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1.
Chem Sci ; 12(5): 1907-1914, 2020 Dec 22.
Artigo em Inglês | MEDLINE | ID: mdl-34163954

RESUMO

On-demand electrospray ionization from different liquid channels in the same emitter was realized using filamented capillary and gas phase charge supply. The solution sub-channel was formed when back-filling solution to the emitter tip by capillary action along the filament. Gas phase charge carriers were used to trigger electrospray ionization from the solution meniscus at the tip. The meniscus at the tip opening may be fully filled or partially empty to generate electrospray ionization in main-channel regime and sub-channel regime, respectively. For emitters with 4 µm tip opening, the two nested electrospray (nested-ESI) channels accommodated ESI flow rates ranging from 50 pL min-1 to 150 nL min-1. The platform enabled on-demand regime alternations within one sample run, in which the sub-channel regime generated smaller charged droplets. Ionization efficiencies for saccharides, glycopeptide, and proteins were enhanced in the sub-channel regime. Non-specific salt adducts were reduced and identified by regime alternation. Surprisingly, the sub-channel regime produced more uniform responses for a peptide mixture whose relative ionization efficiencies were insensitive to ESI conditions in previous picoelectrospray study. The nested channels also allowed effective washing of emitter tip for multiple sampling and analysis operations.

2.
Anal Chem ; 92(1): 1041-1049, 2020 01 07.
Artigo em Inglês | MEDLINE | ID: mdl-31769661

RESUMO

Ultraviolet photodissociation (UVPD) produces rich and informative fragmentation of intact protein ions, but in the case of high mass proteins (>30 kDa) the spectra are congested with overlapping isotope patterns of highly charged fragment ions. In the most congested regions, many fragments cannot be confidently identified even when high-resolution mass analyzers and modern deconvolution algorithms are used. Gas-phase ion-ion proton transfer reactions (PTR), which reduce the charge states of highly charged ions, can be used to alleviate this congestion and facilitate the identification of additional fragment ions when performed following UVPD. We have developed protocols for sequentially performing PTR on multiple populations of ions generated by UVPD in a way that can be tailored to balance the depth of characterization with speed and throughput. The improvements in sequence coverage and fragment identifications are demonstrated for four proteins ranging in size from 29 to 56 kDa. Sequence coverages up to 80% were achieved for carbonic anhydrase (29 kDa), 50% for aldolase (39 kDa), 46% for enolase (46 kDa), and 27% for glutamate dehydrogenase (56 kDa), and up to 74% sequence coverage was obtained for 25 kDa antibody drug conjugate subunits in online LC-MS experiments.


Assuntos
Enzimas/química , Imunoconjugados/química , Prótons , Sequência de Aminoácidos , Animais , Bovinos , Cromatografia Líquida/métodos , Enzimas/efeitos da radiação , Imunoconjugados/efeitos da radiação , Limite de Detecção , Proteólise/efeitos da radiação , Coelhos , Saccharomyces cerevisiae/enzimologia , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/efeitos da radiação , Espectrometria de Massas em Tandem/métodos , Raios Ultravioleta
3.
Anal Chem ; 90(14): 8583-8591, 2018 07 17.
Artigo em Inglês | MEDLINE | ID: mdl-29927232

RESUMO

Ultraviolet photodissociation (UVPD) is a nonselective activation method in which both precursor and fragment ions may absorb photons and dissociate. Photoactivation of fragment ions may result in secondary or multiple generations of dissociation, which decreases the signal-to-noise ratio (S/N) of larger fragment ions owing to the prevalent subdivision of the ion current into many smaller, often less informative, fragment ions. Here we report the use of dipolar excitation waveforms to displace fragment ions out of the laser beam path, thus alleviating the extent of secondary dissociation during 193 nm UVPD. This fragment ion protection (FIP) strategy increases S/N of larger fragment ions and improves the sequence coverage obtained for proteins via retaining information deeper into the midsection of protein sequences.

