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1.
J Am Soc Mass Spectrom ; 32(8): 2019-2032, 2021 Aug 04.
Artigo em Inglês | MEDLINE | ID: mdl-33835810

RESUMO

Characterization and monitoring of post-translational modifications (PTMs) by peptide mapping is a ubiquitous assay in biopharmaceutical characterization. Often, this assay is coupled to reversed-phase liquid chromatographic (LC) separations that require long gradients to identify all components of the protein digest and resolve critical modifications for relative quantitation. Incorporating ion mobility (IM) as an orthogonal separation that relies on peptide structure can supplement the LC separation by providing an additional differentiation filter to resolve isobaric peptides, potentially reducing ambiguity in identification through mobility-aligned fragmentation and helping to reduce the run time of peptide mapping assays. A next-generation high-resolution ion mobility (HRIM) technique, based on structures for lossless ion manipulations (SLIM) technology with a 13 m ion path, provides peak capacities and higher resolving power that rivals traditional chromatographic separations and, owing to its ability to resolve isobaric peptides that coelute in faster chromatographic methods, allows for up to 3× shorter run times than conventional peptide mapping methods. In this study, the NIST monoclonal antibody IgG1κ (NIST RM 8671, NISTmAb) was characterized by LC-HRIM-MS and LC-HRIM-MS with collision-induced dissociation (HRIM-CID-MS) using a 20 min analytical method. This approach delivered a sequence coverage of 96.5%. LC-HRIM-CID-MS experiments provided additional confidence in sequence determination. HRIM-MS resolved critical oxidations, deamidations, and isomerizations that coelute with their native counterparts in the chromatographic dimension. Finally, quantitative measurements of % modification were made using only the m/z-extracted HRIM arrival time distributions, showing good agreement with the reference liquid-phase separation. This study shows, for the first time, the analytical capability of HRIM using SLIM technology for enhancing peptide mapping workflows relevant to biopharmaceutical characterization.


Assuntos
Espectrometria de Massas/métodos , Mapeamento de Peptídeos/métodos , Peptídeos/análise , Peptídeos/metabolismo , Anticorpos Monoclonais/análise , Anticorpos Monoclonais/química , Produtos Biológicos/análise , Produtos Biológicos/química , Ensaios de Triagem em Larga Escala , Espectrometria de Mobilidade Iônica , Íons/química , Isomerismo , Peptídeos/química , Processamento de Proteína Pós-Traducional , Controle de Qualidade
2.
Biochemistry ; 59(4): 436-449, 2020 02 04.
Artigo em Inglês | MEDLINE | ID: mdl-31814404

RESUMO

Huntington's disease is a genetic neurodegenerative disorder characterized by the formation of amyloid fibrils of the huntingtin protein (htt). The 17-residue N-terminal region of htt (Nt17) has been implicated in the formation of early phase oligomeric species, which may be neurotoxic. Because tertiary interactions with a downstream (C-terminal) polyproline (polyP) region of htt may disrupt the formation of oligomers, which are precursors to fibrillar species, the effect of co-incubation of a region of htt with a 10-residue polyP peptide on oligomerization and fibrillization has been examined by atomic force microscopy. From multiple, time-course experiments, morphological changes in oligomeric species are observed for the protein/peptide mixture and compared with the protein alone. Additionally, an overall decrease in fibril formation is observed for the heterogeneous mixture. To consider potential sites of interaction between the Nt17 region and polyP, mixtures containing Nt17 and polyP peptides have been examined by ion mobility spectrometry and gas-phase hydrogen-deuterium exchange coupled with mass spectrometry. These data combined with molecular dynamics simulations suggest that the C-terminal region of Nt17 may be a primary point of contact. One interpretation of the results is that polyP may possibly regulate Nt17 by inducing a random coil region in the C-terminal portion of Nt17, thus decreasing the propensity to form the reactive amphipathic α-helix. A separate interpretation is that the residues important for helix-helix interactions are blocked by polyP association.


