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1.
Anal Chem ; 94(10): 4141-4145, 2022 03 15.
Artículo en Inglés | MEDLINE | ID: mdl-35234449

RESUMEN

Mass spectrometry (MS) allows for automated analysis of complex samples at high resolution without the need for labeling/derivatization. Liquid atmospheric pressure matrix-assisted laser desorption/ionization (LAP-MALDI) enables rapid sample preparation and MS analysis using microtiter-plate formats and high-performing mass spectrometers. We present a step change in high-speed, large-scale MS sample analysis of peptides at 20 samples/s and an enzymatic assay at 40 samples/s, i.e., an order of magnitude faster than current MS platforms. LAP-MALDI requires only low amounts of sample volume (<2 µL), of which only a fraction (<1%) is typically consumed, and allows for multiplexing and high-speed MS/MS analysis, demonstrated at ∼10 samples/s. Its high ion signal stability and similarity to electrospray ionization enables CVs below 10% and the analysis of multiply charged peptide ions at these extreme speeds. LAP-MALDI MS fulfills the speed requirements for large-scale population diagnostics and compound screening with the potential of analyzing >1 million samples per day.


Asunto(s)
Presión Atmosférica , Espectrometría de Masas en Tándem , Iones , Rayos Láser , Péptidos/química , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción/métodos
2.
Rapid Commun Mass Spectrom ; 35 Suppl 1: e8246, 2021 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-30067883

RESUMEN

RATIONALE: Liquid atmospheric pressure matrix-assisted laser desorption/ionisation (AP-MALDI) has been shown to enable the production of electrospray ionisation (ESI)-like multiply charged analyte ions with little sample consumption and long-lasting, robust ion yield for sensitive analysis by mass spectrometry (MS). Previous reports have focused on positive ion production. Here, we report an initial optimisation of liquid AP-MALDI for ESI-like negative ion production and its application to the analysis of peptides/proteins, DNA and lipids. METHODS: The instrumentation employed for this study is identical to that of earlier liquid AP-MALDI MS studies for positive analyte ion production with a simple non-commercial AP ion source that is attached to a Waters Synapt G2-Si mass spectrometer and incorporates a heated ion transfer tube. The preparation of liquid MALDI matrices is similar to positive ion mode analysis but has been adjusted for negative ion mode by changing the chromophore to 3-aminoquinoline and 9-aminoacridine for further improvements. RESULTS: For DNA, liquid AP-MALDI MS analysis benefited from switching to 9-aminoacridine-based MALDI samples and the negative ion mode, increasing the number of charges by up to a factor of 2 and the analyte ion signal intensities by more than 10-fold compared with the positive ion mode. The limit of detection was recorded at around 10 fmol for ATGCAT. For lipids, negative ion mode analysis provided a fully orthogonal set of detected lipids. CONCLUSIONS: Negative ion mode is a sensitive alternative to positive ion mode in liquid AP-MALDI MS analysis. In particular, the analysis of lipids and DNA benefited from the complementarity of the detected lipid species and the vastly greater DNA ion signal intensities in negative ion mode.