4.
Angew Chem Int Ed Engl ; 55(40): 12417-21, 2016 09 26.
Artigo em Inglês | MEDLINE | ID: mdl-27605434

RESUMO

The first application of light-emitting diodes (LEDs) for ultraviolet photodissociation (UVPD) mass spectrometry is reported. LEDs provide a compact, low cost light source and have been incorporated directly into the trapping cell of an Orbitrap mass spectrometer. MS/MS efficiencies of over 50 % were obtained using an extended irradiation period, and UVPD was optimized by modulating the ion trapping parameters to maximize the overlap between the ion cloud and the irradiation volume.

5.
Anal Chem ; 85(23): 11284-90, 2013 Dec 03.
Artigo em Inglês | MEDLINE | ID: mdl-24171553

RESUMO

A novel differentially pumped dual linear quadrupole ion trap (DLQIT) mass spectrometer was designed and built to facilitate tandem MS experiments free from interfering reactions. The instrument consists of two differentially pumped Thermo Scientific linear quadrupole ion trap (LQIT) systems that have been connected via an ion transfer octupole encased in a machined manifold. Tandem MS experiments can be performed in the front trap and then the resulting product ions can be transferred via axial ejection into the back trap for further, independent tandem MS experiments in a differentially pumped area. This approach allows the examination of consecutive collision-activated dissociation (CAD) and ion-molecule reactions without unwanted side reactions that often occur when CAD and ion-molecule reactions are examined in the same space. Hence, it greatly facilitates investigations of ion structures. In addition, the overall lower pressure of the DLQIT, as compared to commercial LQIT instruments, results in a reduction of unwanted side reactions with atmospheric contaminants, such as water and oxygen, in CAD and ion-molecule experiments.


Assuntos
Aceleradores de Partículas , Espectrometria de Massas em Tandem/métodos , Troca Iônica , Espectrometria de Massas/métodos
6.
J Am Soc Mass Spectrom ; 24(11): 1623-33, 2013 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-23677544

RESUMO

We describe the implementation and characterization of activated ion electron transfer dissociation (AI-ETD) on a hybrid QLT-Orbitrap mass spectrometer. AI-ETD was performed using a collision cell that was modified to enable ETD reactions, in addition to normal collisional activation. The instrument manifold was modified to enable irradiation of ions along the axis of this modified cell with IR photons from a CO2 laser. Laser power settings were optimized for both charge (z) and mass to charge (m/z) and the instrument control firmware was updated to allow for automated adjustments to the level of irradiation. This implementation of AI-ETD yielded 1.6-fold more unique identifications than ETD in an nLC-MS/MS analysis of tryptic yeast peptides. Furthermore, we investigated the application of AI-ETD on large scale analysis of phosphopeptides, where laser power aids ETD, but can produce b- and y-type ions because of the phosphoryl moiety's high IR adsorption. nLC-MS/MS analysis of phosphopeptides derived from human embryonic stem cells using AI-ETD yielded 2.4-fold more unique identifications than ETD alone, demonstrating a promising advance in ETD sequencing of PTM containing peptides.


Assuntos
Elétrons , Íons/química , Espectrometria de Massas/instrumentação , Fosfopeptídeos/análise , Células-Tronco Embrionárias/química , Humanos , Raios Infravermelhos , Lasers , Peptídeos/efeitos da radiação , Fosfopeptídeos/efeitos da radiação , Espectrometria de Massas em Tandem
7.
J Am Soc Mass Spectrom ; 24(6): 816-27, 2013 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-23609185

