Exploiting ion-mobility mass spectrometry for unraveling proteome complexity.

Structure-elucidation of human CCL5 by integrating trapped ion mobility spectrometry-mass spectrometry (TIMS-MS) with Structure Relaxation Approximation (SRA) analysis

ADVERTISEMENT RETURN TO ISSUEPREVReviewNEXTIon Mobility-Mass Spectrometry: Time-Dispersive InstrumentationJody C. May and John A. McLean*View Author Information Department of Chemistry, Center for Innovative Technology, Vanderbilt Institute for Chemical Biology, Vanderbilt Institute for Integrative Biosystems Research and Education , Vanderbilt University, Nashville, Tennessee 37235, United States*E-mail: [email protected]Cite this: Anal. Chem. 2015, 87, 3, 1422–1436Publication Date (Web):December 19, 2014Publication History Published online9 January 2015Published inissue 3 February 2015https://doi.org/10.1021/ac504720mCopyright © 2014 American Chemical SocietyRIGHTS & PERMISSIONSACS AuthorChoiceArticle Views13748Altmetric-Citations286LEARN ABOUT THESE METRICSArticle Views are the COUNTER-compliant sum of full text article downloads since November 2008 (both PDF and HTML) across all institutions and individuals. These metrics are regularly updated to reflect usage leading up to the last few days.Citations are the number of other articles citing this article, calculated by Crossref and updated daily. Find more information about Crossref citation counts.The Altmetric Attention Score is a quantitative measure of the attention that a research article has received online. Clicking on the donut icon will load a page at altmetric.com with additional details about the score and the social media presence for the given article. Find more information on the Altmetric Attention Score and how the score is calculated. Share Add toView InAdd Full Text with ReferenceAdd Description ExportRISCitationCitation and abstractCitation and referencesMore Options Share onFacebookTwitterWechatLinked InReddit PDF (4 MB) Get e-AlertsSUBJECTS:Collisions,Ions,Mass spectrometers,Optimization,Power Get e-Alerts

Discrimination of isomers is an important and valuable feature in many analytical applications, and the identification of chiral isomers and cis-trans isomers is the current research focus. In this work, a simple method for direct, simultaneous recognition of d-/l-proline (P), d-/l-/cis-/trans-4-hydroxyproline (4-HP), and d-/l-/cis-/trans-N-tert-butoxycarbony (N-Boc-4-HP) was investigated by means of trapped ion mobility spectrometry-mass spectrometry (TIMS-MS). The isomers with cis-/trans-/d-/l-configuration can be directly recognized based on their mobility upon reaction with natamycin (Nat) and metal ions through noncovalent interactions. The results indicate that the recognition of the enantiomers has certain specificity, and the structural difference of the enantiomers was increased in a complex with Nat and metal ions. Herein, d-/l-P can be recognized through the ternary complexes [P + Nat + Mg - H]+, [P + 2Nat + Ca - H]+, [P + 2Nat + Mn - H]+, and [P + Nat + Cu - H]+. Similarly, c-4-HPL, c-4-HPD, t-4-HPL, and t-4-HPD can be recognized by [4-HP + Nat + Ca - H]+, [4-HP + 2Nat + Ca - H]+, and [4-HP + Nat + Cu - H]+, while N-Boc-c-4-HPL, N-Boc-c-4-HPD, N-Boc-t-4-HPL, and N-Boc-t-4-HPD were recognized through the enantiomer complexes [N-Boc-4-HP + Nat + Li]+, [N-Boc-4-HP + Nat + 2Na - H]+, [N-Boc-4-HP + Nat + K]+, [N-Boc-4-HP + Nat + Mn - H]+, and [N-Boc-4-HP + Nat + Ba - H]+. Moreover, tandem mass spectrometry (MS/MS) results indicated that different collision energies were obtained for the same fragment ions, which implied that the enantiomer complexes that contributed to their mobility separation shared identical interaction mode but had different gas-phase rigid geometries. Furthermore, the relative quantification for the enantiomers was performed, and the results were supported by a satisfactory coefficient (R2 > 0.99). The developed method can provide a promising and powerful strategy for the separation of chiral proline and its d-/l-/cis-/trans derivatives, bearing the advantages of higher speed, better accuracy, high selectivity, and no need for chemical derivatization and chromatographic separation.

