RESUMO
Bromodomain proteins (BRD) are key chromatin regulators of genome function and stability as well as therapeutic targets in cancer. Here, we systematically delineate the contribution of human BRD proteins for genome stability and DNA double-strand break (DSB) repair using several cell-based assays and proteomic interaction network analysis. Applying these approaches, we identify 24 of the 42 BRD proteins as promoters of DNA repair and/or genome integrity. We identified a BRD-reader function of PCAF that bound TIP60-mediated histone acetylations at DSBs to recruit a DUB complex to deubiquitylate histone H2BK120, to allowing direct acetylation by PCAF, and repair of DSBs by homologous recombination. We also discovered the bromo-and-extra-terminal (BET) BRD proteins, BRD2 and BRD4, as negative regulators of transcription-associated RNA-DNA hybrids (R-loops) as inhibition of BRD2 or BRD4 increased R-loop formation, which generated DSBs. These breaks were reliant on topoisomerase II, and BRD2 directly bound and activated topoisomerase I, a known restrainer of R-loops. Thus, comprehensive interactome and functional profiling of BRD proteins revealed new homologous recombination and genome stability pathways, providing a framework to understand genome maintenance by BRD proteins and the effects of their pharmacological inhibition.
Assuntos
Instabilidade Genômica , Estruturas R-Loop , Reparo de DNA por Recombinação/genética , Fatores de Transcrição/genética , Acetilação , Linhagem Celular , Quebras de DNA de Cadeia Dupla , DNA Topoisomerases Tipo I/metabolismo , DNA Topoisomerases Tipo II/metabolismo , Células HEK293 , Células HeLa , Humanos , Transativadores/metabolismo , Fatores de Transcrição/análise , Ubiquitinação , Fatores de Transcrição de p300-CBP/genética , Fatores de Transcrição de p300-CBP/metabolismoRESUMO
Chromatin connects DNA damage response factors to sites of damaged DNA to promote the signaling and repair of DNA lesions. The histone H2A variants H2AX, H2AZ, and macroH2A represent key chromatin constituents that facilitate DNA repair. Through proteomic screening of these variants, we identified ZMYM3 (zinc finger, myeloproliferative, and mental retardation-type 3) as a chromatin-interacting protein that promotes DNA repair by homologous recombination (HR). ZMYM3 is recruited to DNA double-strand breaks through bivalent interactions with both histone and DNA components of the nucleosome. We show that ZMYM3 links the HR factor BRCA1 to damaged chromatin through specific interactions with components of the BRCA1-A subcomplex, including ABRA1 and RAP80. By regulating ABRA1 recruitment to damaged chromatin, ZMYM3 facilitates the fine-tuning of BRCA1 interactions with DNA damage sites and chromatin. Consistent with a role in regulating BRCA1 function, ZMYM3 deficiency results in impaired HR repair and genome instability. Thus, our work identifies a critical chromatin-binding DNA damage response factor, ZMYM3, which modulates BRCA1 functions within chromatin to ensure the maintenance of genome integrity.
Assuntos
Proteína BRCA1/metabolismo , Neoplasias Ósseas/metabolismo , Cromatina/metabolismo , Reparo do DNA , Proteínas Nucleares/metabolismo , Osteossarcoma/metabolismo , Sequência de Aminoácidos , Proteína BRCA1/genética , Neoplasias Ósseas/genética , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Cromatina/genética , Quebras de DNA de Cadeia Dupla , Proteínas de Ligação a DNA , Instabilidade Genômica , Células HEK293 , Chaperonas de Histonas , Histonas/genética , Histonas/metabolismo , Recombinação Homóloga , Humanos , Proteínas Nucleares/genética , Osteossarcoma/genética , Homologia de Sequência de Aminoácidos , Células Tumorais CultivadasRESUMO
Broad interest in nucleic acids, both their therapeutic capabilities and understanding the nuances of their structure and resulting function, has increased in recent years. Post-transcriptional modifications, in particular, have become an important analysis target, as these covalent modifications to the sugars, nitrogenous bases, and phosphate backbone impart differential functionality to synthetic and biological nucleic acids. Characterizing these post-transcriptional modifications can be difficult with traditional sequencing workflows; however, advancements in top-down mass spectrometry address these challenges. Online desalting platforms have enabled facile sample cleanup and reliable ionization of increasingly large (100 nt) oligonucleotides, and application of existing tandem mass spectrometry techniques has yielded information-rich spectra which can be used to interrogate primary sequences. To extend the capabilities of top-down MS and its analysis of nucleic acids, we have developed a nanoflow desalting platform for high-throughput and low sample-use desalting coupled with collision-induced dissociation (CID), 213 nm ultraviolet photodissociation (UVPD), and activated-ion electron photodetachment dissociation (a-EPD) to yield high-quality MS/MS spectra. Fragments identified using an m/z-domain isotope matching strategy yielded high sequence coverage (>70%) of a yeast phenylalanine tRNA.
