RESUMO
Ion mobility/mass spectrometry techniques are used to study the chiral preferences of small proline clusters (containing 2 to 23 proline monomers) produced by electrospray ionization. By varying the composition of the electrospray solution from enantiomerically pure (100% L or 100% D) to racemic (50:50 L:D), it is possible to delineate which cluster sizes prefer homochiral (resolved) or heterochiral (antiresolved) compositions. The results show a remarkable oscillation in chiral preference. Singly protonated clusters, [xPro+H](+) (where x corresponds to the number of prolines), favor homochiral assemblies (for x = 4, 6, 11 and 12); heterochiral structures are preferred (although the preferences are not as strong) for x = 5 and 7. Larger, doubly protonated clusters [xPro+2H](2+) favor homochiral assemblies for x = 18, 19, and 23 and heterochiral structures for x = 14, 16, 17, 20, 21, and 22. Some of the variations that are observed can be rationalized through simple structures that would lead to especially stable geometries. It is suggested that some antiresolved clusters, such as [22Pro+2H](2+), may be comprised of resolved D- and L-proline domains.
Assuntos
Prolina/química , Modelos Moleculares , Espectrometria de Massas por Ionização por ElectrosprayRESUMO
A high-capacity ion trap coupled to a time-of-flight (TOF) mass spectrometer has been developed to carry out comprehensive linked scan analysis of all stored ions in the ion trap. The approach involves a novel tapered geometry high-capacity ion trap that can store more than 10(6) ions (range 800-4000 m/z) without degrading its performance. Ions are stored and scanned out from the high-capacity ion trap as a function of m/z, collisionally fragmented and analyzed by TOF. Accurate mass analysis is achieved on both the precursor and fragment ions of all species ejected from the ion trap. We demonstrate the approach for comprehensive linked-scan identification of phosphopeptides in mixtures with their corresponding unphosphorylated peptides.
RESUMO
A desorption electrospray ionization (DESI) source has been coupled to an ion mobility time-of-flight mass spectrometer for the analysis of proteins. Analysis of solid-phase horse heart cytochrome c and chicken egg white lysozyme proteins with different DESI solvents and conditions shows similar mass spectra and charge state distributions to those formed when using electrospray to analyze these proteins in solution. The ion mobility data show evidence for compact ion structures [when the surface is exposed to a spray that favors retention of "nativelike" structures (50:50 water:methanol)] or elongated structures [when the surface is exposed to a spray that favors "denatured" structures (49:49:2 water:methanol:acetic acid)]. The results suggest that the DESI experiment is somewhat gentler than ESI and under appropriate conditions, it is possible to preserve structural information throughout the DESI process. Mechanisms that are consistent with these results are discussed.
Assuntos
Espectrometria de Massas/métodos , Desnaturação Proteica , Proteínas/química , Absorção , Animais , Citocromos c/química , Íons/química , Modelos Químicos , Muramidase/química , Dobramento de Proteína , Soluções/química , Espectrometria de Massas por Ionização por ElectrosprayRESUMO
The folding pathways of gas-phase cytochrome c ions produced by electrospray ionization have been studied by an ion trapping/ion mobility technique that allows conformations to be examined over extended timescales (10 ms to 10 s). The results show that the +9 charge state emerges from solution as a compact structure and then rapidly unfolds into several substantially more open structures, a transition that requires 30-60 ms; over substantially longer timescales (250 ms to 10 s) elongated states appear to refold into an array of folded structures. The new folded states are less compact than those that are apparent during the initial unfolding. Apparently, unfolding to highly open conformations is a key step that must occur before +9 ions can sample more compact states that are stable at longer times.
Assuntos
Citocromos c/química , Citocromos c/ultraestrutura , Gases/química , Espectrometria de Massas por Ionização por Electrospray/instrumentação , Espectrometria de Massas por Ionização por Electrospray/métodos , Cinética , Transição de Fase , Conformação Proteica , Dobramento de Proteína , Renaturação ProteicaRESUMO
The present work seeks to illuminate the underlying principles which control the aggregation of chiral building blocks into larger aggregates by examining the role that entropy plays in this process. Entropic effects are first examined within the confines of a simple model system, and the results are then compared to experimental data on clusters of amino acids. The model system predicts that the formation of a specific structure is more likely to occur from an enantiopure solution because forming a particular structure from a racemic solution is hindered by significant entropic barriers. These predictions are in good agreement with the experimental results. In our examination of clusters of all of the amino acids, clusters which are unusually abundant are found only when enantiopure solutions are sampled. Furthermore, the majority of all clusters exhibit no preference for chiral composition, suggesting that entropic effects negate any changes in enthalpy. Although the experimental data are not comprehensive, our results strongly suggest that specificity in homochiral clusters is entropically advantageous compared to specificity in racemic clusters.