Isomeric oligosaccharides gamma-cyclodextrin (gamma-CD), glucosyl-betaCD (Glc1-betaCD) and maltosyl-alphaCD (Glc2-alphaCD) were analyzed by traveling-wave ion mobility (twIM) mass spectrometry (MS). Their formation of multicharged multimers differed from each other. The ion mobility-mass spectrometry was useful in the self-assembling and complex formation analyses of CD isomers. The drift times of the isomers and their product ions with the same mass were almost the same in collision-induced dissociation (CID) MS/MS. In contrast, the ion mobility peak widths were sensitive to structural differences of the isomeric product ions. The twIM peak width (ms-micros) of the product ions [M-Glc(n) + H]+ (n = 0-6) of gamma-CD correlated linearly with their masses (Da); the large and/or long chain product ions had wider peak widths, which were much wider than those from the general diffusion effect. This was a novel and useful 'trend line' to discriminate between the three isomers. Plots of [M-Glc(2-6) + H]+ of Glc1-betaCD and [M-Glc(3-6) + H]+ of Glc2-alphaCD product ions' plots were on the same trend line as gamma-CD. The plots of [M-Glc1 + H]+ of Glc1-betaCD and [M-Glc(1, 2) + H]+ of Glc2-alphaCD strayed from the gamma-CD line; their peak widths were narrower than those of gamma-CD. These results indicated that product ions from the chemical species of Glc1-beta CD and Glc2-alphaCD retained their CD structure. Analyses of the IM peak widths enable us to elucidate the structures of the product ions.

Chondroitin sulfate (CS) glycosaminoglycans are bioactive sulfated polysaccharides comprising repeating units of uronic acid and N-acetyl galactose sulfated at various positions. The optimal length and sulfation pattern of the CS bioactive sequences remain elusive so that structure-activity relationships cannot be easily established. Development of efficient analytical methods allowing the differentiation of the various sulfation patterns of CS sequences is therefore of particular importance to correlate their biological functions to the sulfation pattern. Discrimination of different oligomers (dp2 to dp6) of synthetic chondroitin sulfate isomers was evaluated by electrospray ionization tandem mass spectrometry (ESI-MS/MS) in the negative-ion mode from deprotonated and alkali adduct species. In addition, ion mobility mass spectrometry (IMS-MS) was used to study the influence of both the degree of polymerization and sulfate group location on the gas-phase conformation of CS oligomers. ESI-MS/MS spectra of chondroitin sulfate isomers show characteristic product ions exclusively from alkali adduct species (Li, Na, K and Cs). Whatever the alkali adducts studied, MS/MS of chondroitin oligosaccharides sulfated at position 6 yields a specific product ion at m/z 139 while CS oligosaccharides sulfated at position 4 show a specific product ion at m/z 154. Being observed for the different CS oligomers di-, tetra- and hexasaccharides, these fragment ions are considered as diagnostic ions for chondroitin 6-O-sulfate and chondroitin 4-O-sulfate, respectively. IMS-MS experiments reveal that collision cross-sections (CCS) of CS oligomers with low charge states evolved linearly with degrees of polymerization indicating a similar gas-phase conformation. This study allows the fast and unambiguous differentiation of CS isomers sulfated at position 6 or 4 for both saturated and unsaturated analogues from MS/MS experiments. In addition, the CCS linear evolution of CS oligomers in function of the degree of polymerization indicates that no folding occurs even for hexasaccharides.