RESUMO

We describe and characterize an improved implementation of ETD on a modified hybrid linear ion trap-Orbitrap instrument. Instead of performing ETD in the mass-analyzing quadrupole linear ion trap (A-QLT), the instrument collision cell was modified to enable ETD. We partitioned the collision cell into a multi-section rf ion storage and transfer device to enable injection and simultaneous separate storage of precursor and reagent ions. Application of a secondary (axial) confinement voltage to the cell end lens electrodes enables charge-sign independent trapping for ion-ion reactions. The approximately 2-fold higher quadrupole field frequency of this cell relative to that of the A-QLT enables higher reagent ion densities and correspondingly faster ETD reactions, and, with the collision cell's longer axial dimensions, larger populations of precursor ions may be reacted. The higher ion capacity of the collision cell permits the accumulation and reaction of multiple full loads of precursor ions from the A-QLT followed by FT Orbitrap m/z analysis of the ETD product ions. This extends the intra-scan dynamic range by increasing the maximum number of product ions in a single MS/MS event. For analyses of large peptide/small protein precursor cations, this reduces or eliminates the need for spectral averaging to achieve acceptable ETD product ion signal-to-noise levels. Using larger ion populations, we demonstrate improvements in protein sequence coverage and aggregate protein identifications in LC-MS/MS analysis of intact protein species as compared to the standard ETD implementation.


Assuntos
Espectrometria de Massas/instrumentação , Proteínas/química , Sequência de Aminoácidos , Proteínas Fúngicas/química , Íons/química , Espectrometria de Massas/métodos , Dados de Sequência Molecular
8.
J Am Soc Mass Spectrom ; 23(5): 880-8, 2012 May.
Artigo em Inglês | MEDLINE | ID: mdl-22351295

RESUMO

Identification of drug metabolites can often yield important information regarding clearance mechanism, pharmacologic activity, or toxicity for drug candidate molecules. Additionally, the identification of metabolites can provide beneficial structure-activity insight to help guide lead optimization efforts towards molecules with optimal metabolic profiles. There are challenges associated with detecting and identifying metabolites in the presence of complex biological matrices, and new LC-MS technologies have been developed to meet these challenges. In this report, we describe the development of an experimental approach that applies unique features of the hybrid linear ion trap Orbitrap mass spectrometer to streamline in vitro and in vivo metabolite identification experiments. The approach, referred to as MS(M), utilizes multiple collision cells, dissociation methods, mass analyzers, and detectors. With multiple scan types and different dissociation modes built into one experimental method, along with flexible post-acquisition analysis options, the MS(M) workflow offers an attractive option to fast and reliable identification of metabolites in different kinds of in vitro and in vivo samples. The MS(M) workflow was successfully applied to metabolite identification analysis of verapamil in both in vitro rat hepatocyte incubations and in vivo rat bile samples.


Assuntos
Espectrometria de Massas/instrumentação , Espectrometria de Massas/métodos , Preparações Farmacêuticas/metabolismo , Animais , Bile/metabolismo , Cromatografia Líquida , Hepatócitos/metabolismo , Masculino , Farmacocinética , Ratos , Ratos Sprague-Dawley , Verapamil/metabolismo , Verapamil/farmacocinética
9.
Anal Chem ; 84(3): 1533-9, 2012 Feb 07.
Artigo em Inglês | MEDLINE | ID: mdl-22192247

RESUMO

We report the implementation of front-end higher energy collision-induced dissociation (fHCD) on a benchtop dual-pressure linear ion trap. Software and hardware modifications were employed, described in detail vide-infra, to allow isolated ions to undergo collisions with ambient gas molecules in an intermediate multipole (q00) of the instrument. Results comparing the performance of fHCD and resonance excitation collision-induced dissociation (RE-CID) in terms of injection time, total number of scans, efficiency, mass measurement accuracy (MMA), unique peptide identifications, and spectral quality of labile modified peptides are presented. fHCD is approximately 23% as efficient as RE-CID, and depending on the search algorithm, it identifies 6.6% more or 15% less peptides (q < 0.01) from a soluble whole-cell lysate ( Caenorhabditis elegans ) than RE-CID using Mascot or Sequest search algorithms, respectively. fHCD offers a clear advantage for the analysis of phosphorylated and glycosylated (O-GlcNAc) peptides as the average cross-correlation score (XCorr) for spectra using fHCD was statistically greater (p < 0.05) than for spectra collected using RE-CID.