3.
Analyst ; 146(22): 6861-6873, 2021 Nov 08.
Artículo en Inglés | MEDLINE | ID: mdl-34632987

RESUMEN

We describe the implementation of a simple three-electrode surface-induced dissociation (SID) cell on a cyclic ion mobility spectrometer (cIMS) and demonstrate the utility of multipass mobility separations for resolving multiple conformations of protein complexes generated during collision-induced and surface-induced unfolding (CIU & SIU) experiments. In addition to CIU and SIU, SID of protein complexes is readily accomplished within the native instrument software and with no additional external power supplies by entering a single SID collision energy, a simplification in user experience compared to prior implementations. A set of cyclic homomeric protein complexes and a heterohexamer with known CID and SID behavior were analyzed to investigate mass and mobility resolution improvements, the latter of which improved by 20-50% (median: 33%) compared to a linear travelling wave device. Multiple passes of intact complexes, or their SID fragments, increased the mobility resolution by an average of 15% per pass, with the racetrack effect being observed after ∼3 or 4 passes, depending on the drift time spread of the analytes. Even with modest improvements to apparent mobility resolving power, multipass experiments were particularly useful for separating conformations produced from CIU and SIU experiments. We illustrate several examples where either (1) multipass experiments revealed multiple overlapping conformations previously unobserved or obscured due to limited mobility resolution, or (2) CIU or SIU conformations that appeared 'native' in a single pass experiment were actually slightly compacted or expanded, with the change only being measurable through multipass experiments. The work conducted here, the first utilization of multipass cyclic ion mobility for CIU, SIU, and SID of protein assemblies and a demonstration of a wholly integrated SIU/SID workflow, paves the way for widespread adoption of SID technology for native mass spectrometry and also improves our understanding of gas-phase protein complex CIU and SIU conformationomes.


Asunto(s)
Proteínas , Programas Informáticos , Espectrometría de Masas
4.
Anal Chem ; 92(4): 2931-2936, 2020 02 18.
Artículo en Inglés | MEDLINE | ID: mdl-31967792

RESUMEN

Label-free high-throughput screening using mass spectrometry has the potential to provide rapid large-scale sample analysis at a speed of more than one sample per second. Such speed is important for compound library, assay and future clinical screening of millions of samples within a reasonable time frame. Herein, we present a liquid atmospheric pressure matrix-assisted laser desorption/ionization (AP-MALDI) setup for high-throughput large-scale sample analysis (>5 samples per second) for three substance classes (peptides, antibiotics, and lipids). Liquid support matrices (LSM) were used for the analysis of standard substances as well as complex biological fluids (milk). Throughput and analytical robustness were mainly dependent on the complexity of the sample composition and the current limitations of the commercial hardware. However, the ultimate limits of liquid AP-MALDI in sample throughput can be conservatively estimated to be beyond 10-20 samples per second. This level of analytical speed is highly competitive compared with other label-free MS methods, including electrospray ionization and solid state MALDI, as well as MS methods using multiplexing by labeling, which in principle can also be used in combination with liquid AP-MALDI MS.

5.
Anal Chem ; 92(5): 3674-3681, 2020 03 03.
Artículo en Inglés | MEDLINE | ID: mdl-31999103

RESUMEN

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.


Asunto(s)
Espectrometría de Masas/métodos , Desnaturalización Proteica , Proteínas/química , Secuencia de Aminoácidos , Animales , Bovinos , Humanos , Modelos Moleculares , Multimerización de Proteína , Estructura Cuaternaria de Proteína
6.
Anal Chem ; 90(13): 8020-8027, 2018 07 03.
Artículo en Inglés | MEDLINE | ID: mdl-29846054

RESUMEN

Tandem mass spectrometry (MS/MS) is an invaluable experimental tool for providing analytical data supporting the identification of small molecules and peptides in mass-spectrometry-based "omics" experiments. Data-dependent MS/MS (DDA) is a real-time MS/MS-acquisition strategy that is responsive to the signals detected in a given sample. However, in analysis of even moderately complex samples with state-of-the-art instrumentation, the speed of MS/MS acquisition is insufficient to offer comprehensive MS/MS coverage of all detected molecules. Data-independent approaches (DIA) offer greater MS/MS coverage, typically at the expense of selectivity or sensitivity. This report describes data-set-dependent MS/MS (DsDA), a novel integration of MS1-data processing and target prioritization to enable comprehensive MS/MS sampling during the initial MS-level experiment. This approach is guided by the premise that in omics experiments, individual injections are typically made as part of a larger set of samples, and feedback between data processing and data acquisition can allow approximately real-time optimization of MS/MS-acquisition parameters and nearly complete MS/MS-sampling coverage. Using a combination of R, Proteowizard, XCMS, and WRENS software, this concept was implemented on a liquid-chromatograph-coupled quadrupole time-of-flight mass spectrometer. The results illustrate comprehensive MS/MS coverage for a set of complex small-molecule samples and demonstrate a strong improvement on traditional DDA.


