛋白、蛋白点gi|13129470、α-微管蛋白,分别与新陈代谢、植物防御、能量代谢、细胞的结构蛋白相关。     

An improved method of sample preparation on AnchorChip targets for MALDI-MS and MS/MS and its application in the liver proteome project

An improved method for sample preparation for MALDI-MS and MS/MS using AnchorChip targets is presented. The method, termed the SMW method (sample, matrix wash), results in better sensitivity for peptide mass fingerprinting as well as for sequencing by MS/MS than previously published methods. The method allows up-concentration and desalting directly on the mass spectrometric target and should be amenable for automation. A draw back caused by extensive oxidation of methionine and tryptophan in the SMW method can be alleviated by the addition of n-octyl glucopyranoside and DTT to the sample solution. The method was validated for protein identification from a 2-DE based liver proteome study. The SMW method resulted in identification of many more proteins and in most cases with a better score than the previously published methods.     

Identification of Plasma Membrane Glycoproteins Specific to Human Glioblastoma Multiforme Cells Using Lectin Arrays and LC‐MS/MS

Glioblastoma, also known as glioblastoma multiforme (GBM), is the most malignant type of brain cancer and has poor prognosis with a median survival of less than one year. While the structural changes of tumor cell surface carbohydrates are known to be associated with invasive behavior of tumor cells, the cell surface glycoproteins to differentiate the low‐ and high‐grade glioma cells can be potential diagnostic markers and therapeutic targets for GBMs. In the present study, lectin arrays consisting of eight lectins were employed to explore cell surface carbohydrate expression patterns on low‐grade oligodendroglioma cells (Hs683) and GBM cells (T98G). Griffonia simplicifolia I (GS I) was found to selectively bind to T98G cells and not to Hs683 cells. For identification of the glioblastoma‐specific cell surface markers, the glycoproteins from each cell type were captured by a GS I lectin column and analyzed by LC‐MS/MS. The identified proteins from the two cell types were quantified using label‐free quantitative analysis based on spectral counting. Of cell surface glycoproteins showing significant increases in T98G cells, five proteins were selected for verification of both protein and glycosylation level changes using Western blot and GS I lectin‐based immunosorbent assay.     

Binding of copper (II) ion to an Alzheimer's tau peptide as revealed by MALDI-TOF MS, CD, and NMR

The tau protein plays an important role in some neurodegenerative diseases including Alzheimer's disease (AD). Neurofibrillary tangles (NFTs), a biological marker for AD, are aggregates of bundles of paired helical filaments (PHFs). In general, the alpha-sheet structure favors aberrant protein aggregates. However, some reports have shown that the alpha-helix structure is capable of triggering the formation of aberrant tau protein aggregates and PHFs have a high alpha-helix content. In addition, the third repeat fragment in the four-repeat microtubule-binding domain of the tau protein (residues 306-336: VQIVYKPVDLSKVTSKCGSLGNIHHKPGGGQ, according to the longest tau protein) adopts a helical structure in trifluoroethanol (TFE) and may be a self-assembly model in the tau protein. In the human brain, there is a very small quantity of copper, which performs an important function. In our study, by means of matrix assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF MS), circular dichroism (CD), and nuclear magnetic resonance (NMR) spectroscopy, the binding properties of copper (II) ion to the R3 peptide derived from the third repeat fragment (residues 318-335: VTSKCGSLGNIHHKPGGG) have been investigated. The results show that copper ions bind to the R3 peptide. CD spectra, ultraviolet (UV)-visible absorption spectra, and MALDI-TOF MS show pH dependence and stoichiometry of Cu2+ binding. Furthermore, CD spectra and NMR spectroscopy elucidate the copper binding sites located in the R3 peptide. Finally, CD spectra reveal that the R3 peptide adopts a mixture structure of random structures, alpha-helices, and beta-turns in aqueous solutions at physiological pH. At pH 7.5, the addition of 0.25 mol eq of Cu2+ induces the conformational change from the mixture mentioned above to a monomeric helical structure, and a beta-sheet structure forms in the presence of 1 mol eq of Cu2+. As alpha-helix and beta-sheet structures are responsible for the formation of PHFs, it is hypothesized that Cu2+ is an inducer of self-assembly of the R3 peptide and makes the R3 peptide form a structure like PHF. Hence, it is postulated that Cu2+ plays an important role in the aggregation of the R3 peptide and tau protein and that copper (II) binding may be another possible involvement in AD.     

应用免疫和质谱法对亚心形扁藻氢酶蛋白进行亚细胞定位和分类

亚心形扁藻(Platymonas subcordiformis)是新发现的一株产氢海洋单细胞绿藻,经过胁迫调控可实现一定时间的持续产氢。氢酶是亚心形扁藻在胁迫条件下进行光合产氢的一个关键酶。但到目前为止,亚心形扁藻氢酶相关信息仍不清楚。利用蛋白合成抑制剂氯霉素和放线菌酮对亚心形扁藻氢酶活性进行考察,同时利用免疫印迹技术和免疫胶体金电镜对亚心形扁藻氢酶蛋白进行亚细胞定位分析。结果表明:亚心形扁藻氢酶蛋白可能由胞浆内合成,在叶绿体行使功能。采用免疫共沉淀技术富集亚心形扁藻细胞氢酶蛋白,SDS-聚丙烯酰胺凝胶电泳(SDS-PAGE)对免疫共沉淀复合物进行分离,从胶中切取目的蛋白条带,胶内酶解后进行基质辅助激光解吸飞行时间质谱(MALDI-TOF-MS)分析,得到相应的肽指纹图谱,通过搜索数据库检索初步断定亚心形扁藻氢酶蛋白为铁氢酶。     

Matrix-assisted Laser Desorption/Ionization Time of Flight (MALDI-TOF) Mass Spectrometric Analysis of Intact Proteins Larger than 100 kDa

Effectively determining masses of proteins is critical to many biological studies (e.g. for structural biology investigations). Accurate mass determination allows one to evaluate the correctness of protein primary sequences, the presence of mutations and/or post-translational modifications, the possible protein degradation, the sample homogeneity, and the degree of isotope incorporation in case of labelling (e.g. ¹³C labelling).Electrospray ionisation (ESI) mass spectrometry (MS) is widely used for mass determination of denatured proteins, but its efficiency is affected by the composition of the sample buffer. In particular, the presence of salts, detergents, and contaminants severely undermines the effectiveness of protein analysis by ESI-MS. Matrix-assisted laser desorption/ionization (MALDI) MS is an attractive alternative, due to its salt tolerance and the simplicity of data acquisition and interpretation. Moreover, the mass determination of large heterogeneous proteins (bigger than 100 kDa) is easier by MALDI-MS due to the absence of overlapping high charge state distributions which are present in ESI spectra.Here we present an accessible approach for analysing proteins larger than 100 kDa by MALDI-time of flight (TOF). We illustrate the advantages of using a mixture of two matrices (i.e. 2,5-dihydroxybenzoic acid and α-cyano-4-hydroxycinnamic acid) and the utility of the thin layer method as approach for sample deposition. We also discuss the critical role of the matrix and solvent purity, of the standards used for calibration, of the laser energy, and of the acquisition time. Overall, we provide information necessary to a novice for analysing intact proteins larger than 100 kDa by MALDI-MS.