Assuntos
Espectrometria de Massas/instrumentação , Proteômica/instrumentação , Animais , Caenorhabditis elegans/metabolismo , Glicopeptídeos/análise , Espectrometria de Massas/métodos , Fosfopeptídeos/análise , Pressão , Software
10.
Mol Cell Proteomics ; 11(3): O111.013698, 2012 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-22159718

RESUMO

Although only a few years old, the combination of a linear ion trap with an Orbitrap analyzer has become one of the standard mass spectrometers to characterize proteins and proteomes. Here we describe a novel version of this instrument family, the Orbitrap Elite, which is improved in three main areas. The ion transfer optics has an ion path that blocks the line of sight to achieve more robust operation. The tandem MS acquisition speed of the dual cell linear ion trap now exceeds 12 Hz. Most importantly, the resolving power of the Orbitrap analyzer has been increased twofold for the same transient length by employing a compact, high-field Orbitrap analyzer that almost doubles the observed frequencies. An enhanced Fourier Transform algorithm-incorporating phase information-further doubles the resolving power to 240,000 at m/z 400 for a 768 ms transient. For top-down experiments, we combine a survey scan with a selected ion monitoring scan of the charge state of the protein to be fragmented and with several HCD microscans. Despite the 120,000 resolving power for SIM and HCD scans, the total cycle time is within several seconds and therefore suitable for liquid chromatography tandem MS. For bottom-up proteomics, we combined survey scans at 240,000 resolving power with data-dependent collision-induced dissociation of the 20 most abundant precursors in a total cycle time of 2.5 s-increasing protein identifications in complex mixtures by about 30%. The speed of the Orbitrap Elite furthermore allows scan modes in which complementary dissociation mechanisms are routinely obtained of all fragmented peptides.


Assuntos
Cromatografia Líquida , Fragmentos de Peptídeos/análise , Fragmentos de Peptídeos/metabolismo , Proteoma/análise , Proteoma/metabolismo , Proteômica/instrumentação , Espectrometria de Massas em Tandem/instrumentação , Células HeLa , Humanos , Proteômica/métodos , Espectrometria de Massas em Tandem/métodos
11.
Anal Chem ; 82(24): 10068-74, 2010 Dec 15.
Artigo em Inglês | MEDLINE | ID: mdl-21062032

RESUMO

Using a modified electron transfer dissociation (ETD)-enabled quadrupole linear ion trap (QLT) mass spectrometer, we demonstrate the utility of IR activation concomitant with ETD ion-ion reactions (activated-ion ETD, AI-ETD). Analyzing 12 strong cation exchanged (SCX) fractions of a LysC digest of human cell protein extract using ETD, collision-activated dissociation (CAD), and AI-ETD, we find that AI-ETD generates 13 405 peptide spectral matches (PSMs) at a 1% false-discovery rate (1% FDR), surpassing both ETD (7 968) and CAD (10 904). We also analyze 12 SCX fractions of a tryptic digest of human cell protein extract and find that ETD produces 6 234 PSMs, AI-ETD 9 130 PSMs, and CAD 15 209 PSMs. Compared to ETD with supplemental collisional activation (ETcaD), AI-ETD generates ∼80% more PSMs for the whole cell lysate digested with trypsin and ∼50% more PSMs for the whole cell lysate digested with LysC.


Assuntos
Extratos Celulares/química , Espectrometria de Massas/métodos , Fragmentos de Peptídeos/isolamento & purificação , Humanos , Raios Infravermelhos , Espectrometria de Massas/instrumentação , Metaloendopeptidases/metabolismo , Fragmentos de Peptídeos/análise , Proteínas/análise , Proteínas/metabolismo , Tripsina/metabolismo
13.
Mol Cell Proteomics ; 8(12): 2759-69, 2009 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-19828875