Semi-automated screen for global protein conformational changes in solution by ion mobility spectrometry–massspectrometry combined with size-exclusion chromatography and differential hydrogen–deuterium exchange

Abstract. Measurement techniques that provide molecular-level information are needed to elucidate the multiphase processes that produce secondary organic aerosol (SOA) species in the atmosphere. Here we demonstrate the application of ion mobility spectrometry-mass spectrometry (IMS–MS) to the simultaneous characterization of the elemental composition and molecular structures of organic species in the gas and particulate phases. Molecular ions of gas-phase organic species are measured online with IMS–MS after ionization with a custom-built nitrate chemical ionization (CI) source. This CI–IMS–MS technique is used to obtain time-resolved measurements (5 min) of highly oxidized organic molecules during the 2013 Southern Oxidant and Aerosol Study (SOAS) ambient field campaign in the forested SE US. The ambient IMS–MS signals are consistent with laboratory IMS–MS spectra obtained from single-component carboxylic acids and multicomponent mixtures of isoprene and monoterpene oxidation products. Mass-mobility correlations in the 2-D IMS–MS space provide a means of identifying ions with similar molecular structures within complex mass spectra and are used to separate and identify monoterpene oxidation products in the ambient data that are produced from different chemical pathways. Water-soluble organic carbon (WSOC) constituents of fine aerosol particles that are not resolvable with standard analytical separation methods, such as liquid chromatography (LC), are shown to be separable with IMS–MS coupled to an electrospray ionization (ESI) source. The capability to use ion mobility to differentiate between isomers is demonstrated for organosulfates derived from the reactive uptake of isomers of isoprene epoxydiols (IEPOX) onto wet acidic sulfate aerosol. Controlled fragmentation of precursor ions by collisionally induced dissociation (CID) in the transfer region between the IMS and the MS is used to validate MS peak assignments, elucidate structures of oligomers, and confirm the presence of the organosulfate functional group.

Since the development of electrospray ionization (ESI) for ion mobility spectrometry mass spectrometry (IMMS), IMMS have been extensively applied for characterization of gas-phase bio-molecules. Conventional ion mobility spectrometry (IMS), defined as drift tube IMS (DT-IMS), is typically a stacked ring design that utilizes a low electric field gradient. Field asymmetric ion mobility spectrometry (FAIMS) is a newer version of IMS, however, the geometry of the system is significantly different than DT-IMS and data are collected using a much higher electric field. Here we report construction of a novel ambient pressure dual gate DT-IMS coupled with a FAIMS system and then coupled to a quadrupole ion trap mass spectrometer (QITMS) to form a hybrid three-dimensional separation instrument, DT-IMS-FAIMS-QITMS. The DT-IMS was operated at ~3 Townsend (electric field/number density (E/N) or (Td)) and was coupled in series with a FAIMS, operated at ~80 Td. Ions were mobility-selected by the dual gate DT-IMS into the FAIMS and from the FAIMS the ions were detected by the QITMS for as either MS or MSn. The system was evaluated using cocaine as an analytical standard and tested for the application of separating three isomeric tri-peptides: tyrosine-glycine-tryptophan (YGW), tryptophan-glycine-tyrosine (WGY) and tyrosine-tryptophan-glycine (YWG). All three tri-peptides were separated in the DT-IMS dimension and each had one mobility peak. The samples were partially separated in the FAIMS dimension but two conformation peaks were detected for the YWG sample while YGW and WGY produced only one peak. Ion validation was achieved for all three samples using QITMS.

Protein post-translational modifications provide critical proteomic details towards elucidating mechanisms of altered protein function due to toxic exposure, altered metabolism, or disease pathogenesis. Lysine propionylation is a recently described modification that occurs due to metabolic alterations in propionyl-CoA metabolism and sirtuin depropionylase activity. Acrolein is a toxic aldehyde generated through exogenous and endogenous pathways, such as industrial exposure, cigarette smoke inhalation, and non-enzymatic lipid peroxidation. Importantly, lysine modifications arising from propionylation and acroleination can be isobaric - indistinguishable by mass spectrometry - and inseparable via reverse-phase chromatography. Here, we present the novel application of trapped ion mobility spectrometry (TIMS) to resolve such competing isobaric lysine modifications. Specifically, the PTM products of a small synthetic peptide were analyzed using a prototype TIMS - time-of-flight mass spectrometer (TIMS-TOF). In that the mobilities of these propionylated and acroleinated peptides differ by only 1%, a high-resolution mobility analysis is required to resolve the two. We were able to achieve more than sufficient resolution in the TIMS analyzer (~170), readily separating these isobars.