RESUMO
Lipopolysaccharides (LPS) and lipooligosaccharides (LOS) are located in the outer membrane of Gram-negative bacteria and are comprised of three distinctive parts: lipid A, core oligosaccharide (OS), and O-antigen. The structure of each region influences bacterial stability, toxicity, and pathogenesis. Here, we highlight the use of targeted activated-electron photodetachment (a-EPD) tandem mass spectrometry to characterize LPS and LOS from two crucial players in the human gut microbiota, Escherichia coli Nissle and Bacteroides fragilis. a-EPD is a hybrid activation method that uses ultraviolet photoirradiation to generate charge-reduced radical ions followed by collisional activation to produce informative fragmentation patterns. We benchmark the a-EPD method for top-down characterization of triacyl LOS from E. coli R2, then focus on characterization of LPS from E. coli Nissle and B. fragilis. Notably, a-EPD affords extensive fragmentation throughout the backbone of the core OS and O-antigen regions of LPS from E. coli Nissle. This hybrid approach facilitated the elucidation of structural details for LPS from B. fragilis, revealing a putative hexuronic acid (HexA) conjugated to lipid A.
Assuntos
Escherichia coli , Lipopolissacarídeos , Lipopolissacarídeos/química , Escherichia coli/química , Bacteroides fragilis/química , Elétrons , Espectrometria de Massas em TandemRESUMO
Gram-negative bacteria develop and exhibit resistance to antibiotics, owing to their highly asymmetric outer membrane maintained by a group of six proteins comprising the Mla (maintenance of lipid asymmetry) pathway. Here, we investigate the lipid binding preferences of one Mla protein, MlaC, which transports lipids through the periplasm. We used ultraviolet photodissociation (UVPD) to identify and characterize modifications of lipids endogenously bound to MlaC expressed in three different bacteria strains. UVPD was also used to localize lipid binding to MlaC residues 130-140, consistent with the crystal structure reported for lipid-bound MlaC. The impact of removing the bound lipid from MlaC on its structure was monitored based on collision cross section measurements, revealing that the protein unfolded prior to release of the lipid. The lipid selectivity of MlaC was evaluated based on titrimetric experiments, indicating that MlaC-bound lipids in various classes (sphingolipids, glycerophospholipids, and fatty acids) as long as they possessed no more than two acyl chains.