RESUMO
Recent improvements in ion mobility/time-of-flight mass spectrometry techniques have made it possible to incorporate nano-flow liquid chromatography and collision induced dissociation techniques. This combination of approaches provides a new strategy for detailed characterization of complex systems--such as, combinatorial libraries. Our work uses this technology to provide a detailed analysis of a tetrapeptide library having the general form Xxx1-Xxx2-Xxx3-Xxx4 where Xxx1 = Glu, Phe, Val, Asn; Xxx2 = Glu, Phe, Val, Tyr; Xxx3 = Glu, Phe, Val, Thr; and Xxx4 = Glu, Phe, Val, Leu--a system that is expected to contain 256 different peptide sequences. The results corroborate the presence of many expected peptide sequences and indicate that some synthetic steps appear to have failed. Particularly interesting is the observation of a t-butyl protecting group on the tyrosine (Tyr) residue. It appears that most Tyr containing peptides that have this t-butyl group attached favor formation of [2M + 2H]2+ dimers, which can be readily distinguished from [M + H]+ monomers based on differences in their gas-phase mobilities. In this case, we demonstrate the use of the mobility differences between [2M + 2H]2+ and [M + H]+ ions as a signature for a failure of a synthetic step.
Assuntos
Oligopeptídeos/química , Biblioteca de Peptídeos , Sequência de Aminoácidos , Aminoácidos , Cromatografia Líquida , Técnicas de Química Combinatória , Bases de Dados de Proteínas , Cromatografia Gasosa-Espectrometria de Massas , Oligopeptídeos/síntese química , Oligopeptídeos/classificação , Espectrometria de Massas por Ionização por Electrospray , Terminologia como AssuntoRESUMO
Chiral enrichment of serine is achieved in experiments that involve formation of serine octamers starting from non-racemic serine solutions. Serine octamers were generated by means of electrospray and sonic spray ionization of aqueous solutions of d(3)-L-serine (108 Da) and D-serine (105 Da) having different molar ratios of enantiomers. A cyclic process involving the formation of chirally-enriched octameric cluster ions and their dissociation, viz. Ser(1) --> Ser(8) --> Ser(1), allows serine monomers to be regenerated with increased enantiomeric excess as shown in two types of experiments: (1) Chiral enrichment in serine was observed in MS/MS/MS experiments in a quadrupole ion trap in which the entire distribution of serine octamers formed from non-racemic solutions was isolated, collisionally activated, and fragmented. Monomeric serine was regenerated with increased enantiomeric excess upon dissociation of octamers when compared with the enantiomeric composition of the original solution. (2) Chiral enrichment was observed in the products of soft-landing of mass-selected protonated serine octamers. These ions were generated by means of electrospray or sonic spray ionization, mass selected, and collected on a gold surface using ion soft-landing. Chiral enrichment of the soft-landed serine was established by redissolving the recovered material and comparing the intensities of protonated molecular ions of d(3)-L-serine and D-serine after APCI-MS analysis. Both of these experiments showed comparable results, suggesting that formation of serine octamers depends only on the enantiomeric composition of the serine solution and that the magnitude of the chiral preference is intrinsic to octamers formed from solutions of given chiral composition.
Assuntos
Serina/química , Íons/química , Marcação por Isótopo , Espectrometria de Massas , Soluções/química , EstereoisomerismoRESUMO
Passive transfer of broadly neutralizing HIV antibodies can prevent infection, which suggests that vaccines that elicit such antibodies would be protective. Thus far, however, few broadly neutralizing HIV antibodies that occur naturally have been characterized. To determine whether these antibodies are part of a larger group of related molecules, we cloned 576 new HIV antibodies from four unrelated individuals. All four individuals produced expanded clones of potent broadly neutralizing CD4-binding-site antibodies that mimic binding to CD4. Despite extensive hypermutation, the new antibodies shared a consensus sequence of 68 immunoglobulin H (IgH) chain amino acids and arise independently from two related IgH genes. Comparison of the crystal structure of one of the antibodies to the broadly neutralizing antibody VRC01 revealed conservation of the contacts to the HIV spike.
Assuntos
Anticorpos Neutralizantes/química , Anticorpos Neutralizantes/imunologia , Antígenos CD4/metabolismo , Anticorpos Anti-HIV/química , Anticorpos Anti-HIV/imunologia , Proteína gp120 do Envelope de HIV/imunologia , Sequência de Aminoácidos , Anticorpos Neutralizantes/metabolismo , Afinidade de Anticorpos , Especificidade de Anticorpos , Sítios de Ligação , Sítios de Ligação de Anticorpos , Antígenos CD4/imunologia , Clonagem Molecular , Sequência Consenso , Cristalografia por Raios X , Genes de Cadeia Pesada de Imunoglobulina , Anticorpos Anti-HIV/metabolismo , Proteína gp120 do Envelope de HIV/química , Proteína gp120 do Envelope de HIV/metabolismo , Infecções por HIV/imunologia , Humanos , Fragmentos Fab das Imunoglobulinas/química , Cadeias Pesadas de Imunoglobulinas/química , Cadeias Leves de Imunoglobulina/química , Mimetismo Molecular , Dados de Sequência Molecular , Mutação , Conformação ProteicaRESUMO
Mass spectrometry is a method of choice for quantifying low-abundance proteins and peptides in many biological studies. Here, we describe a range of computational aspects of protein and peptide quantitation, including methods for finding and integrating mass spectrometric peptide peaks, and detecting interference to obtain a robust measure of the amount of proteins present in samples.
