Journal of the American Society for Mass Spectrometry最新文献

{"title":"Characterization of Sugammadex-Related Isomeric Cyclodextrin Impurities Using Cyclic Ion Mobility High-Resolution Mass Spectrometry","authors":"Péter S. Szakály,&nbsp;Dávid Papp,&nbsp;Arnold Steckel,&nbsp;Erzsébet Varga and Gitta Schlosser*,&nbsp;","doi":"10.1021/jasms.4c0024310.1021/jasms.4c00243","DOIUrl":"https://doi.org/10.1021/jasms.4c00243https://doi.org/10.1021/jasms.4c00243","url":null,"abstract":"<p >Cyclic ion mobility–mass spectrometry (cIM-MS) is a powerful technique for separating and identifying isomeric mixtures of compounds. When coupled with chromatography, cIM-MS creates a multidimensional separation system, with high resolving power and peak capacity. In this study, we report the cyclic ion mobility separation and high-resolution mass spectrometry identification of four regioisomers of a Sugammadex-related impurity, abbreviated as Di-OH-SGM. Separation using multipass cyclic ion mobility was achieved by selecting the [M + 2Na]²⁺ ion, while other adducts, such as [M + Na]⁺, [M + 2H]²⁺, [M + H + Na]²⁺, and [M – 2H]^2– did not yield isomer separation. Two methods were developed for ion mobility separation of the isomers: a conventional multipass method and a slicing method. Isomer assignment was based on the characteristic fragment ions. The collision cross section values (^cTWCCS_N2) of the resolved cyclodextrin isomers were also determined. Ion mobility separation of structurally different fragment ions was demonstrated. Additionally, by coupling cIM-MS with reversed-phase liquid chromatography (HPLC-cIM-MS), two-dimensional separation of the isomers was achieved. The isomers, separated using HPLC-cIM-MS, were identified with the same approach as with cIM-MS alone, and their elution order provided insights into their relative hydrophobicity.</p>","PeriodicalId":672,"journal":{"name":"Journal of the American Society for Mass Spectrometry","volume":"36 2","pages":"258–264 258–264"},"PeriodicalIF":3.1,"publicationDate":"2025-01-24","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://pubs.acs.org/doi/epdf/10.1021/jasms.4c00243","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143127202","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"化学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Rigorous Analysis of Multimodal HDX-MS Spectra","authors":"Lisa M. Tuttle,&nbsp;Ellie I. James,&nbsp;Florian Georgescauld,&nbsp;Thomas E. Wales,&nbsp;David D. Weis,&nbsp;John R. Engen,&nbsp;Abhinav Nath,&nbsp;Rachel E. Klevit and Miklos Guttman*,&nbsp;","doi":"10.1021/jasms.4c0047110.1021/jasms.4c00471","DOIUrl":"https://doi.org/10.1021/jasms.4c00471https://doi.org/10.1021/jasms.4c00471","url":null,"abstract":"<p >An inherent strength of hydrogen/deuterium exchange coupled to mass spectrometry (HDX-MS) is its ability to detect the presence of multiple conformational states of a protein, which often manifest as multimodal isotopic envelopes. However, the statistical considerations for accurate analysis of multimodal spectra have yet to be established. Here we outline an unrestrained binomial distribution fitting approach with the corresponding statistical tests to accurately detect and, when possible, deconvolute isotopic distributions that contain multiple subpopulations. The algorithms have been incorporated into an updated version of the freely available software, HX-Express, and validated using known mixtures of peptides deuterated to varying degrees. This approach presents a readily accessible tool to fit and interpret bimodal and trimodal behavior in HDX-MS data for mixed populations, EX1 kinetics, and pulse labeling data.</p>","PeriodicalId":672,"journal":{"name":"Journal of the American Society for Mass Spectrometry","volume":"36 2","pages":"416–423 416–423"},"PeriodicalIF":3.1,"publicationDate":"2025-01-21","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143126846","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"化学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Gas-Phase Fragmentation of Coenzyme Q10 Radical Anion Generated by APCI: A Study by High/Low-Resolution Tandem/Sequential Mass Spectrometry","authors":"Mariachiara Bianco,&nbsp;Ilario Losito*,&nbsp;Giovanni Ventura,&nbsp;Beniamino Leoni,&nbsp;Onofrio Davide Palmitessa,&nbsp;Massimiliano Renna,&nbsp;Pietro Santamaria,&nbsp;Cosima Damiana Calvano and Tommaso R.I. Cataldi,&nbsp;","doi":"10.1021/jasms.4c0039910.1021/jasms.4c00399","DOIUrl":"https://doi.org/10.1021/jasms.4c00399https://doi.org/10.1021/jasms.4c00399","url":null,"abstract":"<p >Coenzyme Q₁₀ (CoQ₁₀) and closely related compounds with varying isoprenoid tail lengths (CoQ_<i>n</i>, <i>n</i> = 6–9) are biochemical cofactors involved in many physiological processes, playing important roles in cellular respiration and energy production. Liquid chromatography (LC) coupled with single or tandem mass spectrometry (MS) using electrospray (ESI) or atmospheric