Structural Characterization of Human Histone H4.1 by Tandem Nonlinear and Linear Ion Mobility Spectrometry Complemented with Molecular Dynamics Simulations
Ontology highlight
ABSTRACT:
SUBMITTER: Pham K
PROVIDER: S-EPMC8582071 | biostudies-literature | 2021 Oct
REPOSITORIES: biostudies-literature
Similar Datasets
Project description:Characterization of histone proteoforms with various post-translational modifications (PTMs) is critical for a better understanding of functions of histone proteoforms in epigenetic control of gene expression. Mass spectrometry (MS)-based top-down proteomics (TDP) is a valuable approach for delineating histone proteoforms because it can provide us with a bird's-eye view of histone proteoforms carrying diverse combinations of PTMs. Here, we present the first example of coupling capillary zone electrophoresis (CZE), ion mobility spectrometry (IMS), and MS for online multi-dimensional separations of histone proteoforms. Our CZE-high-field asymmetric waveform IMS (FAIMS)-MS/MS platform identified 366 (ProSight PD) and 602 (TopPIC) histone proteoforms from a commercial calf histone sample using a low microgram amount of histone sample as the starting material. CZE-FAIMS-MS/MS improved the number of histone proteoform identifications by about 3 folds compared to CZE-MS/MS alone (without FAIMS). The results indicate that CZE-FAIMS-MS/MS could be a useful tool for comprehensive characterization of histone proteoforms with high sensitivity.
| S-EPMC10922523 | biostudies-literature
Project description:Trapped ion-mobility spectrometry combined with quadrupole time-of-flight mass spectrometry (TIMS-QTOFMS) was evaluated as a tool for resolving linear and branched isomeric polyester oligomers. Solutions of polyester samples were infused directly into the ion source employing electrospray ionization (ESI). TIMS-MS provides both mobility and m/z data on the formed ions, allowing construction of extracted-ion mobilograms (EIMs). EIMs of polyester molecules showed multimodal patterns, indicating conformational differences among isomers. Subsequent TIMS-MS/MS experiments indicated mobility differences to be caused by (degree of) branching. These assignments were supported by liquid chromatography-TIMS-MS/MS analysis, confirming that direct TIMS-MS provided fast (500 ms/scan) distinction between linear and branched small oligomers. Observing larger oligomers (up to 3000 Da) using TIMS required additional molecular charging to ensure ion entrapment within the mobility window. Molecular supercharging was achieved using m-nitrobenzyl alcohol (NBA). The additional charges on the oligomer structures enhanced mobility separation of isomeric species but also added to the complexity of the obtained fragmentation mass spectra. This complexity could be partly reduced by post-TIMS analyte-decharging applying collision-induced dissociation (CID) prior to Q1 with subsequent isolation of the singly charged ions for further fragmentation. The as-obtained EIM profiles were still quite complex as larger molecules possess more possible structural isomers. Nevertheless, distinguishing between linear and symmetrically branched oligomers was possible based on measured differences in collisional cross sections (CCSs). The established TIMS-QTOFMS approach reliably allows branching information on isomeric polyester molecules up to 3000 Da to be obtained in less than 1 min analysis time.
| S-EPMC8176450 | biostudies-literature
Project description:Estradiol is an estrogenic steroid that can undergo glucuronidation at two different sites, which results in two estradiol glucuronide regioisomers. These isomers can be produced by different enzymes and can have different biological activities before being eliminated from the body. Although there have been previous methods that can distinguish the two isomers, these methods often have long acquisition times or high cost per analysis. In this study, traveling wave ion mobility spectrometry (TWIMS) coupled with mass spectrometry (MS) was employed to separate estradiol glucuronides using alkali metal adduction in positive ion mode, where the sodiated dimer adduct provided adequate separation both in single-component standards and in two-component mixtures. Additionally, in negative ion mode, tandem mass spectrometry (MS/MS) was used to quantitatively determine the relative composition of the two isomers. This was possible due to differences in the energetic requirements for loss of the glucuronic acid, which was characterized by energy-resolved collision-induced dissociation (CID). This work demonstrated that the intensity of the glucuronic acid neutral loss product as compared with the intensity of the intact precursor ion can be used to determine the percentage of each isomer present in a mixture. Overall, TWIMS successfully separated estradiol glucuronide isomers in positive ion mode and MS/MS via CID enables relative quantitation of each isomer in negative ion mode.
