RESUMO
The development of new ion-activation/dissociation methods continues to be one of the most active areas of mass spectrometry owing to the broad applications of tandem mass spectrometry in the identification and structural characterization of molecules. This Review will showcase the impact of ultraviolet photodissociation (UVPD) as a frontier strategy for generating informative fragmentation patterns of ions, especially for biological molecules whose complicated structures, subtle modifications, and large sizes often impede molecular characterization. UVPD energizes ions via absorption of high-energy photons, which allows access to new dissociation pathways relative to more conventional ion-activation methods. Applications of UVPD for the analysis of peptides, proteins, lipids, and other classes of biologically relevant molecules are emphasized in this Review.
Assuntos
Lipídeos/análise , Ácidos Nucleicos/análise , Oligossacarídeos/análise , Peptídeos/análise , Proteínas/análise , Sequência de Aminoácidos , Animais , Sequência de Carboidratos , Humanos , Lipídeos/efeitos da radiação , Espectrometria de Massas/métodos , Ácidos Nucleicos/efeitos da radiação , Oligossacarídeos/efeitos da radiação , Peptídeos/metabolismo , Peptídeos/efeitos da radiação , Processamento de Proteína Pós-Traducional , Proteínas/metabolismo , Proteínas/efeitos da radiação , Proteômica , Raios UltravioletaRESUMO
Electron-based fragmentation methods have revolutionized biomolecular mass spectrometry, in particular native and top-down protein analysis. Here, we report the use of a new electromagnetostatic cell to perform electron capture dissociation (ECD) within a quadrupole/ion mobility/time-of-flight mass spectrometer. This cell was installed between the ion mobility and time-of-flight regions of the instrument, and fragmentation was fast enough to be compatible with mobility separation. The instrument was already fitted with electron transfer dissociation (ETD) between the quadrupole and mobility regions prior to modification. We show excellent fragmentation efficiency for denatured peptides and proteins without the need to trap ions in the gas phase. Additionally, we demonstrate native top-down backbone fragmentation of noncovalent protein complexes, leading to comparable sequence coverage to what was achieved using the instrument's existing ETD capabilities. Limited collisional ion activation of the hemoglobin tetramer before ECD was reflected in the observed fragmentation pattern, and complementary ion mobility measurements prior to ECD provided orthogonal evidence of monomer unfolding within this complex. The approach demonstrated here provides a powerful platform for both top-down proteomics and mass spectrometry-based structural biology studies.
Assuntos
Espectrometria de Massas/métodos , Desnaturação Proteica , Proteínas/química , Sequência de Aminoácidos , Animais , Bovinos , Humanos , Modelos Moleculares , Multimerização Proteica , Estrutura Quaternária de ProteínaRESUMO
Cationic antimicrobial peptides (CAMPs) have been known to act as multi-modal weapons against Gram-negative bacteria. As a new approach to investigate the nature of the interactions between CAMPs and the surfaces of bacteria, native mass spectrometry and two MS/MS strategies (ultraviolet photodissociation (UVPD) and higher energy collisional activation (HCD)) are used to examine formation and disassembly of saccharolipid·peptide complexes. Kdo2-lipid A (KLA) is used as a model saccharolipid to evaluate complexation with a series of cationic peptides (melittin and three analogs). Collisional activation of the KLA·peptide complexes results in the disruption of electrostatic interactions, resulting in apo-sequence ions with shifts in the distribution of ions compared to the fragmentation patterns of the apo-peptides. UVPD of the KLA·peptide complexes results in both apo- and holo-sequence ions of the peptides, the latter in which the KLA remains bound to the truncated peptide fragment despite cleavage of a covalent bond of the peptide backbone. Mapping both the N- and C-terminal holo-product ions gives insight into the peptide motifs (specifically an electropositive KRKR segment and a proline residue) that are responsible for mediating the electrostatic interactions between the cationic peptides and saccharolipid.