pressure chemical ionization (APCI) is considered the gold standard for the identification and quantification of CoQ₁₀ in food and biological samples. However, the characteristic fragmentation exhibited by the CoQ₁₀ radical anion ([M]^•^–, <i>m</i>/<i>z</i> 862.684), the prevailing ion generated by APCI in negative polarity, has not been studied in detail. In this work, a systematic study was carried out to clarify this issue, using higher collisional energy dissociation (HCD) with high-resolution tandem FTMS and collision-induced dissociation-low-resolution sequential mass spectrometry (CID-MS^<i>n</i>, <i>n</i> = 2–4). Various fragmentation pathways were successfully interpreted, with some structures proposed for product ions checked using density functional theory (DFT) calculations. Besides the already-known detachments of methyl radicals occurring directly from the CoQ₁₀ radical anion and leading to ions like [M – CH₃]<b>^–</b> and [M – 2CH₃]^•–, the homolytic cleavage of C–C bonds along the oligo-isoprenoid side chain was tentatively proposed to explain some of the observed fragmentations. As a result, the generation of uncommon yet potentially stable distonic biradical anions was hypothesized, with some of them likely undergoing intramolecular cyclization to generate ions without unpaired electrons. Diagnostic product ions emerged from the fragmentation processes of CoQ₁₀ and were found to be common also to the radical anions of other CoQ_<i>n</i> derivatives (<i>n</i> = 7–9), facilitating their identification in extracts of edible <i>Brassicaceae</i> plant microgreens by reversed-phase liquid chromatography (RPLC)-APCI-FTMS.</p>","PeriodicalId":672,"journal":{"name":"Journal of the American Society for Mass Spectrometry","volume":"36 2","pages":"318–328 318–328"},"PeriodicalIF":3.1,"publicationDate":"2025-01-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143127080","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"化学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Revealing Unpredicted Aspartic Acid Isomerization Hotspots by Probing Diagnostic Fragmentation Propensities in Top-Down and Middle-Down Mass Spectrometry.","authors":"Blandine Denefeld, Joanna Hajduk, Jure Cerar, Jean-Michel Rondeau, Jérôme Dayer, Manuel Lang, Wolfram Kern, François Griaud","doi":"10.1021/jasms.4c00443","DOIUrl":"https://doi.org/10.1021/jasms.4c00443","url":null,"abstract":"<p><p>Isomerization of aspartic acid residues is a relevant degradation pathway of protein biopharmaceuticals as it can impair their biological activity. However, the in silico prediction of isomerization hotspots and their consequences remains ambiguous and misleading. We have previously shown that all ion differential analysis (AiDA) of middle-down spectra can be used to reveal diagnostic terminal and internal fragments with more sensitivity than the conventional fragment ion mass matching methodology. In this study, we use AiDA to characterize the degradation of an antibody fragment at three aspartic acid isomerization sites including a novel DW motif directly with electron-transfer/higher-energy collisional dissociation top-down and middle-down mass spectrometry. We show that AiDA methodology is pivotal to probe diagnostic fragmentation propensities of terminal c and z fragments at the N-terminus and vicinity of isomerization sites in addition to the diagnostic c+57 terminal fragments. Furthermore, AiDA can probe remote structural changes in the loop of an antibody complementarity-determining region induced by isomerization and the succinimide intermediate, revealing interactions between residues in agreement with molecular simulations. This study shows that aspartic acid residues at noncanonical DW and DF motifs can be hotspots for isomerization despite being ranked as false positives in physics-based prediction models. We show that the enzyme-free, fast, and sensitive AiDA methodology can be used as an orthogonal technique to fractionation for online variant characterization.</p>","PeriodicalId":672,"journal":{"name":"Journal of the American Society for Mass Spectrometry","volume":" ","pages":""},"PeriodicalIF":3.1,"publicationDate":"2025-01-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142998185","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"化学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Unified Fragmentation Pathways of Lithiated, Longer-Chain Acylglycerols Can Be Identified from Tandem Mass Spectrometry and Density Functional Computations","authors":"J. Stuart Grossert*,&nbsp; and ,&nbsp;Louis Ramaley,&nbsp;","doi":"10.1021/jasms.4c0042310.1021/jasms.4c00423","DOIUrl":"https://doi.org/10.1021/jasms.4c00423https://doi.org/10.1021/jasms.4c00423","url":null,"abstract":"<p >We extend our previous work on the energetics and mechanisms of fragmentation in the mass spectrometry of triacylglycerols (TAGs). Previously, we proposed viable mechanisms for the collision-induced