| S-EPMC6812596 | biostudies-literature
Project description:The dense O-glycosylation of mucins plays an important role in the defensive properties of the mucus hydrogel. Aberrant glycosylation is often correlated with inflammation and pathology such as COPD, cancer, and Crohn's disease. The inherent complexity of glycans and the diversity in the O-core structure constitute fundamental challenges for the analysis of mucin-type O-glycans. Due to coexistence of multiple isomers, multidimensional workflows such as LC-MS are required. To separate the highly polar carbohydrates, porous graphitized carbon is often used as a stationary phase. However, LC-MS workflows are time-consuming and lack reproducibility. Here we present a rapid alternative for separating and identifying O-glycans released from mucins based on trapped ion mobility mass spectrometry. Compared to established LC-MS, the acquisition time is reduced from an hour to two minutes. To test the validity, the developed workflow was applied to sputum samples from cystic fibrosis patients to map O-glycosylation features associated with disease.
| S-EPMC10960840 | biostudies-literature
Project description:Electron capture dissociation mass spectrometry offers several advantages for the analysis of peptides, most notably that backbone c and z fragments typically retain labile modifications such as phosphorylation. We have shown previously that, in some cases, the presence of phosphorylation has a deleterious effect on peptide sequence coverage, and hypothesized that intramolecular interactions involving the phosphate group were preventing separation of backbone fragments. In the present work, we seek to rationalize the observed ECD behavior through a combination of ECD of model peptides, traveling wave ion mobility mass spectrometry and molecular dynamics simulations. The results suggest that for doubly protonated ions of phosphopeptide APLpSFRGSLPKSYVK a salt-bridge structure is favored, whereas for the doubly-protonated ions of APLSFRGSLPKpSYVK ionic hydrogen bonds predominate.
| S-EPMC4422852 | biostudies-literature
Project description:Carbohydrates play important roles in biological processes, but their identification remains a significant analytical problem. While mass spectrometry has increasingly enabled the elucidation of carbohydrates, current approaches are limited in their abilities to differentiate isomeric carbohydrates when these are not separated prior to tandem-mass spectrometry analysis. This analytical challenge takes on increased relevance because of the pervasive presence of isomeric carbohydrates in biological systems. Here, we demonstrate that TIMS2-MS2 workflows enabled by tandem-trapped ion mobility spectrometry-mass spectrometry (tTIMS/MS) provide a general approach to differentiate isomeric, nonseparated carbohydrates. Our analysis shows that (1) cross sections measured by TIMS are sufficiently precise and robust for ion identification; (2) fragment ion cross sections from TIMS2 analysis can be analytically exploited to identify carbohydrate precursors even if the precursor ions are not separated by TIMS; (3) low-abundant fragment ions can be exploited to identify carbohydrate precursors even if the precursor ions are not separated by IMS. (4) MS2 analysis of fragment ions produced by TIMS2 can be used to validate and/or further characterize carbohydrate structures. Taken together, our analysis underlines the opportunities that tandem-ion mobility spectrometry/MS methods offer for the characterization of mixtures of isomeric carbohydrates.
| S-EPMC10126951 | biostudies-literature
Project description:Tandem mass spectrometry (MS/MS) plays an essential role in modern chemical analysis. It is used for differentiating isomers and isobars and suppressing chemical noise, which allows high precision quantitation. The MS/MS analysis has been typically applied by isolating the target precursor ions, while disregarding other ions, followed by a fragmentation process that produces the product ions. In this study, configurations of dual linear ion traps were explored to develop high efficiency MS/MS analysis. The ions trapped in the first linear ion trap were axially, mass-selectively transferred to the second linear ion trap for MS/MS analysis. Ions from multiple compounds simultaneously introduced into the mass spectrometer could be sequentially analyzed. This development enables highly efficient use of the sample. For miniature ion trap mass spectrometers with discontinuous atmospheric pressure interfaces, the analysis speed and the quantitation precision can be significantly improved.