Asunto(s)
Análisis de Datos , Espectrometría de Masas en Tándem , Animales , Bovinos , Hordeum/química , Músculos/química , Cebollas/química
7.
Anal Chem ; 90(2): 1077-1080, 2018 01 16.
Artículo en Inglés | MEDLINE | ID: mdl-29266933

RESUMEN

Hydrogen/deuterium exchange mass spectrometry (HDX-MS) is now a routinely used technique to inform on protein structure, dynamics, and interactions. Localizing the incorporated deuterium content on a single residue basis increases the spatial resolution of this technique enabling detailed structural analysis. Here, we investigate the use of ultraviolet photodissociation (UVPD) at 213 nm to measure deuterium levels at single residue resolution in HDX-MS experiments. Using a selectively labeled peptide, we show that UVPD occurs without H/D scrambling as the peptide probe accurately retains its solution-phase deuterium labeling pattern. Our results indicate that UVPD provides an attractive alternative to electron mediated dissociation for increasing the spatial resolution of the HDX-MS experiment, capable of yielding high fragmentation efficiency, high fragment ion diversity, and low precursor ion charge-state dependency.


Asunto(s)
Espectrometría de Masas/métodos , Péptidos/química , Deuterio/análisis , Medición de Intercambio de Deuterio/métodos , Fotólisis , Rayos Ultravioleta
8.
Rapid Commun Mass Spectrom ; 32(24): 2099-2105, 2018 Dec 30.
Artículo en Inglés | MEDLINE | ID: mdl-30230090

RESUMEN

RATIONALE: In-source decay (ISD) matrix-assisted laser desorption/ionisation (MALDI) mass spectrometry with a 1,5-diaminonaphthalene (1,5-DAN) matrix is used for the structural characterisation of peptides. However, MALDI spectra are intrinsically complicated by the presence of matrix ions, which interfere with the peptide fragments. This may cause false-positive results or reduced sequence coverage. This paper reports investigations of ISD processes in an intermediate pressure MALDI ion source and a protocol for the removal of interfering ions using ion mobility separation (IMS). METHODS: An intermediate pressure MALDI source of a Q-IMS-Q-TOF instrument (Synapt G2) has been employed for the ISD of selected peptides using a 1,5-DAN matrix. RESULTS: Successful coupling of the MALDI source tuned for ISD experiments using IMS is demonstrated. The IMS made it possible to remove interfering matrix ions effectively from the spectra and thus to increase the confidence of spectral interpretation. Extensive fragment series corresponding to N-Cα bond cleavages were observed under optimised conditions; on the other hand, weaker series of ions caused by peptide bond cleavages were prevalent for default conditions and/or the α-hydroxycinnamic acid matrix. CONCLUSIONS: Ion mobility has been used for the elimination of matrix ions. The technique has been applied to top-down sequencing of non-tryptic peptides, such as the human palmitoylated analogue of prolactin-releasing peptide used in recent obesity studies, and human and insect antimicrobial peptides.


Asunto(s)
Espectrometría de Masas/métodos , Péptidos/química , Animales , Péptidos Catiónicos Antimicrobianos/química , Humanos , Insectos , Espectrometría de Masas/instrumentación , Hormona Liberadora de Prolactina/química , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción/métodos
9.
Anal Chem ; 89(1): 916-921, 2017 01 03.
Artículo en Inglés | MEDLINE | ID: mdl-27958700

RESUMEN

Concerted tandem and traveling wave ion mobility mass spectrometry (CTS analysis) is a unique method that results in a four-dimensional data set including nominal precursor ion mass, product ion mobility, accurate mass of product ion, and ion abundance. This nontargeted lipidomics CTS approach was applied in both positive- and negative-ion mode to phospholipids present in human serum, and the data set was used to evaluate the value of product ion mobility in identifying lipids in a complex mixture. It was determined that the combination of diagnostic product ions and unique collisional cross-section values of product ions is a powerful tool in the structural identification of lipids in a complex biological sample.


