含二硫键的蛋白质/多肽的MALDI-TOF MS源内裂解研究

应用基质辅助激光解吸电离飞行时间质谱(MALDI-TOF MS)法研究含二硫键的人胰岛素与甘精胰岛素酶解液的源内裂解(ISD)。比较了不同基质种类及不同结晶状态对含二硫键的人胰岛素与甘精胰岛素酶解液的源内裂解的影响。结果表明,含二硫键的蛋白质的ISD发生受激光点照射位置的影响,在不同基质与结晶形态的条件下,含二硫键的蛋白质的ISD碎片信息不同。通过分析比较,含二硫键的蛋白质的ISD较容易控制,并且其基质的种类及结晶状态作用很关键。需要获得大量碎片时,使激光照射在样品和阿魏酸(FA)基质形成的大结晶处;不希望出现碎片时,可使用2,4,6-三羟基苯乙酮(THAP)为基质,或使激光照射在样品和其他基质形成的细小均匀结晶处。     

Fragmentations of (M-H)- anions of underivatised peptides. Part 2: Characteristic cleavages of Ser and Cys and of disulfides and other post-translational modifications, together with some unusual internal processes

In a previous review (Bowie, Brinkworth, & Dua (2002); Mass Spectrom Rev 21:87-107) we described the characteristic backbone cleavages and side chain fragmentations which occur from (M-H)(-) parent anions of underivatized peptides. This work is briefly summarized in the present review. Cys was not described in the previous review: here we describe the Cys characteristic side chain loss of H(2)S, together with its gamma backbone cleavage. These processes are compared with those of the related Ser. All experimental observations are backed up with theoretical studies at the HF/6-31G(d)//AM1 level of theory, a level of theory which we have shown gives good geometries and acceptable relative energies. The negative ion cleavages of a number of post-translational modifications are described. Negative ion mass spectrometry is the method of choice for identification of disulfides in both peptides and proteins. Intramolecular disulfides are identified by the presence of the fragment anion [(M-H)(-)-H(2)S(2)], and CID MS2 of this fragment normally identifies the positions of the two Cys residues and often the full sequence of the peptide. An unsymmetrically substituted intermolecular disulfide can give up to eight characteristic fragment anions, and CID MS2 of some, or all of these often provides the full sequence of those peptides which form the initial intermolecular disulfide linkage. Negative ion cleavages of disulfides are the most energetically favored of all peptide negative cleavages studied to date. Negative ion mass spectrometry is also valuable for the identification of pyroglutamates, sulfates and phosphates. Finally, some unusual fragmentations are described which involve cyclization/elimination reactions which require the decomposing (M-H)(-) parent anions to adopt the same helical conformation that these peptides have in solution.     

基于质谱技术的二硫键定位分析方法研究进展

蛋白质中的二硫键是一种常见的重要翻译后修饰,对稳定蛋白质的三维空间结构、维持正确的折叠构象、保持和调节生物活性具有重要意义。因此,分析蛋白质中的二硫键对理解生命过程和药物研发至关重要。质谱既可断裂二硫键和肽键,又可作为检测终端,在二硫键定位分析中发挥了重要作用。本文根据断裂二硫键所处介质以及断裂机理进行分类整理,综述了基于质谱技术的主流二硫键定位方法,通过对比各方法的优缺点,为选择合适的质谱分析方式提供参考。最后,提出了二硫键分析领域面临的挑战和方法学研究的发展方向,以促进方法学相关研究者开发更有效的质谱方法。     

Collision-induced fragmentations of the (M-H)- parent anions of underivatized peptides: an aid to structure determination and some unusual negative ion cleavages