Assuntos
Espectrometria de Massas/métodos , Proteínas/análise , Sequência de Aminoácidos , Isótopos , Dados de Sequência Molecular , Peso Molecular , Peptídeos/análise , Peptídeos/química , Proteínas/químicaRESUMO
A series of crown ethers, 12-crown-4, 15-crown-5, 18-crown-6, and dibenzo-30-crown-10, are examined as a possible means of shifting the mobilities of peptide ions. In this approach, a crown ether is added to a solution containing a mixture of peptides and is electrosprayed into the gas phase in order to create distributions of peptide-crown complexes. The ion complexes have different mobilities than the naked peptide ions, and the crown ether molecules appear to interact specifically with basic sites in the peptides thus providing some sequence selectivity. After the peptide-crown complexes are separated by ion mobility spectrometry, the ions can be collisionally activated to dissociate the complex (forming the naked peptide ions) prior to m/z analysis. The overall effect is that complex formation shifts peptide ions to different regions of the mobility spectrum, extending the ability to resolve components. The approach is illustrated by examining isobaric dipeptides as well as a combinatorial library containing 27 tripeptides. Cross sections for the series of crown ether ions and complexes that are observed are reported.
Assuntos
Éteres de Coroa/química , Indicadores e Reagentes/química , Espectrometria de Massas/métodos , Dipeptídeos/químicaRESUMO
Ion mobility measurements, combined with molecular mechanics simulations, are used to study enantiopure and racemic proline clusters formed by electrospray ionization. Broad distributions of cluster sizes and charge states are observed, ranging from clusters containing only a few proline units to clusters that contain more than 100 proline units (i.e., protonated clusters of the form [xPro + nH](n+) with x = 1 to >100 and n = 1-7). As the sizes of clusters increase, there is direct evidence for nanometer scale, chirally induced organization into specific structures. For n = 4 and 5, enantiopure clusters of approximately 50 to 100 prolines assemble into structures that are more elongated than the most compact structure that is observed from the racemic proline clusters. A molecular analogue, cis-4-hydroxy-proline, displays significantly different behavior, indicating that in addition to the rigidity of the side chain ring, intermolecular interactions are important in the formation of chirally directed clusters. This is the first case in which assemblies of chirally selective elongated structures are observed in this size range of amino acid clusters. Relationships between enantiopurity, cluster shape, and overall energetics are discussed.
Assuntos
Prolina/química , Espectrometria de Massas , Modelos Moleculares , Conformação MolecularRESUMO
Here we show experimental evidence for the spontaneous chiral resolution of icosahedral [12Pro+H]+ cluster ion. Molecular simulations reveal that the icosahedron consists of 12 equally spaced prolines where the rigid pyrrolidine ring of each monomer is sticking out of the closed cage. The tightly packed chiral cage traps a single proton in the center cavity. On the other hand, racemic [12Pro+H]+ cluster size exhibits a prismatic structure that can easily incorporate and lose proline monomeric unit sequentially, thus easily forming other geometries. Mechanisms which account for these observations are discussed.
Assuntos
Prolina/química , Ligação de Hidrogênio , Isomerismo , Espectrometria de Massas , Modelos Moleculares , Conformação MolecularRESUMO
In the structure of DL-proline, C5H9NO2, the molecules are connected via classical intermolecular N-H...O hydrogen bonds involving the amine and carboxyl groups [N...O = 2.7129 (15) and 2.8392 (16) A], and form chains along the b-axis direction and parallel to (-101). The chains are linked into sheets via weak non-classical hydrogen bonds. The conformation of the molecule and its packing are notably different from the monohydrated DL-proline form.