Assuntos
Peptídeos Catiônicos Antimicrobianos/química , Lipopolissacarídeos/química , Meliteno/química , Mapeamento de Interação de Proteínas , Peso Molecular , Peptídeos/química , Espectrometria de Massas em Tandem , Raios UltravioletaRESUMO
Mass spectrometry continues to develop as a valuable tool in the analysis of proteins and protein complexes. In protein complex mass spectrometry studies, surface-induced dissociation (SID) has been successfully applied in quadrupole time-of-flight (Q-TOF) instruments. SID provides structural information on noncovalent protein complexes that is complementary to other techniques. However, the mass resolution of Q-TOF instruments can limit the information that can be obtained for protein complexes by SID. Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR MS) provides ultrahigh resolution and ultrahigh mass accuracy measurements. In this study, an SID device was designed and successfully installed in a hybrid FT-ICR instrument in place of the standard gas collision cell. The SID-FT-ICR platform has been tested with several protein complex systems (homooligomers, a heterooligomer, and a protein-ligand complex, ranging from 53 to 85 kDa), and the results are consistent with data previously acquired on Q-TOF platforms, matching predictions from known protein interface information. SID fragments with the same m/z but different charge states are well-resolved based on distinct spacing between adjacent isotope peaks, and the addition of metal cations and ligands can also be isotopically resolved with the ultrahigh mass resolution available in FT-ICR.
Assuntos
Aminoidrolases/análise , Toxina da Cólera/análise , Ciclotrons , Estreptavidina/análise , Aminoidrolases/metabolismo , Análise de Fourier , Espectrometria de Massas , Espectroscopia de Infravermelho com Transformada de Fourier , Propriedades de SuperfícieRESUMO
Determination of structure and folding of certain classes of proteins remains intractable by conventional structural characterization strategies and has spurred the development of alternative methodologies. Mass spectrometry-based approaches have a unique capacity to differentiate protein heterogeneity due to the ability to discriminate populations, whether minor or major, featuring modifications or complexation with non-covalent ligands on the basis of m/z. Cleavage of the peptide backbone can be further utilized to obtain residue-specific structural information. Here, hydrogen elimination monitoring (HEM) upon ultraviolet photodissociation (UVPD) of proteins transferred to the gas phase via nativespray ionization is introduced as an innovative approach to deduce backbone hydrogen bonding patterns. Using well-characterized peptides and a series of proteins, prediction of the engagement of the amide carbonyl oxygen of the protein backbone in hydrogen bonding using UVPD-HEM is demonstrated to show significant agreement with the hydrogen-bonding motifs derived from molecular dynamics simulations and X-ray crystal structures.
Assuntos
Proteínas/química , Hormônio Adrenocorticotrópico/química , Hormônio Adrenocorticotrópico/metabolismo , Sequência de Aminoácidos , Animais , Calmodulina/química , Calmodulina/metabolismo , Cobaias , Hidrogênio/química , Ligação de Hidrogênio , Espectrometria de Massas , Meliteno/química , Meliteno/metabolismo , Fotólise , Estrutura Terciária de Proteína , Proteínas/metabolismo , Raios Ultravioleta , alfa-Sinucleína/química , alfa-Sinucleína/metabolismoRESUMO
Protein-protein interfaces and architecture are critical to the function of multiprotein complexes. Mass spectrometry-based techniques have emerged as powerful strategies for characterization of protein complexes, particularly for heterogeneous mixtures of structures. In the present study, activation and dissociation of three tetrameric protein complexes (streptavidin, transthyretin, and hemoglobin) in the gas phase was undertaken by 193 nm ultraviolet photodissociation (UVPD) for the characterization of higher order structure. High pulse energy UVPD resulted in the production of dimers and low charged monomers exhibiting symmetrical charge partitioning among the subunits (the so-called symmetrical dissociation pathways), consistent with the subunit organization of the complexes. In addition, UVPD promoted backbone cleavages of the monomeric subunits, the abundances of which corresponded to the more flexible loop regions of the proteins.
Assuntos
Espectrometria de Massas , Multimerização Proteica , Proteínas/química , Raios Ultravioleta , Humanos , Estrutura Quaternária de Proteína , Estrutura Secundária de ProteínaRESUMO
The lipid A domain of the endotoxic lipopolysaccharide layer of Gram-negative bacteria is comprised of a diglucosamine backbone to which a variable number of variable length fatty acyl chains are anchored. Traditional characterization of these tails and their linkages by nuclear magnetic resonance (NMR) or mass spectrometry is time-consuming and necessitates databases of pre-existing structures for structural assignment. Here, we introduce an automated de novo approach for characterization of lipid A structures that is completely database-independent. A hierarchical decision-tree MS(n) method is used in conjunction with a hybrid activation technique, UVPDCID, to acquire characteristic fragmentation patterns of lipid A variants from a number of Gram-negative bacteria. Structural assignments are derived from integration of key features from three to five spectra and automated interpretation is achieved in minutes without the need for pre-existing information or candidate structures. The utility of this strategy is demonstrated for a mixture of lipid A structures from an enzymatically modified E. coli lipid A variant. A total of 27 lipid A structures were discovered, many of which were isomeric, showcasing the need for a rapid de novo approach to lipid A characterization.