This article describes the fundamental cleavage reactions of (M-H)(-) anions of underivatized peptides that contain up to 25 amino acid residues. The experimental observations of these cleavages have been backed up by molecular modeling, generally at the AM1 level of theory. The basic cleavages are the ubiquitous alpha- and beta-backbone cleavage reactions, which provide information similar to that of the B and Y + 2 cleavages of MH(+) ions of peptides. The residues Asp and Asn also effect cleavages of the backbone (called delta- and gamma-cleavages), by reactions initiated from side chain enolate anions, causing elimination reactions that cleave the backbone between the Asp (Asn) N bond;C backbone bond. Glu and Gln also direct analogous delta- and gamma-cleavages of the backbone, but in this case the processes are initiated by attack of the side chain CO(2) (-) (CONH(-)) to form a lactone (lactam). Ser and Thr residues undergo characteristic fragmentations of the side chain. These processes, losses of CH(2)O (Ser) and MeCHO (Thr), convert these residues into Gly. In larger peptides, Ser and Thr can effect two backbone cleavage reactions, called gamma- and epsilon -processes. The C-terminal CO(2) (-) (or CONH(-)) forms a hydrogen bond with the side chain OH (of Ser or Thr), placing the C-terminal residue in a position where it may affect S(N) (2) attack at the electrophilic backbone CH of Ser, with concomitant cleavage of the backbone. All of the above negative ion cleavages require the peptide backbone to be conformationally flexible. However, there is a backbone cleavage that requires the peptide to have an alpha-helical conformation in order for the two reacting centers to approach. This cleavage is illustrated for the Glu 23-initiated backbone cleavage at Ile 21 for the (M-H)(-) anion of the antimicrobial peptide caerin 1.1.     

抗肿瘤药物顺铂和血清白蛋白的相互作用研究

Cisplatin is a widely used anticancer agent to treat solid tumours such as ovarian, testicular cancers. The interaction of platinum drugs with human albumin is an important issue that contributes to understanding the transport process of platinum drugs, cell uptake and their side-effects. Here, we applied a bottom-up proteomic approach to study the interaction of cisplatin with recombinant human albumin(rHA). The cisplatin-rHA complexes were prepared under physiological conditions in vitro at different cisplatin/rHA molar ratios for different incubation times. LC/MS analysis enables to identify four platinated peptides and further MS/MS analysis characterizes the platinum binding sites. The results show that cisplatin binds to His67, His128, His247 and Met298 residues in albumin involving an intramolecular crosslinking of His67 to His247 by platinum. Besides, cisplatin is shown to induce the cleavage of the disulfide bond Cys124-Cys169 of albumin and bind to the thiol in the reduced Cys124 residue.     

含二硫键多肽的基质辅助激光解吸电离飞行时间质谱研究

The determination of disulfide bonds becomes an important aspect of obtaining a comprehensive understanding of the chemical structure of a protein. Numerous experimental methods have been developed for the determination of disulfide bonds in proteins. Modern mass spectrometry has developed as an important tool for the analysis of disulfide bond patterns due to its advantages of being simple, rapid and sensitive. The dissociations of the disulfide bonds were detected during the matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) analysis. These fragment ions were attributed to prompt fragmentation or “in-source decay” rather than “post-source decay”. For the double disulfide bonds, ions of plus sulfur and minus sulfur atoms corresponding to cleavages at different sites within the carbon-sulfur-sulfur-carbon disulfide bonds were also observed.     