Assuntos
Espectrometria de Massas por Ionização por Electrospray , Raios Ultravioleta , Temperatura , Lipídeos/química , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Proteínas de Transporte/metabolismo , Proteínas de Transporte/química , Processos FotoquímicosRESUMO
GM1 gangliosidosis is a neurodegenerative disorder caused by mutations in the GLB1 gene, which encodes lysosomal ß-galactosidase. The enzyme deficiency blocks GM1 ganglioside catabolism, leading to accumulation of GM1 ganglioside and asialo-GM1 ganglioside (GA1 glycolipid) in brain. This disease can present in varying degrees of severity, with the level of residual ß-galactosidase activity primarily determining the clinical course. Glb1 null mouse models, which completely lack ß-galactosidase expression, exhibit a less severe form of the disease than expected from the comparable deficiency in humans, suggesting a potential species difference in the GM1 ganglioside degradation pathway. We hypothesized this difference may involve the sialidase NEU3, which acts on GM1 ganglioside to produce GA1 glycolipid. To test this hypothesis, we generated Glb1/Neu3 double KO (DKO) mice. These mice had a significantly shorter lifespan, increased neurodegeneration, and more severe ataxia than Glb1 KO mice. Glb1/Neu3 DKO mouse brains exhibited an increased GM1 ganglioside to GA1 glycolipid ratio compared with Glb1 KO mice, indicating that NEU3 mediated GM1 ganglioside to GA1 glycolipid conversion in Glb1 KO mice. The expression of genes associated with neuroinflammation and glial responses were enhanced in Glb1/Neu3 DKO mice compared with Glb1 KO mice. Mouse NEU3 more efficiently converted GM1 ganglioside to GA1 glycolipid than human NEU3 did. Our findings highlight NEU3's role in ameliorating the consequences of Glb1 deletion in mice, provide insights into NEU3's differential effects between mice and humans in GM1 gangliosidosis, and offer a potential therapeutic approach for reducing toxic GM1 ganglioside accumulation in GM1 gangliosidosis patients.
Assuntos
Gangliosidose GM1 , Animais , Humanos , Camundongos , beta-Galactosidase/genética , beta-Galactosidase/metabolismo , beta-Galactosidase/uso terapêutico , Gangliosídeo G(M1)/metabolismo , Gangliosídeo G(M1)/uso terapêutico , Gangliosidose GM1/genética , Glicolipídeos , Neuraminidase/genética , Neuraminidase/uso terapêuticoRESUMO
Tandem mass spectrometry (MS/MS) spectra of intact proteins can be difficult to interpret owing to the variety of fragment ion types and abundances. This information is crucial for maximizing the information derived from top-down mass spectrometry of proteins and protein complexes. MS-TAFI (Mass Spectrometry Tool for the Analysis of Fragment Ions) is a free Python-based program which offers a streamlined approach to the data analysis and visualization of deconvoluted MS/MS data of intact proteins. The application also contains tools for native mass spectrometry experiments with the ability to search for fragment ions that retain ligands (holo ions) as well as visualize the location of charge sites obtained from 193 nm ultraviolet photodissociation data. The source code and complete application for MS-TAFI is available for download at https://github.com/kylejuetten.
Assuntos
Proteômica , Espectrometria de Massas em Tandem , Espectrometria de Massas em Tandem/métodos , Proteômica/métodos , Proteínas/análise , Software , ÍonsRESUMO
193 nm ultraviolet photodissociation (UVPD) allows high sequence coverage to be obtained for intact proteins using terminal fragments alone. However, internal fragments, those that contain neither N- nor C- terminus, are typically ignored, neglecting their potential to bolster characterization of intact proteins. Here, we explore internal fragments generated by 193 nm UVPD for proteins ranging in size from 17-47 kDa and using the ClipsMS algorithm to facilitate searches for internal fragments. Internal fragments were only retained if identified in multiple replicates in order to reduce spurious assignments and to explore the reproducibility of internal fragments generated by UVPD. Inclusion of internal fragment improved sequence coverage by an average of 18% and 32% for UVPD and HCD, respectively, across all proteins and charge states studied. However, only an average of 18% of UVPD internal fragments were identified in two out of three replicates relative to the average number identified across all replicates for all proteins studied. Conversely, for HCD, an average of 63% of internal fragments were retained across replicates. These trends reflect an increased risk of false-positive identifications and a need for caution when considering internal fragments for UVPD. Additionally, proton-transfer charge reduction (PTCR) reactions were performed following UVPD or HCD to assess the impact on internal fragment identifications, allowing up to 20% more fragment ions to be retained across multiple replicates. At this time, it is difficult to recommend the inclusion of the internal fragment when searching UVPD spectra without further work to develop strategies for reducing the possibilities of false-positive identifications. All mass spectra are available in the public repository jPOST with the accession number JPST001885.