fragmentation of lithiated tripropionylglycerol using triple-quadrupole mass spectrometry. In this work, we used a QqLIT mass spectrometer to study both double- and triple-stage spectra from a range of TAGs having acid chains of types AAA (identical acid chains), AAB, ABA, and ABC, with chain lengths of 6–18 carbon atoms; we also studied some TAGs having a single double bond in the Δ-9 position. Detailed computations on fragmentation pathways were carried out on lithiated trihexanoylglycerol and on a limited number of longer chain ions. Second-stage fragmentations led to the formation of a lithiated ion after the loss of a neutral acid. With a further input of energy, this ion could rearrange into two other ions, one being formed more easily than the other. In a triple-stage fragmentation, these three ions give a defined series of product ions, some of which are characteristic of the substitution pattern on the glycerol core of the TAG. Computed reaction energies for product ion formation showed comparable trends to the relative intensities of the observed product ion spectra. These results have enabled us to propose unified mechanisms for the double- and triple-stage fragmentation of lithiated TAGs. The mechanisms have reasonable energetics, and all ions, as well as saddle points, have viable geometries. In addition, the fragmentation mechanisms are in accord with all the published experimental mass spectra.</p>","PeriodicalId":672,"journal":{"name":"Journal of the American Society for Mass Spectrometry","volume":"36 2","pages":"368–378 368–378"},"PeriodicalIF":3.1,"publicationDate":"2025-01-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143127082","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"化学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Investigation into Drug-Induced Liver Damage Using Multimodal Mass Spectrometry Imaging","authors":"Bryn Flinders,&nbsp;Lennart Huizing,&nbsp;Bhanu Singh,&nbsp;Heng-Keang Lim,&nbsp;Marjolein van Heerden,&nbsp;Filip Cuyckens,&nbsp;Ron M. A. Heeren and Rob J. Vreeken*,&nbsp;","doi":"10.1021/jasms.4c0031310.1021/jasms.4c00313","DOIUrl":"https://doi.org/10.1021/jasms.4c00313https://doi.org/10.1021/jasms.4c00313","url":null,"abstract":"<p >Drug toxicity during the development of candidate pharmaceuticals is the leading cause of discontinuation in preclinical drug discovery and development. Traditionally, the cause of the toxicity is often determined by histological examination, clinical pathology, and the detection of drugs and/or metabolites by liquid chromatography–mass spectrometry (LC-MS). While these techniques individually provide information on the pathological effects of the drug and the detection of metabolites, they cannot provide specific molecular spatial information without additional experiments. Matrix-assisted laser desorption/ionization-mass spectrometry imaging (MALDI-MSI) is a powerful, label-free technique for the simultaneous detection of pharmaceuticals, metabolites, and endogenous chemical species in tissue sections, which makes it suitable for mechanistic toxicological studies to directly correlate the distribution of the drug and its metabolites with histological findings. This capability was demonstrated by the analysis of the liver from dogs dosed with discontinued drug compound B and its N-desmethyl metabolite, compound A. Histological examination showed multifocal hepatocellular necrosis, bile duct hyperplasia, periportal fibrosis, and chronic inflammation. MALDI-MSI analysis of liver tissue dosed with only compound A indicated that liver lesions were associated exclusively with the parent compound, whereas liver lesions with compound B showed the presence of the parent compound and its two metabolites (compound A and an N-oxide metabolite). Using both positive and negative ion modes, simultaneous detection and identification of endogenous molecular markers of the connective tissue, blood vessels, liver parenchyma, and bile duct epithelium was achieved, allowing optimal visualization of histological lesions by mass spectrometry imaging.</p>","PeriodicalId":672,"journal":{"name":"Journal of the American Society for Mass Spectrometry","volume":"36 2","pages":"265–276 265–276"},"PeriodicalIF":3.1,"publicationDate":"2025-01-17","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://pubs.acs.org/doi/epdf/10.1021/jasms.4c00313","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143127064","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"化学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Mass Spectrometry Analysis of Chemically and Collisionally Dissociated Molecular Glue- and PROTAC-Mediated Protein Complexes Informs on Disassembly Pathways","authors":"Edvaldo V. S. Maciel,&nbsp;Jonathan Eisert,&nbsp;Julian Müller,&nbsp;Tanja Habeck and Frederik Lermyte*,&nbsp;","doi":"10.1021/jasms.4c0042910.1021/jasms.4c00429","DOIUrl":"https://doi.org/10.1021/jasms.4c00429https://doi.org/10.1021/jasms.4c00429","url":null,"abstract":"<p >Molecular glues (MGs) and proteolysis-targeting chimeras (PROTACs) are used to modulate protein–protein interactions (PPIs), via induced proximity between compounds that have little or no affinity for each other naturally. They promote either reversible inhibition or selective degradation of a target protein, including ones deemed undruggable by traditional therapeutics. Though native MS (nMS) is capable of analyzing multiprotein complexes, the behavior of these artificially induced compounds in the gas phase is still not fully understood, and the number of publications over the past few years is still rather limited. Here, we studied two MG-induced complexes between mTOR_FRB and FKBP12 as well as a PROTAC-induced complex between FKBP51_FK1 and the von Hippel-Lindau E3 ligase (VHL). Native MS combined with collision-induced dissociation (CID) provided a way of measuring not only the formation of these complexes but also their dissociation pathways. Both protein complexes seem to eject preferably the centrally located small (compared to the mass of the proteins) ligand upon CID, rather than dissociating a peripheral subunit, as is often observed for naturally occurring protein complexes. In contrast, chemically induced dissociation in solution generated complementary data to CID, by disrupting the PPI surface, which resulted in more diverse MS spectra that preserved the stronger interactions in solution.</p>","PeriodicalId":672,"journal":{"name":"Journal of the American Society for Mass Spectrometry","volume":"36 2","pages":"355–367 355–367"},"PeriodicalIF":3.1,"publicationDate":"2025-01-15","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143127140","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"化学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"New Library-Based Methods for Nontargeted Compound Identification by GC-EI-MS","authors":"Deborah F. McGlynn,&nbsp;Lindsay D. Yee,&nbsp;H. Martin Garraffo,&nbsp;Lewis Y. Geer,&nbsp;Tytus D. Mak,&nbsp;Yuri A. Mirokhin,&nbsp;Dmitrii V. Tchekhovskoi,&nbsp;Coty N. Jen,&nbsp;Allen H. Goldstein,&nbsp;Anthony J. Kearsley and Stephen E. Stein*,&nbsp;","doi":"10.1021/jasms.4c0045110.1021/jasms.4c00451","DOIUrl":"https://doi.org/10.1021/jasms.4c00451https://doi.org/10.1021/jasms.4c00451","url":null,"abstract":"<p >While gas chromatography mass spectrometry (GC-MS) has long been used to identify compounds in complex mixtures, this process is often subjective and time-consuming and leaves a large fraction of seemingly good-quality spectra unidentified. In this work, we describe a set of new mass spectral library-based methods to assist compound identification in complex mixtures. These methods employ mass spectral uniqueness and compound ubiquity of library entries alongside noise reduction and automated comparison of retention indices to library compounds. As a test data set, we used a publicly available electron ionization mass spectrometry data set consisting of 4833 spectra of particulate organic compounds emitted by combustion of wildland fuels. In the present work, spectra in this data set were first identified using the NIST 2023 EI-MS Library and associated batch process identification software (NIST MS PepSearch) using retention-index corrected Identity Search scoring. Resulting identifications and related information were then employed to parametrize other factors that correlate with identification. A method for identifying compounds absent from but related to those present in mass spectral libraries using the Hybrid Similarity Search is illustrated. Nevertheless, some 90% of the spectra remain unidentified. Through comparison of unidentified to identified mass spectra in this data set, a new simple measure, namely median relative abundance, was developed for evaluating the likelihood of identification.</p>","PeriodicalId":672,"journal":{"name":"Journal of the American Society for Mass Spectrometry","volume":"36 2","pages":"389–399 389–399"},"PeriodicalIF":3.1,"publicationDate":"2025-01-13","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143127316","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"化学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"The Enzyme Effect: Broadening the Horizon of MS Optimization to Nontryptic Digestion in Proteomics","authors":"Kinga Nagy,&nbsp;Péter Sándor,&nbsp;Károly Vékey,&nbsp;László Drahos and Ágnes Révész*,&nbsp;","doi":"10.1021/jasms.4c0039610.1021/jasms.4c00396","DOIUrl":"https://doi.org/10.1021/jasms.4c00396https://doi.org/10.1021/jasms.4c00396","url":null,"abstract":"<p >In recent years, alternative enzymes with varied specificities have gained importance in MS-based bottom-up proteomics, offering orthogonal information about biological samples and advantages in certain applications. However, most mass spectrometric workflows are optimized for tryptic digests. This raises the questions of whether enzyme specificity impacts