Relative quantification of tau-related peptides using guanidino-labeling derivatization (GLaD) with online-LC on a hybrid ion trap (IT) time-of-flight (ToF) mass spectrometer

Development of a quantification method based on isotopic variants of O-methyl isourea (OMIU) in conjunction with reversed-phase (RP) liquid chromatography (LC) electrospray mass spectrometry is described for determining the relative quantification of tau-related peptides Ac-VQIVXK-NH2. Extracted ion chromatograms of the mass spectrometric data derived from online microcapillary LC separation identifies the retention times of the isotopically derivatized peptides together with their ion abundances. Data-dependent MSMS analysis of both derivatized variants of the same peptide provides a complementary method for identification and resolution between isobaric species. In addition, with respect to offline LC MALDI a larger number of analogues are detected and formation of amyloid is also observed for the aspartic acid and histidine-containing peptides.     

Comprehensive analysis of glycated human serum albumin tryptic peptides by off-line liquid chromatography followed by MALDI analysis on a time-of-flight/curved field reflectron tandem mass spectrometer

Glycated peptides arising from in vivo digestion of glycated proteins, usually called advanced glycation end (AGE) product peptides, are biologically relevant compounds due to their reactivity towards circulating and tissue proteins. To investigate their structures, in vitro glycation of human serum albumin (HSA) has been performed and followed by enzymatic digestion. Using different MALDI based approaches the digestion products obtained have been compared with those arising from enzymatic digestion of the protein. Results obtained using 2,5-dihydroxybenzoic acid (DHB) indicate this as the most effective matrix, leading to an increase in the coverage of the glycated protein. Off-line microbore liquid chromatography prior to MALDI analysis reveals that 63% of the free amino groups amenable to glycation are modified. In addition, the same approach shows the co-presence of underivatised peptides. This indicates that, regardless of the high glucose concentration employed for HSA incubation, glycation does not go to completion. Tandem mass spectrometric data suggest that the collision induced dissociation of singly charged glycated peptides leads to specific fragmentation pathways related to the condensed glucose molecule. The specific neutral losses derived from the activated glycated peptides can be used as signature for establishing the occurrence of glycation processes.     

Development of novel guanidino-labelling derivatisation (GLaD) reagents for liquid chromatography/matrix-assisted laser desorption/ionisation analysis

A new generation of guanidino-labelling (GLaD) reagents were developed for quantitative proteomics using offline microcapillary liquid chromatography (LC) matrix-assisted laser desorption/ionisation mass spectrometry (MALDI-MS). In order to reduce the unwanted overlapping between the isotopic envelopes of the two differentially labelled peptide ions, a novel synthetic route was described for production of both (13)C- and (15)N-containing isotopomers of N,O-dimethylisourea. The use of these types of isotopes has no deleterious effect on the retention times of both differentially labelled peptides during offline microbore reversed-phase LC. In addition, the possibility to incorporate a mass difference of 4 Da can be exploited during post-source decay analysis to generate product ion spectra in which fragment ions containing the modifications appear as doublets in the corresponding product ion spectra, thus facilitating identification of the C-terminal fragment ions.     

苯二酚异构体的电动毛细管色谱-玻碳微电极柱上安培检测分析方法研究

在玻碳纤维微电极上研究了电解液中β－环糊精（ＣＤ）的浓度对邻、间、对苯二酚的循环伏安特性的影响,从实验结果和分子结构水平方面考察了β－ＣＤ与邻、间、对苯二酚的包结作用。研究结果表明,β－ＣＤ与邻、间、对苯二酚的包结作用强弱顺序为间苯二酚、邻苯二酚、对苯二酚。讨论了电泳缓冲液中ΝΗ４Ｃｌ,ＳＤＳ,β－ＣＤ的浓度及ｐＨ值对邻、间、对苯二酚迁移时间的影响。建立了苯二酚异构体胶束电动毛细管色谱－玻碳微电极柱上安培检测分析方法,方法的回收率在９８％～１０３％之间。     

Mass spectra of aminoacyl adenylate pentacoordinated phosphorus compounds

Aminoacyl adenylate pentacoordinated phosphorus compounds were analyzed by field desorption (FD) and fast atom bombardment (FAB) mass spectrometries, together with the B/E linked scan technique, and their mass spectral fragmentation pathways were investigated. For the five bonds (one P-N bond, three P-O bonds and one mixed anhydride bond P-O-CO), the cleavage usually occurred more on the P-N bond, the mixed anhydride bond and the O-C bond adjacent to the P-O bond, and less on the P-O bond. Ion YH(+), corresponding to water loss from protonated 2',3'-O-isopropylidene-adenosine, was the base peak. The results reflect the structural characteristics of aminoacyl adenylate pentacoordinated phosphorus compounds. Copyright 1999 John Wiley & Sons, Ltd.     

Sizing up large protein complexes by electrospray ionisation-based electrophoretic mobility and native mass spectrometry: morphology selective binding of Fabs to hepatitis B virus capsids

The capsid of hepatitis B virus (HBV) is a major viral antigen and important diagnostic indicator. HBV capsids have prominent protrusions (‘spikes’) on their surface and are unique in having either T?=?3 or T?=?4 icosahedral symmetry. Mouse monoclonal and also human polyclonal antibodies bind either near the spike apices (historically the ‘α-determinant’) or in the ‘floor’ regions between them (the ‘β-determinant’). Native mass spectrometry (MS) and gas-phase electrophoretic mobility molecular analysis (GEMMA) were used to monitor the titration of HBV capsids with the antigen-binding domain (Fab) of mAb 3120, which has long defined the β-determinant. Both methods readily distinguished Fab binding to the two capsid morphologies and could provide accurate masses and dimensions for these large immune complexes, which range up to ~8 MDa. As such, native MS and GEMMA provide valuable alternatives to a more time-consuming cryo-electron microscopy analysis for preliminary characterisation of virus-antibody complexes.

奥伯斯佯谬浅析

给出满足宇宙学原理的宇宙模型中计算星体视光度的一般公式，作为特例，导出“稳恒态”和“大爆炸”模型中星体视光度的表达式，结果表明这些模型是可以避免奥伯斯佯谬的。