Asunto(s)
Fosfolípidos/sangre , Humanos , Espectrometría de Masas , Estructura Molecular , Espectrometría de Masas en Tándem
10.
Rapid Commun Mass Spectrom ; 31(17): 1415-1423, 2017 Sep 15.
Artículo en Inglés | MEDLINE | ID: mdl-28590551

RESUMEN

RATIONALE: The position of C=C within fatty acyl chains affects the biological function of lipids. Ozone-induced dissociation mass spectrometry (OzID-MS) has great potential in determination of lipid double-bond position, but has generally been implemented on low-resolution ion trap mass spectrometers. In addition, most of the OzID-MS experiments carried out so far were focused on the sodiated adducts of lipids; fragmentation of the most commonly observed protonated ions generated in LC/MS-based lipidomics workflow has been less explored. METHODS: Ozone generated in line from an ozone generator was connected to the trap and transfer gas supply line of a Synapt G2 high-resolution mass spectrometer. Protonated ions of different phosphatidylcholines (PC) were generated by electrospray ionization through direct infusion. Different parameters, including traveling wave height and velocity, trap entrance and DC potential, were adjusted to maximize the OzID efficiency. sn-positional isomers and cis/trans isomers of lipids were compared for their reactivity with ozone. RESULTS: Traveling wave height and velocity were tuned to prolong the encounter time between lipid ions and ozone, and resulted in improved OzID efficiency, as did increasing trapping region DC and entrance potential. Under optimized settings, at least 1000 times enhancement in OzID efficiency was achieved compared to that under default settings for monounsaturated PC standards. Monounsaturated C=C in the sn-2 PC isomer reacted faster with ozone than the sn-1 isomer. Similarly, the C=C in trans PC reacted faster than in cis PC. CONCLUSIONS: This is the first implementation of OzID in the trap and transfer region of a traveling wave enabled high-resolution mass spectrometer. The OzID reaction efficiency is significantly improved by slowing down ions in the trap region for their prolonged interaction with ozone. This will facilitate application of high-resolution OzID-MS in lipidomics.


Asunto(s)
Lípidos/análisis , Lípidos/química , Ozono/química , Isomerismo , Conformación Molecular , Fosfatidilcolinas/análisis , Fosfatidilcolinas/química , Sodio/química
11.
Methods ; 104: 11-20, 2016 07 15.
Artículo en Inglés | MEDLINE | ID: mdl-26827934

RESUMEN

Liquid matrix-assisted laser desorption/ionization (MALDI) allows the generation of predominantly multiply charged ions in atmospheric pressure (AP) MALDI ion sources for mass spectrometry (MS) analysis. The charge state distribution of the generated ions and the efficiency of the ion source in generating such ions crucially depend on the desolvation regime of the MALDI plume after desorption in the AP-to-vacuum inlet. Both high temperature and a flow regime with increased residence time of the desorbed plume in the desolvation region promote the generation of multiply charged ions. Without such measures the application of an electric ion extraction field significantly increases the ion signal intensity of singly charged species while the detection of multiply charged species is less dependent on the extraction field. In general, optimization of high temperature application facilitates the predominant formation and detection of multiply charged compared to singly charged ion species. In this study an experimental set-up and optimization strategy is described for liquid AP-MALDI MS which improves the ionization efficiency of selected ion species up to 14 times. In combination with ion mobility separation, the method allows the detection of multiply charged peptide and protein ions for analyte solution concentrations as low as 2fmol/µL (0.5µL, i.e. 1fmol, deposited on the target) with very low sample consumption in the low nL-range.