大肠杆菌O77中wabD基因编码的β-1,3-甘露糖基转移酶产物结构的质谱表征

The O77 antigens of Escherichia coli contains a Man-β-1,3-GlcNAc linkage within the repeating unit. A synthetic substrate analog of the natural acceptor substrate undecaprenol-pyrophosphate-lipid [GlcNAc-α-PO₃-PO₃-(CH₂)₁₁-O-phenyl] was used as an acceptor and GDP-Man as a donor substrate. Electrospray ionization tandem mass spectrometry(ESI-MS/MS) is applied for the detailed structural characterization of the enzyme product. A systematic study was conducted on enzyme product to allow rationalization of the fragmentation processes. The major fragments observed in the ESI-MS/MS spectra result from cleavage of glycosidic bond and diphosphate moiety. The fragment originating from the nonreducing end of the product yields information on sequence. Cross-ring cleavages, which are very informative of the linkages of the monosaccharide residues constituting the product, and ‘internal’ cleavage ions which are derived from elimination of substituents from around the pyranose ring, were also observed. This extensive fragmentation shows the expected Man-β-1,3-GlcNAc linkage in the product, confirming that wabD is form of GDP-Man: GlcNAc-pyrophosphate-lipid β-1,3-mannosyltransferase.     

Detection and localization of protein modifications by high resolution tandem mass spectrometry

For interrogation of peptides with diverse modifications, no other instrument is as versatile as the Fourier-transform mass spectrometer (FTMS). Particularly using electrospray ionization (ESI), many intact proteins and their proteolytic products harboring post-translational and chemical modifications (PTMs) have been studied by high resolution tandem mass spectrometry (MS/MS). The widely touted analytical figures of merit for FTMS in fact have translated into clarity when analyzing PTMs from phosphorylations to disulfides, oxidations, methylations, acetylations, and even exotic PTMs found in the biosynthesis of antibiotics and other natural products. A top down approach to PTM detection and localization is proving extensible to an increasing variety of PTMs, some of which are stable to MS/MS at the protein level but unstable to amide bond cleavage by threshold dissociations at the level of small peptides <3 kDa. In contrast, MS/MS using electron capture dissociation (ECD) allows precise localization of even labile PTMs given enough sample and abundant molecular ions. Finally, this brief synopsis of recent literature highlights specific PTMs that perturb the protein backbone therefore altering MS/MS fragmentation patterns. Thus, FTMS will continue its expansion into more laboratories in part because of its ability to detect and deconvolute the regulatory mechanisms of biology written in the language of PTMs.     

UPLC-ESI-TOF MS/MS分析太子参中环肽类成分

建立超高效液相色谱-电喷雾飞行时间串联质谱（UPLC-ESI -TOF MS/MS）分析太子参中环肽类成分的方法。实验采用反相C18色谱柱,梯度洗脱,分离并检测太子参中11种环肽类成分。通过信息关联采集（IDA）模式,运用动态背景扣除(DBS),消除背景离子干扰,在获得11种环肽类成分MS/MS图谱的同时,也得到其精确的分子质量信息。     

基质辅助激光解吸电离质谱用于生物组织的质谱成像应用进展

基质辅助激光解吸电离质谱(MALDI MS)用于分析组织切片已成为质谱学的一个新领域。质谱成像技术通过直接对组织切片表面的质谱扫描,可以快速直观地分析组织中的分子,如蛋白质、多肽、药物分子、代谢产物等及其空间分布信息。本工作综述了组织切片的质谱成像原理,方法学和相关应用。     

肝素/硫酸乙酰肝素四糖同分异构体的制备与表征

肝素/硫酸乙酰肝素（Hep/HS）寡糖结构多样,且与生物学、病理学功能息息相关,表征这些结构对解析它们的功能具有重要意义。本实验以市售猪肠肝素钠为原料,制备了9种肝素四糖,并用二糖组分分析方法结合反相离子对试剂色谱-串联离子阱飞行时间质谱(IPRP-LC/IT-TOF MS)进行结构解析。结果表明：9种肝素四糖中有两组同分异构体,且含己胺离子对试剂的RP-LC/MS能够成功分离肝素寡糖及其同分异构体;采用LC-MS/MS方法可以鉴定其中一组同分异构体的结构。该方法可为HS/Hep寡糖的研究提供标准品,并为解析HS/Hep同分异构体结构提供一种简单有效的方法,进而为更好地理解生物体内HS/Hep结构和生理生化功能的关系提供帮助。     