Assuntos
Proteínas , Espectrometria de Massas em Tandem , Reprodutibilidade dos Testes , Íons , Prótons , Raios UltravioletaRESUMO
Polyubiquitination is an important post-translational modification (PTM) that regulates various biological functions. The linkage sites and topologies of polyubiquitination chains are important factors in determining the fate of polyubiquitinated proteins. Characterization of polyubiquitin chains is the first step in understanding the biological functions of protein ubiquitination, but it is challenging owing to the repeating nature of the ubiquitin chains and the difficulty in deciphering linkage positions. Here, we combine ultraviolet photodissociation (UVPD) mass spectrometry and gas-phase proton transfer charge reduction (PTCR) to facilitate the assignment of product ions generated from Lys6-, Lys11-, Lys29-, Lys33-, Lys48-, and Lys63-linked ubiquitin tetramers. UVPD results in extensive fragmentation of intact proteins in a manner that allows the localization of PTMs. However, UVPD mass spectra of large proteins (>30 kDa) are often congested due to the overlapping isotopic distribution of highly charged fragment ions. UVPD + PTCR improved the identification of PTM-containing fragment ions, allowing the localization of linkage sites in all six tetramers analyzed. UVPD + PTCR also increased the sequence coverage obtained from the PTM-containing fragment ions in each of the four chains of each tetramer by 7 to 44% when compared to UVPD alone.
Assuntos
Prótons , Ubiquitina , Processamento de Proteína Pós-Traducional , Ubiquitinação , LuzRESUMO
Top-down characterization of histones, proteins that are critical participants in an array of DNA-dependent processes, offers the potential to examine the relationship between histone structure and mechanisms of genetic regulation. Mapping patterns of post-translational modifications (PTMs) of histones requires extensive backbone cleavages to bracket the sites of mass shifts corresponding to specific PTMs. Ultraviolet photodissociation (UVPD) causes substantial fragmentation of proteins, which is well-suited for PTM localization, but the resulting spectra are congested with fragment ions that may have overlapping isotopic distributions that confound deconvolution. Gas-phase proton transfer charge reduction (PTCR) decreases the charge states of highly charged ions, thus alleviating this congestion and facilitating the identification of additional sequence-determining and PTM-localizing fragment ions. By integrating UVPD with PTCR for histone proteoform analyses, sequence coverages up to 91% were achieved for calf thymus histone H4 containing acetylation marks at the N-terminus and Lys12 as well as a dimethylation at Arg3. UVPD-PTCR exhibited large gains in characterization for other histones, such as histone H2A, increasing the sequence coverage from 59 to 77% for monoacetylated H2A.
Assuntos
Histonas , Prótons , Humanos , Histonas/química , Espectrometria de Massas em Tandem/métodos , Processamento de Proteína Pós-Traducional , Íons/metabolismo , Raios UltravioletaRESUMO
Complete O-glycosite characterization, including identification of the peptides, localization of the glycosites, and mapping of the glycans, has been a persistent challenge in O-glycoproteomics owing to the technical challenges surrounding O-glycan analysis. Multi-glycosylated peptides pose an even greater challenge owing to their potential heterogeneity. Ultraviolet photodissociation (UVPD) can localize multiple post-translational modifications and is well-suited for the characterization of glycans. Three glycoproteins were assessed based on a strategy combining the use of O-glycoprotease IMPa and HCD-triggered UVPD for the complete characterization of O-glycopeptides. This approach localized multiple adjacent or proximal O-glycosites on individual glycopeptides and identified a previously unknown glycosite on etanercept at S218. Nine different glycoforms were characterized as a multi-glycosylated peptide from etanercept. The performance of UVPD was compared to that of HCD and EThcD for the localization of O-glycosites and the characterization of the constituent peptides and glycans.
