RESUMO
UPLC-ion mobility spectrometry separations combined with mass spectrometry (UPLC-IM-MS) and tandem mass spectrometry (UPLC-IM-MS/MS) have been investigated for the simultaneous determination of testosterone and epitestosterone glucuronides in urine. The glucuronide epimers of testosterone and epitestosterone were separated by ion mobility spectrometry prior to mass analysis on the basis of differences in their collision cross sections, which have been measured in nitrogen. Combining ion mobility separation with UPLC/MS enhances the analysis of these low-abundance steroids in urine by selective interrogation of specific retention time, mass-to-charge and mobility regions. Detection limits for the UPLC-IM-MS/MS analysis of TG and ETG were 9.9 ng mL(-1) and 98 ng mL(-1) respectively, equivalent to 0.7 ng mL(-1) and 7.4 ng mL(-1) in urine, with linear dynamic ranges corresponding to 0.7-108 ng mL(-1) and 7.4-147 ng mL(-1) in urine. Repeatability (%RSD) for urine extracts was 0.64% and 2.31% for TG and ETG respectively.
Assuntos
Testosterona/análogos & derivados , Cromatografia Líquida de Alta Pressão , Humanos , Espectrometria de Massas , Conformação Molecular , Sensibilidade e Especificidade , Estereoisomerismo , Testosterona/química , Testosterona/urinaRESUMO
We have combined ion mobility spectrometry-mass spectrometry with tandem mass spectrometry to characterise large, non-covalently bound macromolecular complexes in terms of mass, shape (cross-sectional area) and stability (dissociation) in a single experiment. The results indicate that the quaternary architecture of a complex influences its residual shape following removal of a single subunit by collision-induced dissociation tandem mass spectrometry. Complexes whose subunits are bound to several neighbouring subunits to create a ring-like three-dimensional (3D) architecture undergo significant collapse upon dissociation. In contrast, subunits which have only a single neighbouring subunit within a complex retain much of their original shape upon complex dissociation. Specifically, we have determined the architecture of two transient, on-pathway intermediates observed during in vitro viral capsid assembly. Knowledge of the mass, stoichiometry and cross-sectional area of each viral assembly intermediate allowed us to model a range of potential structures based on the known X-ray structure of the coat protein building blocks. Comparing the cross-sectional areas of these potential architectures before and after dissociation provided tangible evidence for the assignment of the topologies of the complexes, which have been found to encompass both the 3-fold and the 5-fold symmetry axes of the final icosahedral viral shell. Such insights provide unique information about virus assembly pathways that could allow the design of anti-viral therapeutics directed at the assembly step. This methodology can be readily applied to the structural characterisation of many other non-covalently bound macromolecular complexes and their assembly pathways.
Assuntos
Proteínas do Capsídeo/química , Modelos Moleculares , Complexos Multiproteicos/química , Multimerização Proteica , Espectrometria de Massas por Ionização por Electrospray/métodos , Espectrometria de Massas em Tandem/métodos , Animais , Proteínas do Capsídeo/metabolismo , Bovinos , Cristalografia por Raios X , Cavalos , Humanos , Levivirus , Conformação Molecular , Método de Monte Carlo , Complexos Multiproteicos/metabolismo , Proteínas/química , Proteínas/metabolismoRESUMO
Detailed knowledge of the tertiary and quaternary structure of proteins and protein complexes is of immense importance in understanding their functionality. Similarly, variations in the conformational states of proteins form the underlying mechanisms behind many biomolecular processes, numerous of which are disease-related. Thus, the availability of reliable and accurate biophysical techniques that can provide detailed information concerning these issues is of paramount importance. Ion mobility spectrometry (IMS) coupled to mass spectrometry (MS) offers a unique opportunity to separate multi-component biomolecular entities and to measure the molecular mass and collision cross-section of individual components in a single, rapid (= 2 min) experiment, providing 3D- architectural information directly. Here we report a method of calibrating a commercially available electrospray ionisation (ESI)-travelling wave ion mobility spectrometry (TWIMS)-mass spectrometer using known cross-sectional areas determined for a range of biomolecules by conventional IMS-MS. Using this method of calibration, we have analysed a range of proteins of differing mass and 3D architecture in their native conformations by ESI-TWIMS-MS and found that the cross-sectional areas measured in this way compare extremely favourably with cross-sectional areas calculated using an in-house computing method based on Protein Data Bank NMR-derived co-ordinates. This not only provides a high degree of confidence in the calibration method, but also suggests that the gas phase ESI- TWIMS-MS measurements relate well to solution-based measurements derived from other biophysical techniques. In order to determine which instrumental parameters affect the ESI-TWIMS-MS cross-sectional area calibration, a systematic study of the parameters used to optimise TWIMS drift time separations has been carried out, observing the effect each parameter has on drift times and IMS resolution. Finally, the ESI-TWIMS-MS cross-sectional area calibration has been applied to the analysis of the amyloidogenic protein beta(2)-microglobulin and measurements for three co-populated conformational families, present under denaturing conditions, have been made: the folded, partially unfolded and unfolded states.