Asunto(s)
Péptidos/aislamiento & purificación , Proteínas/aislamiento & purificación , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción/métodos , Presión Atmosférica , Iones/química , Péptidos/química , Proteínas/clasificación
12.
Anal Chem ; 88(20): 9964-9971, 2016 Oct 18.
Artículo en Inglés | MEDLINE | ID: mdl-27631466

RESUMEN

We demonstrate the capabilities of a laser-coupled ion mobility mass spectrometer for analysis of peptide sequence and structure showing ultraviolet photodissociation (UVPD) spectra of mass and mobility selected ions. A Synapt G2-S mass spectrometer has been modified to allow photointeraction of ions post the mobility cell. For this work, we have employed a single wavelength laser, which irradiates at 266 nm. We present the unique capabilities of this instrument and demonstrate several key features. Irradiation of luteinizing hormone releasing hormone (LHRH), growth hormone releasing hexapeptide (GHRP-6), and TrpCage (sequence NLYIQWLKDGGPSSGRPPPS) yields extensive b- and y-type fragmentation as well as a- and c-type ions. In addition, we observe side chain losses, including the indole group from tryptophan, and immonium ions. For negatively charged ions, we show the advantage of using collision-induced dissociation (CID) post-UVPD: radical ions are produced following irradiation, and these fragment with higher efficiency. Further, we have incorporated ion mobility and subsequent drift time gating into the UVPD method allowing the separate analysis of m/z-coincident species, both conformers and multimers. To demonstrate, we selectively dissociate the singly charged dimer or doubly charged monomer of the peptide gramicidin A and conformers of the [M + 5H]5+ form of the peptide melittin. Each mobility selected form has a different "fingerprint" dissociation spectrum, both predominantly containing b and y fragments. Differences in the intensities of various loss channels between the two species were revealed. The smaller conformer of melittin has fewer cleavage sites along the peptide backbone than the larger conformer suggesting considerable structural differences. For gramicidin, a single laser shot UVPD discriminates between primary photodissociation and subsequent fragmentation of fragments. We also show how this modified instrument facilitates activated electron photodissociation. UVPD-IM-MS analysis serves both as a method for peptide sequencing for peptides of similar (or identical) m/z and a method for optical analysis of mobility separated species.

13.
Anal Chem ; 88(2): 1218-21, 2016 Jan 19.
Artículo en Inglés | MEDLINE | ID: mdl-26641730

RESUMEN

Mass spectrometry has emerged as a useful tool in the study of proteins and protein complexes. It is of fundamental interest to explore how the structures of proteins and protein complexes are affected by the absence of solvent and how this alters with increasing time in the gas phase. Here we demonstrate that a range of protein and protein complexes can be confined within the Trap T-wave region of a modified Waters Synapt G2S instrument, including monomeric (ß-lactoglobulin), dimeric (ß-lactoglobulin and enolase), tetrameric (streptavidin, concanavalin A, and pyruvate kinase), and pentameric (C-reactive protein) complexes, ranging in size up to 237 kDa. We demonstrate that complexes can be confined within the Trap region for varying lengths of time over the range 1-60 s and with up to 86% trapping efficiency for 1 s trapping. Furthermore, using model systems, we show that these noncovalent complexes can also be fragmented by surface-induced dissociation (SID) following trapping. SID reveals similar dissociation patterns over all trapping times studied for unactivated protein complexes, suggesting that any conformational changes occurring over this time scale are insufficient to cause substantial differences in the SID spectra of these complexes. Intentional alteration of structure by cone activation produces a distinct SID spectrum, with the differences observed being conserved, in comparison to unactivated complex, after trapping. However, subtle differences in the SID spectra of the activated complex are also observed as a function of trapping time.


