蛋白质组研究中离子阱串联质谱数据搜库结果解释方法

基于离子阱串联质谱仪的鸟枪法是一种高通量的蛋白质鉴定方法。得到的数据一般使用软件SEQUEST搜索蛋白质序列数据库,得到肽段鉴定列表以及相应的打分。为了得到蛋白质鉴定列表,还需要进行肽段鉴定结果的过滤和假阳性率的计算,然后根据肽段鉴定结果组装蛋白质列表。这两个问题目前还没有很好地解决。对已有的方法进行总结和比较,可以给搜库结果解释方法的选择提供参考,对数据质量控制方法的改进也有所帮助。     

后基因组时代的质谱技术

人类基因组测序已完成 ,生命科学研究进入了后基因组时代。今后的人类基因组研究发展趋势是解决基因的功能和不同种族间基因的差异等更为复杂的问题。随之而来的繁重任务迫切需要各种先进技术[1,2 ] 。质谱技术因其具有超高分辨率、灵敏度 ,以及高通量、良好的自动化前景等优点而备受关注。1 .质谱技术的现状质谱仪一般由离子源 ,质量分析器和离子检测器 3部分组成。质谱仪操作可简化为 :产生气相离子 ;按离子的质荷比 (ｍａｓｓｔｏｃｈａｒｇｅｒａｔｉｏ ,ｍ/ｚ)将离子在空间或时间上分离 ;测定不同质荷比下离子的数量。如今针对多肽、蛋…     

整体蛋白质电喷雾质谱原位解析

蛋白质组学系统研究了生物体蛋白质组,尤其是一定生理、病理条件下差异表达的蛋白;对蛋白质序列、翻译后修饰及其位置的定性鉴定可以帮助我们系统地了解蛋白质的结构和功能。随着软电离技术(如电喷雾电离技术)及高质量测量精度、高质量分辨质谱仪(如轨道阱质谱仪)的发展与相对普及,完整蛋白质的质谱表征(即所谓的自上而下蛋白质组学)已成为可能且渐渐流行起来;相应的数据库搜索引擎和蛋白质鉴定生物信息学工具也有了一定的进展。本文对作者研发的蛋白质电喷雾质谱原位解析算法"同位素质荷比及轮廓指纹比对"及整体蛋白质数据库搜索引擎"Protein Goggle2.0"(http://proteingoggle.tongji.edu.cn/)做一个概述。     

中科院上海植物逆境生物学研究中心招聘蛋白质组学研究人员

正中科院上海植物逆境生物学研究中心蛋白质组学平台应用现代色谱-质谱技术对植物蛋白质翻译后修饰及其相互作用进行分析鉴定,以揭示蛋白质在生命过程中结构和功能的相互关系。在中科院的大力支持下,蛋白质组学实验室装备了高分辨的组合轨道阱质谱仪(Orbitrap Fusion)、四极杆轨道阱质谱仪(Q Exactive),三重四极杆飞行时间质谱仪(TripleTOF 5600)、基质辅助解吸电离串联飞行时间质谱仪(MALDI TOF/TOF)、四极杆气质联用仪(GCMS)和电感耦合等离子体质谱仪(ICP-MS)等先进仪器设备。平台隶属于中组部"千人计划"顶尖人才与创新团队,现承担支持多项国家重大科研项目,包括优秀青年     

植物和土壤δ13C的分析误差校正与数据标准化

植物和土壤中稳定碳同位素(δ¹³C)可用于指示、示踪和整合碳循环关键过程与功能。双路和连续流气体稳定同位素比值质谱仪(IRMS)是δ¹³C的两种测试技术,其精度和准确度受分析误差和数据标准化方法影响。分析误差包括记忆效应、时间漂移和信号强度依赖性等。将测定序列按样品δ¹³C从低到高排列或延长气路冲洗时间消除或降低记忆效应;在测样序列中内插标准物质并建立测定时间与分析误差的函数关系校正时间漂移;信号强度依赖性应先校正样品空白效应,再校正仪器非线性响应。数据标准化包括标准物质和数据标准化方法选择。选择的标准物质δ¹³C应涵盖待测样品δ¹³C,且遵循同等处理原则;标准物质数量≥4或每个标准物质重复测定次数≥4;数据标准化体系应长期稳定,并添加监测标准物质。     

实用性同位体标记技术

同位体标记技术．诸如iTRAQ（相对和绝对定量的同位体标签）．可以通过报道子离子信号对多元肽进行定量。因为用RAQ允许对多达8个不同的标签集进行分析．所以比只容纳2—3个标记物的代谢标记技术（重同位素标记）更为优越。虽然如此．直至最近在用离子阱质谱仪进行的串联质谱分析中低质量片段的回收率都很差．所以在许多分析平台中很难应用ITRAQ．     