mass spectrometry and if current methods for nontryptic digests are suboptimal. The success of peptide and protein identifications relies on the information content of MS/MS spectra, influenced by collision energy in collision-induced dissociation. We investigated this by conducting LC-MS/MS measurements with different enzymes, including trypsin, Arg-C, Glu-C, Asp-N, and chymotrypsin, at varying collision energies. We analyzed peptide scores for thousands of peptides and determined optimal collision energy (CE) values. Our results showed a linear <i>m</i>/<i>z</i> dependence for all enzymes, with Glu-C, Asp-N, and chymotrypsin requiring significantly lower energies than trypsin and Arg-C. We proposed a tailored CE selection method for these alternative enzymes, applying ca. 20% lower energy compared to tryptic peptides. This would result in a 10–15 eV decrease on a Bruker QTof instrument and a 5–6 NCE% (normalized collision energy) difference on an Orbitrap. The optimized method improved bottom-up proteomics performance by 8–32%, as measured by peptide identification and sequence coverage. The different trends in fragmentation behavior were linked to the effects of C-terminal basic amino acids for Arg-C and trypsin, stabilizing y fragment ions. This optimized method boosts the performance and provides insight into the impact of enzyme specificity. Data sets are available in the MassIVE repository (MSV000095066).</p>","PeriodicalId":672,"journal":{"name":"Journal of the American Society for Mass Spectrometry","volume":"36 2","pages":"299–308 299–308"},"PeriodicalIF":3.1,"publicationDate":"2025-01-13","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://pubs.acs.org/doi/epdf/10.1021/jasms.4c00396","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143127405","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"化学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Gas-Phase Fluorescence Excitation Experiments on Cryogenically Cold Rhodamine B Cations Linked to Various Amino Acid Esters","authors":"Iden Djavani-Tabrizi,&nbsp;Ziqi Yuan,&nbsp;Thomas Toft Lindkvist,&nbsp;Jiamin Xing,&nbsp;Li Chen* and Steen Bro̷ndsted Nielsen*,&nbsp;","doi":"10.1021/jasms.4c0046910.1021/jasms.4c00469","DOIUrl":"https://doi.org/10.1021/jasms.4c00469https://doi.org/10.1021/jasms.4c00469","url":null,"abstract":"<p >Förster resonance energy transfer (FRET) is becoming a valuable technique in gas-phase structural biology for identifying local structural motifs and conformations of biological molecules, such as peptides and proteins. This method involves labeling the biomolecule with two dyes, a donor dye and an acceptor dye, that are commonly charged rhodamines. Here we examine how different amino acid (AA) methyl esters linked to the dye via amide linkages can influence the dye transition energy and, consequently, the energy-transfer efficiency, using cryogenic ion fluorescence spectroscopy. Absorption spectra were recorded for rhodamine B⁺-labeled AA esters (RB⁺-AA) through fluorescence–excitation experiments at the LUNA2 setup in Aarhus, which operates at cryogenic temperatures (down to approximately 100 K). The AAs studied include aliphatic ones (alanine (A), leucine (L), <i>tert</i>-leucine (<i>tert</i>-L), and methionine (M)), aromatic ones (phenylalanine (F) and tryptophan (W)), and two with polar side chains (serine (S) and threonine (T)). Results show that the band maximum either remains unchanged compared to RB⁺ or red shifts by over 3 nm in the case of RB⁺-M and RB⁺-F. While the spectra of RB⁺-A and RB⁺-L closely resemble that of RB⁺, RB⁺-<i>tert</i>-L shows a distinct red shift of about 1.4 nm. Spectral variations do not appear to be more influenced by the presence of aromatic AA side chains than other types, as differences observed between aliphatic AAs are comparable to those between the three groups. Instead, these variations appear to arise from differing conformations where the dihedral angle between the xanthene moiety and the pendant phenyl group varies, as influenced by the linked AA side chain. The angle determines the π-overlap between the two aromatic moieties, and according to TD-DFT calculations, an angle larger than 90° can easily account for red shifts due to larger delocalization of the π-electron cloud. Another factor is the polarizability of the side chain that could also contribute to the red shift. RB⁺-F and RB⁺-W spectra exhibit red-shifted, narrower absorption profiles, which is likely associated with the large aromatic side chains that limit the number of contributing structural configurations.</p>","PeriodicalId":672,"journal":{"name":"Journal of the American Society for Mass Spectrometry","volume":"36 2","pages":"409–415 409–415"},"PeriodicalIF":3.1,"publicationDate":"2025-01-06","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143127533","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"化学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}