Assuntos
Glicopeptídeos , Peptídeos , Glicopeptídeos/química , Etanercepte , Glicosilação , PolissacarídeosRESUMO
The measurement of collision cross sections (CCS, σ) offers supplemental information about sizes and conformations of ions beyond mass analysis alone. We have previously shown that CCSs can be determined directly from the time-domain transient decay of ions in an Orbitrap mass analyzer as ions oscillate around the central electrode and collide with neutral gas, thus removing them from the ion packet. Herein, we develop the modified hard collision model, thus deviating from the prior FT-MS hard sphere model, to determine CCSs as a function of center-of-mass collision energy in the Orbitrap analyzer. With this model, we aim to increase the upper mass limit of CCS measurement for native-like proteins, characterized by low charge states and presumed to be in more compact conformations. We also combine CCS measurements with collision induced unfolding and tandem mass spectrometry experiments to monitor protein unfolding and disassembly of protein complexes and measure CCSs of ejected monomers from protein complexes.
Assuntos
Proteínas , Proteínas/química , Íons/químicaRESUMO
Spontaneous conversion of aspartic acid (Asp) to isoaspartic acid (isoAsp) is a ubiquitous modification that influences the structure and function of proteins. This modification of Asp impacts the stability of biotherapeutics and has been linked to the development of neurodegenerative diseases. We explored the use of 193 nm ultraviolet photodissociation (UVPD) to distinguish Asp and isoAsp in the protonated and deprotonated peptides. The differences in the relative abundances of several fragment ions uniquely generated by UVPD were used to differentiate isomeric peptide standards containing Asp or isoAsp. These fragment ions result from the cleavage of bonds N-terminal to Asp/isoAsp residues in addition to the side-chain losses from Asp/isoAsp or the losses of COOH, CO2, CO, or H2O from y-ions. Fragmentation of Asp-containing tryptic peptides using UVPD resulted in more enhanced w/w + 1/y - 1/x ions, while isoAsp-containing peptides yielded more enhanced y - 18/y - 45/y - 46 ions. UVPD was also used to identify an isomerized peptide from a tryptic digest of a monoclonal antibody. Moreover, UVPD of a protonated nontryptic peptide resulted in more enhanced y ions N- and C-terminal to isoAsp and differences in b/y ion ratios that were used to identify the isoAsp peptide.
Assuntos
Ácido Isoaspártico , Peptídeos , Ácido Isoaspártico/análise , Ácido Isoaspártico/química , Sequência de Aminoácidos , Espectrometria de Massas/métodos , Peptídeos/química , Ácido Aspártico/química , Íons , Raios UltravioletaRESUMO
Posttranslational modifications (PTMs) are covalent modifications of proteins that modulate the structure and functions of proteins and regulate biological processes. The development of various mass spectrometry-based proteomics workflows has facilitated the identification of hundreds of PTMs and aided the understanding of biological significance in a high throughput manner. Improvements in sample preparation and PTM enrichment techniques, instrumentation for liquid chromatography-tandem mass spectrometry (LC-MS/MS), and advanced data analysis tools enhance the specificity and sensitivity of PTM identification. Highly prevalent PTMs like phosphorylation, glycosylation, acetylation, ubiquitinylation, and methylation are extensively studied. However, the functions and impact of less abundant PTMs are not as well understood and underscore the need for analytical methods that aim to characterize these PTMs. This review focuses on the advancement and analytical challenges associated with the characterization of three less common but biologically relevant PTMs, specifically, adenosine diphosphate-ribosylation, tyrosine sulfation, and tyrosine nitration. The advantages and disadvantages of various enrichment, separation, and MS/MS techniques utilized to identify and localize these PTMs are described.
RESUMO
The extracellular polysaccharide capsule of Klebsiella pneumoniae resists penetration by antimicrobials and protects the bacteria from the innate immune system. Host antimicrobial peptides are inactivated by the capsule as it impedes their penetration to the bacterial membrane. While the capsule sequesters most peptides, a few antimicrobial peptides have been identified that retain activity against encapsulated K. pneumoniae, suggesting that this bacterial defense can be overcome. However, it is unclear what factors allow peptides to avoid capsule inhibition. To address this, we created a peptide analog with strong antimicrobial activity toward several K. pneumoniae strains from a previously inactive peptide. We characterized the effects of these two peptides on K. pneumoniae, along with their physical interactions with K. pneumoniae capsule. Both peptides disrupted bacterial cell membranes, but only the active peptide displayed this activity against capsulated K. pneumoniae Unexpectedly, the active peptide showed no decrease in capsule binding, but did lose secondary structure in a capsule-dependent fashion compared with the inactive parent peptide. We found that these characteristics are associated with capsule-peptide aggregation, leading to disruption of the K. pneumoniae capsule. Our findings reveal a potential mechanism for disrupting the protective barrier that K. pneumoniae uses to avoid the immune system and last-resort antibiotics.