招聘启事

正中科院上海植物逆境生物学研究中心蛋白质和代谢组学平台招聘ICP-MS工作人员中国科学院上海植物逆境生物学研究中心蛋白质和代谢组学平台利用超高效液相色谱和高分辨的质谱仪器,对植物中的痕量无机元素组分、有机代谢产物和蛋白质进行全面系统的分离、定性和定量分析研究。该平台配备了电感耦合等离子体质谱仪(ICP-MS)、飞行时间质谱仪(Triple TOF 5600,TOF/TOF 5800)、气质联用仪(GCMS)、轨道阱质谱仪(Orbitrap Fusin,Q Exactive)和超高效液相色谱等植物蛋白质和代     

MALDI-TOF质谱技术对克罗诺杆菌的鉴定与分型

以基质辅助激光解析电离飞行时间质谱(MALDI-TOFMS)技术用于克罗诺杆菌的鉴定与分型。通过对获得的克罗诺杆菌属典型菌株、阴沟肠杆菌和产气肠杆菌近似菌株以及克罗诺杆菌分离株的蛋白质质量图谱进行对比分析,找出克罗诺杆菌特征性离子峰,将其作为鉴定克罗诺杆菌的生物标识物;对全细菌蛋白质质量图谱进行聚类分析,将克罗诺杆菌属进一步划分为不同类型,结果显示,4株克罗诺杆菌参考菌株质量图谱约在5740(m/z)离子质荷比处出现1个相近离子峰,28株克罗诺杆菌分离株中27株(占96.4%)表现出相同结果;32株克罗诺杆菌被分为6种类型(以50%距离水平为分类界限)。MALDI-TOFMS作为一种新的技术,不仅能够用于克罗诺杆菌的鉴定,而且根据获得的细菌蛋白质质量图谱可将克罗诺杆菌划分为不同类型。     

蛋白质二级结构预测的系统误差

目前蛋白质二级结构的预测准确率徘徊在75%左右,难以作进一步提高。本文通过统计学的方法,对蛋白质的冗余数据库进行了分析。并由此证明,目前影响预测准确率继续的真正原因是蛋白质数据库本身的系统误差,系统误差大约为25%。而该误差是由于实验条件的客观原因带来的。     

规模化蛋白质鉴定中母离子的准确检测技术研究

蛋白质组学的基础研究之一是蛋白质鉴定.规模化的蛋白质鉴定通常采用"鸟枪法",即选择一些酶切肽段(母离子)碎裂生成二级谱图,通过二级谱图及其母离子质量鉴定肽段,再推断对应的蛋白质.在鉴定过程中,母离子质量是一个关键参数.母离子是否是肽段的单同位素峰决定了正确肽段是否能进入候选,母离子的质量精度决定了候选肽段的数目.本文从判断单同位素峰和系统误差校准这两个角度研究了母离子的准确检测技术.判断单同位素峰的技术在蛋白质上已有研究,包括电荷判断、单同位素峰判断和重叠同位素峰判断.可以借鉴蛋白质水平的技术研究母离子的单同位素峰判断方法.同时母离子的系统误差校准也有较为成熟的方法.这两个角度的研究有助于提高规模化蛋白质的鉴定率.     

Micrometric molecular histology of lipids by mass spectrometry imaging

Time-Of-Flight Secondary Ion Mass Spectrometry is compared to other mass spectrometry imaging techniques, and recent improvements of the experimental methods, driven by biological and biomedical applications, are described and discussed. This review shows that this method that can be considered as a micrometric molecular histology is particularly efficient for obtaining images of various lipid species at the surface of a tissue sample, without sample preparation, and with a routine spatial resolution of 1μm or less.     

Parts per million mass accuracy on an Orbitrap mass spectrometer via lock mass injection into a C-trap

Mass accuracy is a key parameter of mass spectrometric performance. TOF instruments can reach low parts per million, and FT-ICR instruments are capable of even greater accuracy provided ion numbers are well controlled. Here we demonstrate sub-ppm mass accuracy on a linear ion trap coupled via a radio frequency-only storage trap (C-trap) to the orbitrap mass spectrometer (LTQ Orbitrap). Prior to acquisition of a spectrum, a background ion originating from ambient air is first transferred to the C-trap. Ions forming the MS or MS(n) spectrum are then added to this species, and all ions are injected into the orbitrap for analysis. Real time recalibration on the "lock mass" by corrections of mass shift removes mass error associated with calibration of the mass scale. The remaining mass error is mainly due to imperfect peaks caused by weak signals and is addressed by averaging the mass measurement over the LC peak, weighted by signal intensity. For peptide database searches in proteomics, we introduce a variable mass tolerance and achieve average absolute mass deviations of 0.48 ppm (standard deviation 0.38 ppm) and maximal deviations of less than 2 ppm. For tandem mass spectra we demonstrate similarly high mass accuracy and discuss its impact on database searching. High and routine mass accuracy in a compact instrument will dramatically improve certainty of peptide and small molecule identification.     