Mass spectrometry in bioinorganic analytical chemistry

A considerable momentum has recently been gained by in vitro and in vivo studies of interactions of trace elements in biomolecules due to advances in inductively coupled plasma mass spectrometry (ICP MS) used as a detector in chromatography and capillary and planar electrophoresis. The multi-isotopic (including non-metals such as S, P, or Se) detection capability, high sensitivity, tolerance to matrix, and large linearity range regardless of the chemical environment of an analyte make ICP MS a valuable complementary technique to electrospray MS and MALDI MS. This review covers different facets of the recent progress in metal speciation in biochemistry, including probing in vitro interactions between metals and biomolecules, detection, determination, and structural characterization of heteroatom-containing molecules in biological tissues, and protein monitoring and quantification via a heteroelement (S, Se, or P) signal. The application areas include environmental chemistry, plant and animal biochemistry, nutrition, and medicine.     

电喷雾萃取电离-串联质谱中N,N-二乙基苯胺类化合物的特征碎裂反应

掌握串联质谱中的裂解反应是深刻理解质谱裂解规律,从而对复杂基体中相关化合物进行快速、精准结构解析的关键。本文以电喷雾萃取电离-串联质谱（EESI-MS/MS）为手段,研究N,N-二乙基苯胺类化合物的串联质谱行为,考察不同的取代基模式对裂解反应的影响,揭示裂解反应机理,总结该类化合物发生裂解反应的特征和基本规律。研究结果不仅丰富了气相离子化学的研究内容,还从根本上降低了检测的假阳性率,为实际样品中相关化合物的结构分析鉴定提供了科学依据。     

LIQUID CHROMATOGRAPHY–MASS SPECTROMETRY BOTTOM‐UP PROTEOMIC METHODS IN ANIMAL SPECIES ANALYSIS OF PROCESSED MEAT FOR FOOD AUTHENTICATION AND THE DETECTION OF ADULTERATIONS

This review offers an overview of the current status and the most recent advances in liquid chromatography–mass spectrometry (LC‐MS) techniques with both high‐resolution and low‐resolution tandem mass analyzers applied to the identification and detection of heat‐stable species‐speci?c peptide markers of meat in highly processed food products. We present sets of myofibrillar and sarcoplasmic proteins, which turned out to be the source of 105 heat‐stable peptides, detectable in processed meat using LC‐MS/MS. A list of heat‐stable species‐specific peptides was compiled for eleven types of white and red meat including chicken, duck, goose, turkey, pork, beef, lamb, rabbit, buffalo, deer, and horse meat, which can be used as markers for meat authentication. Among the 105 peptides, 57 were verified by multiple reaction monitoring, enabling identification of each species with high specificity and selectivity. The most described and monitored species by LC‐MS/MS so far are chicken and pork with 26 confirmed heat‐stable peptide markers for each meat. In thermally processed samples, myosin, myoglobin, hemoglobin, l ‐lactase dehydrogenase A and β‐enolase are the main protein sources of heat‐stable markers. © 2019 John Wiley & Sons Ltd. Mass Spec Rev     

碰撞诱导解离和高能碰撞解离方式进行蛋白质组学分析的比较

为了比较碰撞诱导解离（collision induced dissociation,CID）和高能碰撞解离（high energy collision dissociation,HCD）两种离子裂解模式在蛋白质组学分析方面的差异,分别应用这两种模式对牛血清白蛋白和细胞裂解物进行分析。通过比较所鉴定到的多肽和蛋白数目,发现在牛血清白蛋白中,HCD碎裂方法所得的谱图匹配率和多肽Mascot得分均较高,表明其质谱图质量较好。在复杂样品细胞裂解物分析中,CID碎裂方法鉴定到的谱图数、多肽数和蛋白数目均较多,表明该模式的灵敏度较高;HCD碎裂方法所得的谱图匹配率和Mascot得分均较高,表明其质谱图质量较好。因此,这两种模式均可用于大规模蛋白质组学分析,CID模式可提供更高的灵敏度,而HCD模式则可获得更高质量的质谱谱图。     