Assuntos
Prolina/química , Cristalização , Cristalografia por Raios X , Hidrogênio/química , Ligação de Hidrogênio , Modelos Moleculares , Conformação MolecularRESUMO
A field modulation approach for high-throughput ion mobility/time-of-flight analyses of complex mixtures has been developed using a split-field drift tube. In this approach, complex mixtures of peptides, such as those that arise from tryptic digestion of protein mixtures, are separated by nanocolumn liquid chromatography, ionized by electrospray ionization, and analyzed by ion mobility/time-of-flight techniques. The split-field drift tube allows parent ions to be separated based on differences in their low-field mobilities through the first-field region before entering the second region. For increased throughput, the magnitude of the field in the second region can be modulated throughout an LC separation in order to favor transmission of different types of ions: parent ions at low fields; fragments from primarily [M+3H]3+ peptides at moderate fields; or, fragmentation of [M+3H]3+ and [M+2H]2+ species at higher fields. We demonstrate the approach with two examples: a mixture of tryptic peptides from digestion of hemoglobin; and a complex mixture of tryptic peptides from digestion of human plasma.
Assuntos
Cromatografia Líquida de Alta Pressão/instrumentação , Proteômica/métodos , Espectrometria de Massas por Ionização por Electrospray/instrumentação , Proteínas Sanguíneas/análise , Proteínas Sanguíneas/metabolismo , Hemoglobinas/análise , Hemoglobinas/metabolismo , Humanos , Métodos , Fragmentos de Peptídeos/análise , Proteômica/instrumentação , Tripsina/metabolismoRESUMO
Chirality plays an important role in the formation and stability of noncovalent clusters which are made of chiral molecules. It is shown that clusters can exhibit a preference for both homochirality and heterochirality. Serine cluster formation is dominated by the formation of heterochiral aggregates, with the exception of the previously observed homochiral serine octamer. Thus, the majority of serine clusters lead to chiral anti-resolution, or the racemic mixing of enantiomers.
RESUMO
This work describes a novel technique for the digestion of metals in solid matrices. The technique is called pressure assisted chelating extraction (PACE). In a typical procedure, a solid sample is placed in a stainless steel cell and is mixed with appropriate chelating agents. Using a programmed sequence of temperature, static time, pressure and thermal equilibration available in ASE 200, the metal is removed under moderate temperature (up to 200 degrees C) and pressure (up to 3000 psi). PACE achieves metal recovery that is equivalent to that of wet digestion techniques and also provides for a clean and safe operation by substituting the strong acids commonly used during wet digestion with chelating agents. It uses less solvents and significantly less time (minutes vs. hours) for metal digestion. PACE has been validated using certified standard reference materials (SRMs) including industrial sludge, buffalo river sediments and coal fly ash. The total time required to remove metals was approximately 20 min. Results show that the PACE system provides an ideal platform for efficient, rapid, and safe metal digestion. Good agreement between measured and reference values for Pb, Mn, and Cu were found with recoveries averaging between 80 and 101% and a relative standard deviation of less than 5%. This approach may provide an alternative digestion technique for environmental samples, alloys, biological materials and samples of geological importance. The potential advantage offered lies in non-destruction of the sample, automation and the exclusion of concentrated mineral acids during the digestion procedure.
Assuntos
Quelantes , Poluentes Ambientais/análise , Oligoelementos/análise , Metais , Pressão , Sensibilidade e Especificidade , Espectrofotometria Atômica , TemperaturaRESUMO
A prototype linear octopole ion trap/ion mobility/tandem mass spectrometer has been coupled with a nanoflow liquid chromatography separation approach and used to separate and characterize a complicated peptide mixture from digestion of soluble proteins extracted from human urine. In this approach, two dimensions of separation (nanoflow liquid chromatography and ion mobility) are followed by collision induced dissociation (CID) and mass spectrometry (MS) analysis. From a preliminary analysis of the most intense CID-MS features in a part of the dataset, it is possible to assign 27 peptide ions which correspond to 13 proteins. The data contain many additional CID-MS features for less intense ions. A limited discussion of these features and their potential utility in identifying complicated peptide mixtures required for proteomics study is presented.
Assuntos
Cromatografia Líquida/métodos , Espectrometria de Massas/métodos , Proteínas/análise , Proteinúria , Proteômica , Urina/química , Sequência de Aminoácidos , Cromatografia Líquida/instrumentação , Humanos , Íons/análise , Espectrometria de Massas/instrumentaçãoRESUMO
A linear octopole trap interface for an ion mobility time-of-flight mass spectrometer has been developed for focusing and accumulating continuous beams of ions produced by electrospray ionization. The interface improves experimental efficiencies by factors of approximately 50-200 compared with an analogous configuration that utilizes a three-dimensional Paul geometry trap (Hoaglund-Hyzer, C. S.; Lee, Y. J.; Counterman, A. E.; Clemmer, D. E. Anal. Chem. 2002, 74, 992-1006). With these improvements, it is possible to record nested drift (flight) time distributions for complex mixtures in fractions of a second. We demonstrate the approach for several well-defined peptide mixtures and an assessment of the detection limits is given. Additionally, we demonstrate the utility of the approach in the field of proteomics by an on-line, three-dimensional nano-LC-ion mobility-TOF separation of tryptic peptides from the Drosophila proteome.