Transformation and other factors of the peptide mass spectrometry pairwise peak-list comparison process

Background:  

Biological Mass Spectrometry is used to analyse peptides and proteins. A mass spectrum generates a list of measured mass to charge ratios and intensities of ionised peptides, which is called a peak-list. In order to classify the underlying amino acid sequence, the acquired spectra are usually compared with synthetic ones. Development of suitable methods of direct peak-list comparison may be advantageous for many applications.     

Combinatorial approaches for mass spectra recalibration

Mass spectrometry has become one of the most popular analysis techniques in Proteomics and Systems Biology. With the creation of larger datasets, the automated recalibration of mass spectra becomes important to ensure that every peak in the sample spectrum is correctly assigned to some peptide and protein. Algorithms for recalibrating mass spectra have to be robust with respect to wrongly assigned peaks, as well as efficient due to the amount of mass spectrometry data. The recalibration of mass spectra leads us to the problem of finding an optimal matching between mass spectra under measurement errors. We have developed two deterministic methods that allow robust computation of such a matching: The first approach uses a computational geometry interpretation of the problem, and tries to find two parallel lines with constant distance that stab a maximal number of points in the plane. The second approach is based on finding a maximal common approximate subsequence, and improves existing algorithms by one order of magnitude exploiting the sequential nature of the matching problem. We compare our results to a computational geometry algorithm using a topological line-sweep.     

Identification and determination of propafenone and its principal metabolites in human urine using capillary gas chromatography/mass spectrometry

The capillary gas chromatography/mass spectrometry of trimethylsilyl-trifluoroacetyl, trifluoroacetyl and pentafluoropropionyl (PFP) derivatives of the antiarrhythmic agent propafenone (Rytmonorm), as well as its main metabolites N-despropyl-propafenone and 5-hydroxy-propafenone, have been investigated. Both electron impact and positive isobutane chemical ionization mass spectrometry using the Ion Trap Detector have been evaluated. The presence of propafenone and its co-extracted metabolites in human urine at time intervals after the oral administration of 150 mg Rytmonorm to healthy volunteers was established, and the urinary excretion of propafenone and 5-hydroxy-propafenone was calculated using selective chemical ionization mass spectrometric detection. Only a few per cent of the dose was excreted unchanged in the urine. Large intersubject variabilities had been observed also. The large dynamic range of the Ion Trap Detector and the high correlation coefficients (0.92-0.99) of the calibration curves were striking.     

Analysis of long-chain bases in sphingolipids by positive ion fast atom bombardment or matrix-assisted secondary ion mass spectrometry

The structures of long-chain bases are expressed as [CH2C(NH2) = CHR]+ (Z+) in the positive ion mode spectra obtained on fast atom bombardment (FAB) mass spectrometry or liquid-matrix-assisted secondary ion mass spectrometry (SIMS) [Benninghoven, A., Ed. (1983) Ion Formation from Organic Solids, Springer, Berlin]. This phenomenon is common to sphingolipids in general: glycosphingolipids [see reviews by Sweeley and Nunez [Sweeley, C. C., & Nunez, H. A. (1985) Annu. Rev. Biochem. 54, 765] and Kanfer and Hakomori [Kanfer, J. N., & Hakomori, S. (1983) Handb. Lipid Res. 3]] and phosphonosphingolipids [Hayashi, A., & Matsubara, T. (1982) in New Vistas in Glycolipid Research (Makita, A., Handa, S., Taketomi, T., & Nagai, Y., Eds.) p 103, Plenum, New York], inclusive. Phytosphingosine compounds show the same type of fragmentation without additional dehydration if a neutral matrix is used. A Z+ ion is easily detected in the lower mass region (m/z 200-400) as an even mass number fragment ion, and confirmation is made by means of B/E constant and B2/E constant linked scan techniques [Boyd, R. K., & Beynon, J. H. (1977) Org. Mass Spectrom. 12, 163; Boyd, R. K., & Shushan, B. (1981) Int. J. Mass Spectrom. Ion Phys. 37, 355; Macdonald, C. G., & Lacey, M. J. (1984) Org. Mass Spectrom. 19, 55]. [Principles of linked scannings are explicitly summarized by Jennings and Mason [Jennings, K. R., & Mason, R. S. (1983) in Tandem Mass Spectrometry (McLafferty, F. W., Ed.) p 197, Wiley, New York] besides the cited literature.]     