Assuntos
Proteína Huntingtina/química , Doença de Huntington/metabolismo , Sequência de Aminoácidos , Amiloide/química , Amiloide/metabolismo , Humanos , Proteína Huntingtina/genética , Proteína Huntingtina/metabolismo , Doença de Huntington/genética , Cinética , Microscopia de Força Atômica/métodos , Simulação de Dinâmica Molecular , Proteínas do Tecido Nervoso/metabolismo , Proteínas Nucleares/metabolismo , Peptídeos/química , Conformação Proteica em alfa-Hélice , Estrutura Secundária de Proteína
3.
Biophys J ; 111(2): 349-362, 2016 Jul 26.
Artigo em Inglês | MEDLINE | ID: mdl-27463137

RESUMO

Huntington's disease (HD) is a genetic neurodegenerative disorder caused by an expanded polyglutamine (polyQ) domain near the N-terminus of the huntingtin (htt) protein. Expanded polyQ leads to htt aggregation. The first 17 amino acids (Nt(17)) in htt comprise a lipid-binding domain that undergoes a number of posttranslational modifications that can modulate htt toxicity and subcellular localization. As there are three lysines within Nt(17), we evaluated the impact of lysine acetylation on htt aggregation in solution and on model lipid bilayers. Acetylation of htt-exon1(51Q) and synthetic truncated htt-exon 1 mimicking peptides (Nt(17)-Q35-P10-KK) was achieved using a selective covalent label, sulfo-N-hydroxysuccinimide (NHSA). With this treatment, all three lysine residues (K6, K9, and K15) in Nt(17) were significantly acetylated. N-terminal htt acetylation retarded fibril formation in solution and promoted the formation of larger globular aggregates. Acetylated htt also bound lipid membranes and disrupted the lipid bilayer morphology less aggressively compared with the wild-type. Computational studies provided mechanistic insights into how acetylation alters the interaction of Nt(17) with lipid membranes. Our results highlight that N-terminal acetylation influences the aggregation of htt and its interaction with lipid bilayers.


Assuntos
Éxons , Proteína Huntingtina/química , Proteína Huntingtina/metabolismo , Bicamadas Lipídicas/metabolismo , Agregados Proteicos , Acetilação , Sequência de Aminoácidos , Animais , Linhagem Celular , Membrana Celular/metabolismo , Proteína Huntingtina/genética , Camundongos , Peptídeos/metabolismo
4.
Biochemistry ; 54(28): 4285-96, 2015 Jul 21.
Artigo em Inglês | MEDLINE | ID: mdl-26098795

RESUMO

Early stage oligomer formation of the huntingtin protein may be driven by self-association of the 17-residue amphipathic α-helix at the protein's N-terminus (Nt17). Oligomeric structures have been implicated in neuronal toxicity and may represent important neurotoxic species in Huntington's disease. Therefore, a residue-specific structural characterization of Nt17 is crucial to understanding and potentially inhibiting oligomer formation. Native electrospray ion mobility spectrometry-mass spectrometry (IMS-MS) techniques and molecular dynamics simulations (MDS) have been applied to study coexisting monomer and multimer conformations of Nt17, independent of the remainder of huntingtin exon 1. MDS suggests gas-phase monomer ion structures comprise a helix-turn-coil configuration and a helix-extended-coil region. Elongated dimer species comprise partially helical monomers arranged in an antiparallel geometry. This stacked helical bundle may represent the earliest stages of Nt17-driven oligomer formation. Nt17 monomers and multimers have been further probed using diethylpyrocarbonate (DEPC). An N-terminal site (N-terminus of Threonine-3) and Lysine-6 are modified at higher DEPC concentrations, which led to the formation of an intermediate monomer structure. These modifications resulted in decreased extended monomer ion conformers, as well as a reduction in multimer formation. From the MDS experiments for the dimer ions, Lys6 residues in both monomer constituents interact with Ser16 and Glu12 residues on adjacent peptides; therefore, the decrease in multimer formation could result from disruption of these or similar interactions. This work provides a structurally selective model from which to study Nt17 self-association and provides critical insight toward Nt17 multimerization and, possibly, the early stages of huntingtin exon 1 aggregation.