| S-EPMC4148301 | biostudies-literature
Project description:RationaleIsobaric tandem mass tags are an attractive alternative to mass difference tags and label-free approaches for quantitative proteomics due to the high degree of multiplexing that can be performed with their implementation. A drawback of tandem mass tags are that the co-isolation and co-fragmentation of labeled peptide precursors can result in chimeric tandem mass (MS/MS) spectra that can underestimate the fold-change expression of each peptide. Ion mobility (IM) separations coupled to quadrupole time-of-flight (Q-TOF) instruments have the potential to mitigate MS/MS spectra chimeracy since IM-MS has the ability to separate ions based on charge, m/z, and collision cross section (CCS).MethodsTwo complex protein mixtures, labeled with DiLeu isobaric tandem mass tags in opposite ratios, were mixed together and analyzed by data-dependent LC/IM-MS/MS. The accuracy of reporters from interfering pairs was compared with and without IM separation.ResultsIM separation was able to mitigate isobaric interference from differentially charged interfering ion pairs, as well as pairs of the same charge. Of the eight example precursors shown, only one had reporters that remained compressed below the significance threshold after IM separation.ConclusionsThe results of this investigation demonstrate proof-of-principle that IM separation of tagged precursors prior to MS/MS fragmentation can help mitigate quantitative inaccuracies caused by isobaric interference. Future improvements of the method would include software for automated correction and use of higher resolution IM instrumentations.
| S-EPMC4000571 | biostudies-literature
Project description:One of the limiting factors in determining the sensitivity of tandem mass spectrometry using hybrid quadrupole orthogonal acceleration time-of-flight instruments is the duty cycle of the orthogonal ion injection system. As a consequence, only a fraction of the generated fragment ion beam is collected by the time-of-flight analyzer. Here we describe a method utilizing postfragmentation ion mobility spectrometry of peptide fragment ions in conjunction with mobility time synchronized orthogonal ion injection leading to a substantially improved duty cycle and a concomitant improvement in sensitivity of up to 10-fold for bottom-up proteomic experiments. This enabled the identification of 7500 human proteins within 1 day and 8600 phosphorylation sites within 5 h of LC-MS/MS time. The method also proved powerful for multiplexed quantification experiments using tandem mass tags exemplified by the chemoproteomic interaction analysis of histone deacetylases with Trichostatin A.
| S-EPMC4256517 | biostudies-literature
Project description:A high resolution ion mobility spectrometer was interfaced to a Synapt G2 high definition mass spectrometer (HDMS) to produce IMMS-IMMS analysis. The hybrid instrument contained an electrospray ionization source, two ion gates, an ambient pressure linear ion mobility drift tube, a quadrupole mass filter, a traveling wave ion mobility spectrometer (TWIMS), and a time-of-flight mass spectrometer. The dual gate drift tube ion mobility spectrometer (DTIMS) could be used to acquire traditional IMS spectra but also could selectively transfer specific mobility selected precursor ions to the Synapt G2 HDMS for mass filtration (quadrupole). The mobility and mass selected ions could then be introduced into a collision cell for fragmentation followed by mobility separation of the fragment ions with the traveling wave ion mobility spectrometer. These mobility separated fragment ions are finally mass analyzed using a time-of-flight mass spectrometer. This results in an IMMS-IMMS analysis and provides a method to evaluate the isomeric heterogeneity of precursor ions by both DTIMS and TWIMS to acquire a mobility-selected and mass-filtered fragmentation pattern and to additionally obtain traveling wave ion mobility spectra of the corresponding product ions. This new IMMS(2) instrument enables the structural diversity of carbohydrates to be studied in greater detail. The physical separation of isomeric oligosaccharide mixtures was achieved by both DTIMS and TWIMS, with DTIMS demonstrating higher resolving power (70-80) than TWIMS (30-40). Mobility selected MS/MS spectra were obtained, and TWIMS evaluation of product ions showed that isomeric forms of fragment ions existed for identical m/z values.
| S-EPMC3633474 | biostudies-literature