Asunto(s)
Proteína C-Reactiva/química , Concanavalina A/química , Gases/química , Lactoglobulinas/química , Fosfopiruvato Hidratasa/química , Piruvato Quinasa/química , Estreptavidina/química , Espectrometría de Masas , Fosfopiruvato Hidratasa/metabolismo , Piruvato Quinasa/metabolismo , Propiedades de Superficie
14.
Methods ; 89: 22-9, 2015 Nov 01.
Artículo en Inglés | MEDLINE | ID: mdl-26014039

RESUMEN

Top-down sequencing methods are becoming increasingly relevant for protein characterization, in particular electron capture (ECD) and electron transfer dissociation (ETD) which allow for extensive backbone cleavage with minimal side reactions. The ability to obtain sequence-specific fragments while maintaining aspects of the higher-order structure, as well as the position of deuterium labels in H/D exchange, has attracted interest from scientists in the field of structural proteomics. Recently, ETD has also been combined with ion mobility on commercially available quadrupole/time-of-flight instruments, and this implementation paves the way to novel structural studies and investigation of the ETD process itself. In the current work, we investigate the use of ETD for fragmentation of standard peptides and proteins and provide a detailed description of the effect of the parameters controlling the time and efficiency of the reaction. We also highlight how the combination with ion mobility separation after electron transfer provides extended analytical benefits, such as assignment of fragments to a specific charge-reduced state of the precursor.


Asunto(s)
Flavoproteínas Transportadoras de Electrones/análisis , Electrones , Espectrometría de Masas/métodos , Animales , Bovinos , Flavoproteínas Transportadoras de Electrones/química , Caballos , Mioglobina/análisis , Mioglobina/química , Sustancia P/análisis , Sustancia P/química , Ubiquitina/análisis , Ubiquitina/química
15.
Proteomics ; 15(16): 2842-50, 2015 Aug.
Artículo en Inglés | MEDLINE | ID: mdl-25603979

RESUMEN

The study of protein conformation by solution-phase hydrogen/deuterium exchange (HDX) coupled to MS is well documented. This involves monitoring the exchange of backbone amide protons with deuterium and provides details concerning the protein's tertiary structure. However, undesired back-exchange during post-HDX analyses can be difficult to control. Here, gas-phase HDX-MS, during which labile hydrogens on amino acid side chains are exchanged in sub-millisecond time scales, has been employed to probe changes within protein structures. Addition of the solvent 2,2,2-trifluoroethanol to a protein in solution can affect the structure of the protein, resulting in an increase in secondary and/or tertiary structure which is detected using circular dichroism. Using a Synapt G2-S ESI-mass spectrometer modified to allow deuterated ammonia into the transfer ion guide (situated between the ion mobility cell and the TOF analyser), gas-phase HDX-MS is shown to reflect minor structural changes experienced by the proteins ß-lactoglobulin and ubiquitin, as observed by the reduction in the level of deuterium incorporation. Additionally, the use of gas-phase HDX-MS to distinguish between co-populated proteins conformers within a solution is demonstrated with the disordered protein calmodulin; the gas-phase HDX-MS results correspond directly with complementary data obtained by use of ion mobility spectrometry-MS.


Asunto(s)
Medición de Intercambio de Deuterio/métodos , Conformación Proteica , Proteínas/química , Modelos Moleculares , Pliegue de Proteína , Proteínas/análisis , Solventes
16.
Anal Chem ; 86(9): 4439-46, 2014 May 06.
Artículo en Inglés | MEDLINE | ID: mdl-24694021