RESUMO

Since its introduction a few years ago, the linear ion trap Orbitrap (LTQ Orbitrap) instrument has become a powerful tool in proteomics research. For high resolution mass spectrometry measurements ions are accumulated in the linear ion trap and passed on to the Orbitrap analyzer. Simultaneously with acquisition of this signal, the major peaks are isolated in turn, fragmented and recorded at high sensitivity in the linear ion trap, combining the strengths of both mass analyzer technologies. Here we describe a next generation LTQ Orbitrap system termed Velos, with significantly increased sensitivity and scan speed. This is achieved by a vacuum interface using a stacked ring radio frequency ion guide with 10-fold higher transfer efficiency in MS/MS mode and 3-5-fold in full scan spectra, by a dual pressure ion trap configuration, and by reduction of overhead times between scans. The first ion trap efficiently captures and fragments ions at relatively high pressure whereas the second ion trap realizes extremely fast scan speeds at reduced pressure. Ion injection times for MS/MS are predicted from full scans instead of performing automatic gain control scans. Together these improvements routinely enable acquisition of up to ten fragmentation spectra per second. Furthermore, an improved higher-energy collisional dissociation cell with increased ion extraction capabilities was implemented. Higher-collision energy dissociation with high mass accuracy Orbitrap readout is as sensitive as ion trap MS/MS scans in the previous generation of the instrument.


Assuntos
Espectrometria de Massas/instrumentação , Pressão , Proteômica/instrumentação , Análise de Sequência de Proteína/instrumentação , Sequência de Aminoácidos , Animais , Anidrases Carbônicas/química , Bovinos , Células HeLa , Humanos , Dados de Sequência Molecular , Peptídeos/química , Soroalbumina Bovina/química , Fatores de Tempo
14.
Anal Chem ; 81(21): 8809-17, 2009 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-19874053

RESUMO

A hybrid linear ion trap/orthogonal time-of-flight (TOF) mass spectrometer has been developed to observe time-dependent vacuum ultraviolet photodissociation product ions. In this apparatus, a reflectron TOF mass analyzer is orthogonally interfaced to an LTQ using rf-only octopole and dc quadrupole ion guides. Precursor ions are generated by electrospray ionization and isolated in the ion trap. Subsequently they are directed to the TOF source where photodissociation occurs and product ions are extracted for mass analysis. To detect photodissociation product ions having axially divergent trajectories, a large rectangular detector is utilized. With variation of the time between photodissociation and orthogonal extraction in the TOF source, product ions formed over a range of times after photoexcitation can be sampled. Time-dependent observation of product ions following 157 nm photodissociation of a singly charged tryptic peptide ion (NWDAGFGR) showed that prompt photofragment ions (x- and v-type ions) dominate the tandem mass spectrum up to 1 micros after the laser shot, but the intensities of low energy thermal fragment ions (y-type ions) become comparable several microseconds later. Different proton mobilization time scales were observed for arginine- and lysine-terminated tryptic peptides.


Assuntos
Íons/química , Peptídeos/análise , Espectrometria de Massas em Tandem/métodos , Raios Ultravioleta , Sequência de Aminoácidos , Espectrometria de Massas em Tandem/instrumentação , Fatores de Tempo
15.
Anal Chem ; 81(21): 8677-86, 2009 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-19785447

RESUMO

Infrared multiphoton dissociation (IRMPD) was implemented in a novel dual pressure linear ion trap for rapid top-down proteomics. The high pressure cell provided improved trapping and isolation efficiencies while the isotopic profiles of 10+ charged ions could be resolved by mass analysis in the low pressure cell that enabled effective top down protein identification. Striking differences between IRMPD in the low pressure cell and CID in the high pressure cell were observed for proteins ranging from 8.6 to 29 kDa. Because of secondary dissociation, IRMPD yielded product ions in significantly lower charge states as compared to CID, thus facilitating more accurate mass identification and streamlining product ion assignment. This outcome was especially useful for database searching of larger proteins (approximately 29 kDa) as IRMPD substantially improved protein identification and scoring confidence. Also, IRMPD showed an increased selectivity toward backbone cleavages N-terminal to proline and C-terminal to acidic residues (especially for the lowest charge states), which could be useful for a priori spectral predictions and enhanced database searching for protein identification.