Respective contributions of atomic emission spectrometry (AES) and secondary ion mass spectrometry (SIMS) to trace element quantification

By means of Secondary Ion Mass Spectrometry (SIMS) it is possible to measure in situ the relative concentration of a given element in a volume of 1 micron 3. Atomic Emission Spectrometry (AES) allows absolute quantitation of tissue homogenates. The use of both techniques lead to correlate relative and absolute elemental concentrations. These methods have been applied to lithium and manganese quantitation after treatments at a therapeutic dose. The results assess the sensibility of SIMS analysis, around 0.1 ppm in biological specimens, and confirm the adequacy of the instrument to trace elements study.     

Towards high-throughput metabolomics using ultrahigh-field Fourier transform ion cyclotron resonance mass spectrometry

With unmatched mass resolution, mass accuracy, and exceptional detection sensitivity, Fourier Transform Ion Cyclotron Resonance Mass Spectrometry (FTICR-MS) has the potential to be a powerful new technique for high-throughput metabolomic analysis. In this study, we examine the properties of an ultrahigh-field 12-Tesla (12T) FTICR-MS for the identification and absolute quantitation of human plasma metabolites, and for the untargeted metabolic fingerprinting of inbred-strain mouse serum by direct infusion (DI). Using internal mass calibration (mass error ≤1 ppm), we determined the rational elemental compositions (incorporating unlimited C, H, N and O, and a maximum of two S, three P, two Na, and one K per formula) of approximately 250 out of 570 metabolite features detected in a 3-min infusion analysis of aqueous extract of human plasma, and were able to identify more than 100 metabolites. Using isotopically-labeled internal standards, we were able to obtain excellent calibration curves for the absolute quantitation of choline with sub-pmol sensitivity, using 500 times less sample than previous LC/MS analyses. Under optimized serum dilution conditions, chemical compounds spiked into mouse serum as metabolite mimics showed a linear response over a 600-fold concentration range. DI/FTICR-MS analysis of serum from 26 mice from 2 inbred strains, with and without acute trichloroethylene (TCE) treatment, gave a relative standard deviation (RSD) of 4.5%. Finally, we extended this method to the metabolomic fingerprinting of serum samples from 49 mice from 5 inbred strains involved in an acute alcohol toxicity study, using both positive and negative electrospray ionization (ESI). Using these samples, we demonstrated the utility of this method for high-throughput metabolomics, with more than 400 metabolites profiled in only 24 h. Our experiments demonstrate that DI/FTICR-MS is well-suited for high-throughput metabolomic analysis. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

微生物学研究中的软电离质谱技术

包括基质辅助激光解吸电离（ＭＡＬＤＩ）和电喷雾（ＥＳＩ）在内的软电离质谱是最近发展起来的质谱技术,由于这些电离方式对样品的破坏性小,质量测定范围大,分子量测定准确,样品纯度要求不高很适合分析成分复杂的微生物样品,ＭＡＬＩＤＩ－ＴＯＦ－ＭＳ结合高分辨率的二维ＳＤＳ－ＰＡＧＥ可以分析１０＾－１２摩尔水平的蛋白,是细菌蛋白质研究过程中必不可少的工具。最近的研究工作表明,通过ＭＡＩＤＩ－ＴＯＦ－ＭＳ或ＨＰ     

Identification of hnRNP P2 as TLS/FUS using electrospray mass spectrometry.

Protein complexes assembled on mRNA precursors can be separated by gel filtration chromatography to yield spliceosomal and H complex fractions (Reed R, Griffith J, Maniatis T, 1988, Cell 53:949-961; Reed R, 1990, Proc Natl Acad Sci USA 87:8031-8035.). Here we use Nano electrospray mass spectrometry (Wilm M, Mann M, 1994, Int J Mass Spectrometry Ion Processes 136:167-180) to identify proteins complexed with Adeno-pre-mRNA in the H complex peak. Four of the major hnRNP proteins, A1, B1, C1, and G, were identified by database analysis based on peptide mass and sequence information. A fifth protein in the H complex peak, corresponding to hnRNP P2, is shown to be the product of the TLS/FUS gene. This was originally identified as a chimeric oncogene formed by the chromosome translocation t(12;16) that is responsible for myxoid liposarcoma. The involvement of hnRNP P2 in oncogenesis provides a clear example of the importance of hnRNP proteins in molecular disease.