傅立叶变换离子回旋共振质谱仪在石油组成分析中的应用

傅立叶变换离子回旋共振质谱仪(FT-ICR MS)是一种具有超高质量分辨能力的质谱仪,在石油组分相对分子质量范围(200～1 000 u)内,其分辨率能够达到几十万甚至上百万,这种分辨能力可以精确地确定由C、H、S、N、O以及它们主要同位素所组成的各种元素组合,真正从分子元素组成层次上研究石油组成。电喷雾(ESI)电离源可以从高浓度复杂烃类基质中选择性地电离石油组分中微量的杂原子极性化合物,ESI与FT-ICR MS相结合已经成为重质油非烃化合物分析的一种重要手段。本文介绍了FT-ICR MS的基本原理及近年来的发展趋势,石油组分的电离化方法及数据处理技术,综述了近期利用FT-ICR MS对石油组成取得的新认识,以及该仪器手段在石油有机地球化学、油田化学、炼油化工等领域的应用情况。尽管在重质油组成定量分析方面还存在一些技术问题,FT-ICR MS无疑将成为石油化学研究和石油工业生产过程的重要检测工具。     

Microorganism characterization by single particle mass spectrometry

In recent years a major effort by several groups has been undertaken to identify bacteria by mass spectrometry at the single cell level. The intent of this review is to highlight the recent progress made in the application of single particle mass spectrometry to the analysis of microorganisms. A large portion of the review highlights improvements in the ionization and mass analysis of bio-aerosols, or particles that contain biologically relevant molecules such as peptides or proteins. While these are not direct applications to bacteria, the results have been central to a progression toward single cell mass spectrometry. Developments in single particle matrix-assisted laser desorption/ionization (MALDI) are summarized. Recent applications of aerosol laser desorption/ionization (LDI) to the analysis of single microorganisms are highlighted. Successful applications of off-line and on-the-fly aerosol MALDI to microorganism detection are discussed. Limitations to current approaches and necessary future achievements are also addressed.     

Discovering new invertebrate neuropeptides using mass spectrometry

Neuropeptides are a complex set of messenger molecules controlling a wide array of regulatory functions and behaviors within an organism. These neuromodulators are cleaved from longer protein molecules and often experience numerous post-translational modifications to achieve their bioactive form. As a result of this complexity, sensitive and versatile analysis schemes are needed to characterize neuropeptides. Mass spectrometry (MS) through a variety of approaches has fueled the discovery of hundreds of neuropeptides in invertebrate species in the last decade. Particularly successful are direct tissue and single neuron analyses by matrix-assisted laser desorption/ionization (MALDI) MS, which has been used to elucidate approximately 440 neuropeptides, and examination of neuronal homogenates by electrospray ionization techniques (ESI), also leading to the characterization of over 450 peptides. Additional MS methods with great promise for the discovery of neuropeptides are MS imaging and large-scale peptidomics studies in combination with a sequenced genome.     

基质辅助激光解吸电离-飞行时间质谱在糖类化合物研究中的应用

综述了基质辅助激光解吸电离-飞行时间质谱(MALDI-TOF-MS)的发展、在糖类化合物结构研究时常选用的基质,以及在不同类型糖化合物分析中的应用。MALDI-TOF-MS在糖类分析中通常采用的是N2激光源,基质多为有机小分子如2,5-二羟基苯甲酸、2,4,5-三羟基苯乙酮、1-羟基异喹啉或2-羟基-5-甲氧基苯甲酸、α-氰基-4-羟基-苯丙烯酸等,基质类型的选择则要取决于糖类的存在形式。糖类化合物如中性糖、酸性糖、硫酸化糖、糖蛋白、蛋白聚糖及糖脂等均可利用适合的基质而进行MALDI-TOF-MS分析。