Assuntos
Proteínas do Tecido Nervoso/química , Dietil Pirocarbonato/química , Humanos , Proteína Huntingtina , Lisina/química , Espectrometria de Massas , Simulação de Dinâmica Molecular , Peptídeos/química , Conformação Proteica , Multimerização Proteica , Estabilidade Proteica , Estrutura Secundária de Proteína , Treonina/química
5.
Anal Chem ; 87(10): 5247-54, 2015 May 19.
Artigo em Inglês | MEDLINE | ID: mdl-25893550

RESUMO

Online deuterium hydrogen exchange (DHX) and pepsin digestion (PD) is demonstrated using drift tube ion mobility spectrometry (DTIMS) coupled with linear ion trap (LTQ) mass spectrometry (MS) with electron transfer dissociation (ETD) capabilities. DHX of deuterated ubiquitin, followed by subsequent quenching and digestion, is performed within ∼60 s, yielding 100% peptide sequence coverage. The high reproducibility of the IMS separation allows spectral feature matching between two-dimensional IMS-MS datasets (undeuterated and deuterated) without the need for dataset alignment. Extracted ion drift time distributions (XIDTDs) of deuterated peptic peptides are mobility-matched to corresponding XIDTDs of undeuterated peptic peptides that were identified using collision-induced dissociation (CID). Matching XIDTDs allows a straightforward identification and deuterium retention evaluation for labeled peptides. Aside from the mobility separation, the ion trapping capabilities of the LTQ, combined with ETD, are demonstrated to provide single-residue resolution. Deuterium retention for the c- series ions across residues M(1)-L(15) and N(25)-R(42) are in good agreement with the known secondary structural elements within ubiquitin.


Assuntos
Medição da Troca de Deutério/instrumentação , Espectrometria de Massas em Tandem/instrumentação , Ubiquitina/química , Sequência de Aminoácidos , Animais , Bovinos , Deutério/química , Desenho de Equipamento , Hidrogênio/química , Íons/química , Espectrometria de Massas/instrumentação , Dados de Sequência Molecular , Pepsina A/metabolismo , Peptídeos/análise , Peptídeos/metabolismo , Proteólise , Suínos , Ubiquitina/metabolismo
6.
Biomol Concepts ; 6(1): 33-46, 2015 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-25741791

RESUMO

Huntington's disease (HD) is caused by a polyglutamine (polyQ) domain that is expanded beyond a critical threshold near the N-terminus of the huntingtin (htt) protein, directly leading to htt aggregation. While full-length htt is a large (on the order of ∼350 kDa) protein, it is proteolyzed into a variety of N-terminal fragments that accumulate in oligomers, fibrils, and larger aggregates. It is clear that polyQ length is a key determinant of htt aggregation and toxicity. However, the flanking sequences around the polyQ domain, such as the first 17 amino acids on the N terminus (Nt17), influence aggregation, aggregate stability, influence other important biochemical properties of the protein and ultimately its role in pathogenesis. Here, we review the impact of Nt17 on htt aggregation mechanisms and kinetics, structural properties of Nt17 in both monomeric and aggregate forms, the potential role of posttranslational modifications (PTMs) that occur in Nt17 in HD, and the function of Nt17 as a membrane targeting domain.


Assuntos
Doença de Huntington/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Peptídeos/metabolismo , Animais , Humanos , Proteína Huntingtina , Doença de Huntington/patologia , Proteínas do Tecido Nervoso/química , Agregação Patológica de Proteínas/metabolismo , Processamento de Proteína Pós-Traducional , Estrutura Terciária de Proteína
7.
J Mass Spectrom ; 50(1): 117-26, 2015 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-25601683

RESUMO

Huntington's disease is a genetic neurodegenerative disorder caused by an expansion in a polyglutamine domain near the N-terminus of the huntingtin (htt) protein that results in the formation of protein aggregates. Here, htt aggregate structure has been examined using hydrogen-deuterium exchange techniques coupled with tandem mass spectrometry. The focus of the study is on the 17-residue N-terminal flanking region of the peptide that has been shown to alter htt aggregation kinetics and morphology. A top-down sequencing strategy employing electron transfer dissociation is utilized to determine the location of accessible and protected hydrogens. In these experiments, peptides aggregate in a deuterium-rich solvent at neutral pH and are subsequently subjected to deuterium-hydrogen back-exchange followed by rapid quenching, disaggregation, and tandem mass spectrometry analysis. Electrospray ionization of the peptide solution produces the [M + 5H](5+) to [M + 10H](10+) charge states and reveals the presence of multiple peptide sequences differing by single glutamine residues. The [M + 7H](7+) to [M + 9](9+) charge states corresponding to the full peptide are used in the electron transfer dissociation analyses. Evidence for protected residues is observed in the 17-residue N-terminal tract and specifically points to lysine residues as potentially playing a significant role in htt aggregation.