Assuntos
Lipídeo A/química , Raios Ultravioleta , Escherichia coli/química , Espectrometria de Massas , Conformação ProteicaRESUMO
Characterization of all gas-phase charge sites of natively sprayed proteins and peptides is demonstrated using 193 nm UVPD. The high sequence coverage offered by UVPD is exploited for the accurate determination of charge sites in protein systems up to 18 kDa, allowing charge site to be studied as a function of protein conformation and the presence of disulfide bonds. Charging protons are found on both basic sidechains and on the amide backbone of less basic amino acids such as serine, glutamine, and proline. UVPD analysis was performed on the 3+ charge state of melittin, the 5+ to 8+ charge states of ubiquitin, and the 8+ charge state of reduced and oxidized ß-lactoglobulin. The location of charges in gas-phase proteins is known to impact structure; molecular modeling of different charge site motifs of 3+ melittin demonstrates how placement of protons in simulations can dramatically impact the predicted structure of the molecule. The location of positive charge sites in ubiquitin and ß-lactoglobulin are additionally found to depend on the presence or absence of salt-bridges, columbic repulsion across the length of the peptide, and protein conformation. Charge site isomers are demonstrated for ubiquitin and ß-lactoglobulin but found to be much less numerous than previously predicted.
Assuntos
Espectrometria de Massas/métodos , Fotólise , Proteínas/química , Raios Ultravioleta , Sequência de Aminoácidos , Modelos Moleculares , Conformação ProteicaRESUMO
In solution, α-helices are stabilized at the termini by a variety of different capping interactions. Study of these interactions in the gas phase provides a unique means to explore the intrinsic properties that cause this stabilization. Evidence of helical and globular conformations is presented here for gas-phase, doubly charged peptides of sequence XAnK, wherein X is D, N, Q, or L. The relative abundance of the helical conformation is found to vary as a function of peptide length and the identity of the first amino acid, consistent with solution phase studies that have looked at the identity of the first amino acid. The N-terminal, b ion fragments of the doubly charged precursor peptides are shown to form helical and globular conformations. The stability of the helical fragments is examined as a function of fragment length, N-terminal amino acid, precursor conformation, and the activation energy used to generate the fragment. At lower collision energies, helical b ions preferentially form, particularly from helical precursors. The abundance of the helical b ion population is observed to dramatically decrease for NAn and DAn b ions smaller than the b10; simulations suggest this feature is due to the b10 having two complete turns of the helix, while the b9 and smaller ions have only a partial second turn, suggesting the b10 is the lower limit for stable helical conformations in b ions. Use of higher collision energies promotes the formation of globular structures in the b ions. This characteristic is attributed to increased conformational dynamics and subsequently improved proton transfer kinetics from the b ion's C-terminal oxazolone ring to the N-terminus.
Assuntos
Gases , Peptídeos/química , Simulação de Dinâmica Molecular , Estabilidade Proteica , Estrutura Secundária de Proteína , SoluçõesRESUMO
Lipid A molecules consist of a diglucosamine sugar core with a number of appended acyl chains that vary in their length and connectivity. Because of the challenging nature of characterizing these molecules and differentiating between isomeric species, an energy-resolved MS/MS strategy was undertaken to track the fragmentation trends and map genealogies of product ions originating from consecutive cleavages of acyl chains. Generalizations were developed based on the number and locations of the primary and secondary acyl chains as well as variations in preferential cleavages arising from the location of the phosphate groups. Secondary acyl chain cleavage occurs most readily for lipid A species at the 3' position, followed by primary acyl chain fragmentation at both the 3' and 3 positions. In the instances of bisphosphorylated lipid A variants, phosphate loss occurs readily in conjunction with the most favorable primary and secondary acyl chain cleavages. Graphical Abstract á .
Assuntos
Lipídeo A/química , Espectrometria de Massas em Tandem/métodos , Lipídeo A/metabolismo , Fosfatos/química , Fosforilação , Raios UltravioletaRESUMO
Dissociation of proteins and peptides by 193 nm ultraviolet photodissociation (UVPD) has gained momentum in proteomic studies because of the diversity of backbone fragments that are produced and subsequent unrivaled sequence coverage obtained by the approach. The pathways that form the basis for the production of particular ion types are not completely understood. In this study, a statistical approach is used to probe hydrogen atom elimination from a + 1 radical ions, and different extents of elimination are found to vary as a function of the identity of the C-terminal residue of the a product ions and the presence or absence of hydrogen bonds to the cleaved residue. Graphical Abstract á .