RESUMEN

Atmospheric pressure electron capture dissociation (AP-ECD) is an emerging technique capable of being adopted to virtually any electrospray mass spectrometer, without modification of the main instrument. To date, however, because the electron capture reactions occur in the ion source, AP-ECD has been limited by its apparent inability to select precursors prior to fragmentation, i.e., to perform tandem mass spectrometry (MS/MS) experiments. In this paper we demonstrate a novel AP-ECD-MS/MS method using an AP-ECD source on a Xevo G2-S quadrupole time-of-flight (Q-TOF) mass spectrometer from Waters Micromass. The method takes advantage of the tendency for electron capture reactions to generate charge-reduced "ECnoD" products, species that have captured an electron and have had a covalent bond cleaved yet do not immediately dissociate into separate products and so retain the mass of the precursor ion. In the method, ECnoD products from the AP-ECD source are isolated in the quadrupole mass filter and induced to dissociate through supplemental activation in the collision cell, and then the liberated ECD fragment ions are mass analyzed using the high-resolution TOF. In this manner, true MS/MS spectra may be obtained with AP-ECD even though all of the precursors in the source are subjected to electron capture reactions in parallel. Here, using a late-model Q-TOF instrument otherwise incapable of performing electron-based fragmentation, we present AP-ECD-MS/MS results for a group of model peptides and show that informative, high-sequence-coverage spectra are readily attainable with the method.


Asunto(s)
Espectrometría de Masas en Tándem/métodos , Secuencia de Aminoácidos , Presión Atmosférica , Fibrina/química , Datos de Secuencia Molecular , Fragmentos de Péptidos/química
17.
Anal Chem ; 86(23): 11868-76, 2014 Dec 02.
Artículo en Inglés | MEDLINE | ID: mdl-25375223

RESUMEN

Gas-phase hydrogen/deuterium exchange (HDX) is a fast and sensitive, yet unharnessed analytical approach for providing information on the structural properties of biomolecules, in a complementary manner to mass analysis. Here, we describe a simple setup for ND3-mediated millisecond gas-phase HDX inside a mass spectrometer immediately after ESI (gas-phase HDX-MS) and show utility for studying the primary and higher-order structure of peptides and proteins. HDX was achieved by passing N2-gas through a container filled with aqueous deuterated ammonia reagent (ND3/D2O) and admitting the saturated gas immediately upstream or downstream of the primary skimmer cone. The approach was implemented on three commercially available mass spectrometers and required no or minor fully reversible reconfiguration of gas-inlets of the ion source. Results from gas-phase HDX-MS of peptides using the aqueous ND3/D2O as HDX reagent indicate that labeling is facilitated exclusively through gaseous ND3, yielding similar results to the infusion of purified ND3-gas, while circumventing the complications associated with the use of hazardous purified gases. Comparison of the solution-phase- and gas-phase deuterium uptake of Leu-Enkephalin and Glu-Fibrinopeptide B, confirmed that this gas-phase HDX-MS approach allows for labeling of sites (heteroatom-bound non-amide hydrogens located on side-chains, N-terminus and C-terminus) not accessed by classical solution-phase HDX-MS. The simple setup is compatible with liquid chromatography and a chip-based automated nanoESI interface, allowing for online gas-phase HDX-MS analysis of peptides and proteins separated on a liquid chromatographic time scale at increased throughput. Furthermore, online gas-phase HDX-MS could be performed in tandem with ion mobility separation or electron transfer dissociation, thus enabling multiple orthogonal analyses of the structural properties of peptides and proteins in a single automated LC-MS workflow.


Asunto(s)
Medición de Intercambio de Deuterio , Gases/química , Péptidos/análisis , Péptidos/química , Cromatografía Liquida , Transporte de Electrón , Espectrometría de Masas , Conformación Proteica , Factores de Tiempo
18.
Anal Chem ; 86(19): 9644-52, 2014 Oct 07.
Artículo en Inglés | MEDLINE | ID: mdl-25188777

RESUMEN

We introduce a new atmospheric pressure charge stripping (AP-CS) method for the electrospray ionization mass spectrometry (ESI-MS) analysis of heterogeneous mixtures, utilizing ion/ion proton transfer reactions within an experimental ion source to remove excess charge from sample ions and thereby reduce spectral congestion. The new method enables the extent of charge stripping to be easily controlled, independent of primary ionization, and there are no complications due to adduct formation. Here, we demonstrate AP-CS with a Xevo G2-S Q-TOF from Waters-Micromass using an ion source originally designed for atmospheric pressure-electron capture dissociation (AP-ECD) experiments; repurposing the AP-ECD ion source for AP-CS requires only adding a supplemental reagent (e.g., a perfluorocompound) to scavenge the electrons and generate anions for the charge-stripping reactions. Results from model peptides are first presented to demonstrate the basic method, including differences between the AP-CS and AP-ECD operating modes, and how the extent of charge stripping may be controlled. This is followed by a demonstration of AP-CS for the ESI-MS analysis of several large poly(ethylene glycol)s (PEGs), up to 40 kDa, typical of those used in biopharmaceutical development.