Peptide and protein aggregation involves the formationof oligomericspecies, but the complex interplay between oligomers of differentconformations and sizes complicates their structural elucidation.Using ion mobility mass spectrometry (IM-MS), we aim to reveal theseearly steps of aggregation for the Ac-PHF6-NH2 peptidesegment from tau protein, thereby distinguishing between differentoligomeric species and gaining an understanding of the aggregationpathway. An important factor that is often neglected, but which canalter the aggregation propensity of peptides, is the terminal cappinggroups. Here, we demonstrate the use of IM-MS to probe the early stagesof aggregate formation of Ac-PHF6-NH2, Ac-PHF6, PHF6-NH2, and uncapped PHF6 peptide segments. The aggregation propensityof the four PHF6 segments is confirmed using thioflavin T fluorescenceassays and transmission electron microscopy. A novel approach basedon post-IM fragmentation and quadrupole selection on the TIMS-Qq-ToF(trapped ion mobility) spectrometer was developed to enhance oligomerassignment, especially for the higher-order aggregates. This approachpushes the limits of IM identification of isobaric species, whosesignatures appear closer to each other with increasing oligomer size,and provides new insights into the interpretation of IM-MS data. Inaddition, TIMS collision cross section values are compared with travelingwave ion mobility (TWIMS) data to evaluate potential instrumentalbias in the trapped ion mobility results. The two IM-MS instrumentalplatforms are based on different ion mobility principles and havedifferent configurations, thereby providing us with valuable insightinto the preservation of weakly bound biomolecular complexes suchas peptide aggregates.

Measurements of the mobility of gaseous ions carried out in the Wills Laboratory at Bristol have shown that a positive ion which at birth is a charged atom or molecule remains in its monomolecular state when the gas is pure. The relation between the mass of the ion and its mobility has been determined in a number of gases. In the presence of polar impurities each ion collects a cluster of molecules of impurity, so that it moves more slowly in an electric field. The purpose of this paper is to describe experiments made to study the formation of the clusters and the number of molecules they contain. Early experimenters measured the mobility of clustered ions, but thenvalues cannot be used for this purpose because the nature and concentration of the impurities present were unknown and uncontrollable. The only way of studying the problem is to use an apparatus made entirely of glass and metal, cleaned by a “ bake out ” and with tap grease and other contamination excluded. Controlled amounts of a known polar impurity can then be added to the pure gas. The experiments here described have been made at room temperature on the alkali positive ions in the inert gases with water vapour as the polar impurity.

Discriminating quinine stereoisomers through collision cross-section differences enhanced by cyclodextrin complexation

In the present work, a novel workflow based on complementary gas-phase separations for the identification of isomeric PAHs from complex mixtures is described. This is the first report on the coupling of gas chromatography (GC), atmospheric pressure laser ionization (APLI), and trapped ion mobility spectrometry-mass spectrometry (TIMS-MS) for the characterization of polycyclic aromatic hydrocarbons. Over a hundred known unknowns are uniquely identified based on the molecular ion retention indices I (5%), mobility (RSD < 0.6% and R = 50-90 with Sr = 0.18 V/ms), mobility-based theoretical candidate assignment (<3%), accurate mass chemical formula assignment (<2 ppm), and electron impact fragmentation pattern and database search. The advantages of theoretical modeling of PAHs and similar compounds were evaluated using candidate structures ranked by retention indices and fragmentation pattern from GC-EI-MS data sets. Over 20 PAH isomeric and deuterated standards were utilized for the GC-APLI-TIMS-TOF MS workflow validation. Noteworthy is the analytical capability for untargeted screening of isomeric and isobaric compounds with additional characterization metrics not available in traditional GC-EI-MSn workflows.

Data associated with "Liquid extraction surface analysis field asymmetric waveform ion mobility spectrometry mass spectrometry for the analysis of dried blood spots: DOI:10.1039/C5AN00933B"

Characterization of native protein structure with ion mobility mass spectrometry, multiplexed fragmentation strategies and multivariant analysis