Assuntos
Antibacterianos/farmacologia , Peptídeos Catiônicos Antimicrobianos/farmacologia , Cápsulas Bacterianas/efeitos dos fármacos , Infecções por Klebsiella/tratamento farmacológico , Klebsiella pneumoniae/efeitos dos fármacos , Animais , Antibacterianos/uso terapêutico , Peptídeos Catiônicos Antimicrobianos/imunologia , Peptídeos Catiônicos Antimicrobianos/uso terapêutico , Cápsulas Bacterianas/metabolismo , Permeabilidade da Membrana Celular/efeitos dos fármacos , Modelos Animais de Doenças , Farmacorresistência Bacteriana Múltipla , Feminino , Células HEK293 , Interações Hospedeiro-Patógeno/imunologia , Humanos , Imunidade Inata , Infecções por Klebsiella/microbiologia , Klebsiella pneumoniae/citologia , Camundongos , Testes de Sensibilidade Microbiana , Polissacarídeos Bacterianos/metabolismoRESUMO
How chromatin shapes pathways that promote genome-epigenome integrity in response to DNA damage is an issue of crucial importance. We report that human bromodomain (BRD)-containing proteins, the primary "readers" of acetylated chromatin, are vital for the DNA damage response (DDR). We discovered that more than one-third of all human BRD proteins change localization in response to DNA damage. We identified ZMYND8 (zinc finger and MYND [myeloid, Nervy, and DEAF-1] domain containing 8) as a novel DDR factor that recruits the nucleosome remodeling and histone deacetylation (NuRD) complex to damaged chromatin. Our data define a transcription-associated DDR pathway mediated by ZMYND8 and the NuRD complex that targets DNA damage, including when it occurs within transcriptionally active chromatin, to repress transcription and promote repair by homologous recombination. Thus, our data identify human BRD proteins as key chromatin modulators of the DDR and provide novel insights into how DNA damage within actively transcribed regions requires chromatin-binding proteins to orchestrate the appropriate response in concordance with the damage-associated chromatin context.
Assuntos
Cromatina/metabolismo , Dano ao DNA , Recombinação Homóloga/genética , Receptores de Superfície Celular/metabolismo , Autoantígenos/metabolismo , Linhagem Celular Tumoral , Regulação da Expressão Gênica , Inativação Gênica , Humanos , Complexo Mi-2 de Remodelação de Nucleossomo e Desacetilase/metabolismo , Ligação Proteica , Transporte Proteico/genética , Receptores de Quinase C Ativada , Receptores de Superfície Celular/genética , Proteínas Supressoras de TumorRESUMO
The direct correlation between proteoforms and biological phenotype necessitates the exploration of mass spectrometry (MS)-based methods more suitable for proteoform detection and characterization. Here, we couple nano-hydrophobic interaction chromatography (nano-HIC) to ultraviolet photodissociation MS (UVPD-MS) for separation and characterization of intact proteins and proteoforms. High linearity, sensitivity, and sequence coverage are obtained with this method for a variety of proteins. Investigation of collisional cross sections of intact proteins during nano-HIC indicates semifolded conformations in low charge states, enabling a different dimension of separation in comparison to traditional, fully denaturing reversed-phase separations. This method is demonstrated for a mixture of intact proteins from Escherichia coli ribosomes; high sequence coverage is obtained for a variety of modified and unmodified proteoforms.