Asunto(s)
Polietilenglicoles/química , Espectrometría de Masa por Ionización de Electrospray/métodos , Presión Atmosférica
19.
Analyst ; 139(24): 6348-51, 2014 Dec 21.
Artículo en Inglés | MEDLINE | ID: mdl-25349872

RESUMEN

An ion mobility mass spectrometer has been modified to allow optical interrogation of ions with different mass-to-charge (m/z) ratios and/or mobilities (K). An ion gating and trapping procedure has been developed which allows us to store ions for several seconds enabling UV photodissociation (UVPD).


Asunto(s)
Mononucleótido de Flavina/química , Iones/química , Espectrometría de Masas/instrumentación , Diseño de Equipo , Procesos Fotoquímicos , Rayos Ultravioleta
20.
Rapid Commun Mass Spectrom ; 27(21): 2383-90, 2013 Nov 15.
Artículo en Inglés | MEDLINE | ID: mdl-24097394

RESUMEN

RATIONALE: Electron transfer dissociation (ETD) within ion trapping mass spectrometers has proven to be a useful tool for the characterisation of post-translational modifications. In this study, we describe the implementation of ETD upon a modified quadrupole time-of-flight (Q-ToF) system and methods for the analysis of glycoproteins. METHODS: Liquid chromatography electrospray ionisation mass spectrometry (LC/ESI-MS) was performed using a hybrid quadrupole/ion mobility/oa-ToF mass spectrometer equipped with ETD functionality. 1,4-Dicyanobenzene reagent anions necessary for the ETD reaction were generated from a glow discharge region located within the ESI source block. ETD reactions occurred in the stacked ring travelling wave ion guide (located after the quadrupole mass filter and prior to the oa-ToF mass analyser). LC/ETD was performed upon 'super-charged' tryptic glycopeptide ions produced from the recombinant monoclonal antibody trastuzumab. LC/ETD was also performed on ions from the smaller glycopeptides obtained from erythropoietin. RESULTS: ETD performed upon the quadruply 'super-charged' N-linked glycopeptide ions of trastuzumab and the triply charged O-linked glycopeptide ions of erythropoietin provided both glycosylation site assignments and full sequence information, respectively. Tandem mass (MS/MS) spectra employing collision-induced dissociation (CID) were dominated by oxonium product ions hampering full peptide sequence characterisation. CONCLUSIONS: LC/ETD on the Q-ToF system proved effective at characterising a number of different N-linked glyco-forms of the tryptic peptide, EEQYNSTYR, from trastuzumab as well as glyco-forms from the O-linked tryptic peptide, EASIPPDAASAAPLR, from erythropoietin. The data demonstrates that the glycopeptide site heterogeneity of trastuzumab and erythropoietin can be accurately characterised. In addition, the post-column mixing of the super-charging reagent, m-NBA, is an effective method to increase the precursor ion charge state and to improve ETD reaction efficiency.


Asunto(s)
Anticuerpos Monoclonales Humanizados/química , Glicoproteínas/química , Espectrometría de Masa por Ionización de Electrospray/métodos , Secuencia de Aminoácidos , Cromatografía Liquida/instrumentación , Cromatografía Liquida/métodos , Electrones , Diseño de Equipo , Glicopéptidos/química , Datos de Secuencia Molecular , Espectrometría de Masa por Ionización de Electrospray/instrumentación , Trastuzumab
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