Assuntos
Medição da Troca de Deutério/métodos , Lisina/química , Proteínas do Tecido Nervoso/química , Proteína Huntingtina , Concentração de Íons de Hidrogênio , Microscopia de Força Atômica , Estrutura Secundária de Proteína , Espectrometria de Massas por Ionização por Electrospray , Espectrometria de Massas em Tandem
8.
J Am Soc Mass Spectrom ; 25(12): 2103-15, 2014 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-25267084

RESUMO

The gas-phase conformations of electrosprayed ions of the model peptide KKDDDDIIKIIK have been examined by ion mobility spectrometry (IMS) and hydrogen deuterium exchange (HDX)-tandem mass spectrometry (MS/MS) techniques. [M+4H](4+) ions exhibit two conformers with collision cross sections of 418 Å(2) and 471 Å(2). [M+3H](3+) ions exhibit a predominant conformer with a collision cross section of 340 Å(2) as well as an unresolved conformer (shoulder) with a collision cross section of ~367 Å(2). Maximum HDX levels for the more compact [M+4H](4+) ions and the compact and partially-folded [M+3H](3+) ions are ~12.9, ~15.5, and ~14.9, respectively. Ion structures obtained from molecular dynamics simulations (MDS) suggest that this ordering of HDX level results from increased charge-site/exchange-site density for the more compact ions of lower charge. Additionally, a new model that includes two distance calculations (charge site to carbonyl group and carbonyl group to exchange site) for the computer-generated structures is shown to better correlate to the experimentally determined per-residue deuterium uptake. Future comparisons of IMS-HDX-MS data with structures obtained from MDS are discussed with respect to novel experiments that will reveal the HDX rates of individual residues.


Assuntos
Medição da Troca de Deutério/métodos , Íons/química , Espectrometria de Massas/métodos , Peptídeos/química , Íons/análise , Simulação de Dinâmica Molecular , Peptídeos/análise , Conformação Proteica
9.
Anal Chem ; 86(16): 8121-8, 2014 Aug 19.
Artigo em Inglês | MEDLINE | ID: mdl-25068446

RESUMO

A new instrument that couples a low-pressure drift tube with a linear ion trap mass spectrometer is demonstrated for complex mixture analysis. The combination of the low-pressure separation with the ion trapping capabilities provides several benefits for complex mixture analysis. These include high sensitivity, unique ion fragmentation capabilities, and high reproducibility. Even though the gas-phase separation and the mass measurement steps are each conducted in an ion filtering mode, detection limits for mobility-selected peptide ions are in the tens of attomole range. In addition to ion separation, the low-pressure drift tube can be used as an ion fragmentation cell yielding mobility-resolved fragment ions that can be subsequently analyzed by multistage tandem mass spectrometry (MS(n)) methods in the ion trap. Because of the ion trap configuration, these methods can be comprised of any number (limited by ion signal) of collision-induced dissociation (CID) and electron transfer dissociation (ETD) processes. The high reproducibility of the gas-phase separation allows for comparison of two-dimensional ion mobility spectrometry (IMS)-MS data sets in a pixel-by-pixel fashion without the need for data set alignment. These advantages are presented in model analyses representing mixtures encountered in proteomics and metabolomics experiments.


Assuntos
Espectrometria de Massas/instrumentação , Metabolômica/instrumentação , Proteômica/instrumentação , Sequência de Aminoácidos , Animais , Proteínas Sanguíneas/análise , Misturas Complexas/análise , Desenho de Equipamento , Humanos , Íons/química , Dados de Sequência Molecular , Fosfopeptídeos/análise , Plasma/química , Pressão
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