Assuntos
Proteínas , Espectrometria de Massas em Tandem , Cromatografia Líquida/métodos , Escherichia coli/genética , Interações Hidrofóbicas e Hidrofílicas , Espectrofotometria Ultravioleta/métodos , Espectrometria de Massas em Tandem/métodos , Raios UltravioletaRESUMO
The recent discovery of asymmetric arrangements of trimers in the tautomerase superfamily (TSF) adds structural diversity to this already mechanistically diverse superfamily. Classification of asymmetric trimers has previously been determined using X-ray crystallography. Here, native mass spectrometry (MS) and ultraviolet photodissociation (UVPD) are employed as an integrated strategy for more rapid and sensitive differentiation of symmetric and asymmetric trimers. Specifically, the unfolding of symmetric and asymmetric trimers initiated by collisional heating was probed using UVPD, which revealed unique gas-phase unfolding pathways. Variations in UVPD patterns from native-like, compact trimeric structures to unfolded, extended conformations indicate a rearrangement of higher-order structure in the asymmetric trimers that are believed to be stabilized by salt-bridge triads, which are absent from the symmetric trimers. Consequently, the symmetric trimers were found to be less stable in the gas phase, resulting in enhanced UVPD fragmentation overall and a notable difference in higher-order re-structuring based on the extent of hydrogen migration of protein fragments. The increased stability of the asymmetric trimers may justify their evolution and concomitant diversification of the TSF. Facilitating the classification of TSF members as symmetric or asymmetric trimers assists in delineating the evolutionary history of the TSF.
Assuntos
Isomerases , Raios Ultravioleta , Cristalografia por Raios X , Isomerases/químicaRESUMO
Cardiolipins (CLs) constitute a structurally complex class of glycerophospholipids with a unique tetraacylated structure accompanied by distinctive functional roles. Aberrations in the composition of this lipid class have been associated with disease states, spurring interest in the development of new approaches to differentiate the structures of diverse CLs in complex mixtures. The structural characterization of these complex lipids using conventional methods, however, suffers from limited resolution and frequently proves unable to discern subtle yet biologically significant features such as unsaturation sites or acyl chain position assignments. Here, we describe the synergistic use of chemical derivatization and hybrid dissociation techniques to characterize CL from complex biological mixtures with both double bond and sn positional isomer resolution in a shotgun mass spectrometry strategy. Utilizing (trimethylsilyl)diazomethane (TMSD), CL phosphate groups were methylated to promote positive-mode ionization by the production of metal-cationized lipids, enabling structural interrogation via hybrid higher-energy collisional activation/ultraviolet photodissociation (HCD/UVPD). This combination of TMSD derivatization and HCD/UVPD fragmentation results in diagnostic product ions that permit distinction and relative quantitation of sn-stereoisomers and the localization of double bonds. Applying this strategy to a total lipid extract from a thyroid carcinoma revealed a previously unreported 18:2/18:1 motif, elucidating a structural feature unique to the lipid class.
Assuntos
Cardiolipinas , Glicerofosfolipídeos , Glicerofosfolipídeos/química , Íons , Espectrometria de Massas/métodos , Espectrofotometria Ultravioleta , Raios UltravioletaRESUMO
Understanding and elucidating the diverse structures and functions of lipids has motivated the development of many innovative tandem mass spectrometry (MS/MS) strategies. Higher-energy activation methods, such as ultraviolet photodissociation (UVPD), generate unique fragment ions from glycerophospholipids that can be used to perform in-depth structural analysis and facilitate the deconvolution of isomeric lipid structures in complex samples. Although detailed characterization is central to the correlation of lipid structure to biological function, it is often impeded by the lack of sufficient instrument sensitivity for highly bioactive but low-abundance phospholipids. Here, we present precursor exclusion (PEx) UVPD, a simple yet powerful technique to enhance the signal-to-noise (S/N) of informative low-abundance fragment ions produced from UVPD of glycerophospholipids. Through the exclusion of the large population of undissociated precursor ions with an MS3 strategy, the S/N of diagnostic fragment ions from PC 18:0/18:2(9Z, 12Z) increased up to an average of 13x for PEx-UVPD compared to UVPD alone. These enhancements were extended to complex mixtures of lipids from bovine liver extract to confidently identify 35 unique structures using liquid chromatography PEx-UVPD. This methodology has the potential to advance lipidomics research by offering deeper structure elucidation and confident identification of biologically active lipids.