Assuntos
Raios Infravermelhos , Íons/química , Proteínas/química , Proteômica/métodos , Espectrometria de Massas em Tandem/métodos , Bases de Dados de Proteínas , Transporte de Íons , Pressão , Prolina/química , Proteômica/instrumentação
16.
Anal Chem ; 81(19): 8109-18, 2009 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-19739654

RESUMO

A dual pressure linear ion trap mass spectrometer was modified to permit infrared multiphoton dissociation (IRMPD) in each of the two cells-the first a high pressure cell operated at nominally 5 x 10(-3) Torr and the second a low pressure cell operated at nominally 3 x 10(-4) Torr. When IRMPD was performed in the high pressure cell, most peptide ions did not undergo significant photodissociation; however, in the low pressure cell peptide cations were efficiently dissociated with less than 25 ms of IR irradiation regardless of charge state. IRMPD of peptide cations allowed the detection of low m/z product ions including the y(1) fragments and immonium ions which are not typically observed by ion trap collision induced dissociation (CID). Photodissociation efficiencies of approximately 100% and MS/MS (tandem mass spectrometry) efficiencies of greater than 60% were observed for both multiply and singly protonated peptides. In general, higher sequence coverage of peptides was obtained using IRMPD over CID. Further, greater than 90% of the product ion current in the IRMPD mass spectra of doubly charged peptide ions was composed of singly charged product ions compared to the CID mass spectra in which the abundances of the multiply and singly charged product ions were equally divided. Highly charged primary product ions also underwent efficient photodissociation to yield singly charged secondary product ions, thus simplifying the IRMPD product ion mass spectra.


Assuntos
Cátions/química , Raios Infravermelhos , Peptídeos/química , Espectrometria de Massas em Tandem/instrumentação , Sequência de Aminoácidos , Dados de Sequência Molecular , Espectrometria de Massas em Tandem/métodos
17.
Anal Chem ; 81(18): 7757-65, 2009 Sep 15.
Artigo em Inglês | MEDLINE | ID: mdl-19689114

RESUMO

The considerable progress in high-throughput proteomics analysis via liquid chromatography-electrospray ionization-tandem mass spectrometry over the past decade has been fueled to a large degree by continuous improvements in instrumentation. High-throughput identification experiments are based on peptide sequencing and are largely accomplished through the use of tandem mass spectrometry, with ion trap and trap-based instruments having become broadly adopted analytical platforms. To satisfy increasingly demanding requirements for depth of characterization and throughput, we present a newly developed dual-pressure linear ion trap mass spectrometer (LTQ Velos) that features increased sensitivity, afforded by a new source design, and demonstrates practical cycle times 2 times shorter than that of an LTQ XL, while improving or maintaining spectral quality for MS/MS fragmentation spectra. These improvements resulted in a substantial increase in the detection and identification of both proteins and unique peptides from the complex proteome of Caenorhabditis elegans, as compared to existing platforms. The greatly increased ion flux into the mass spectrometer in combination with improved isolation of low-abundance precursor ions resulted in increased detection of low-abundance peptides. These improvements cumulatively resulted in a substantially greater penetration into the baker's yeast (Saccharomyces cerevisiae) proteome compared to LTQ XL. Alternatively, faster cycle times on the new instrument allowed for higher throughput for a given depth of proteome analysis, with more peptides and proteins identified in 60 min using an LTQ Velos than in 180 min using an LTQ XL. When mass analysis was carried out with resolution in excess of 25,000 full width at half-maximum (fwhm), it became possible to isotopically resolve a small intact protein and its fragments, opening possibilities for top down experiments.


Assuntos
Cromatografia Líquida de Alta Pressão/métodos , Peptídeos/análise , Proteínas/análise , Espectrometria de Massas por Ionização por Electrospray/instrumentação , Animais , Cavalos , Mioglobina/análise , Proteômica/métodos , Espectrometria de Massas por Ionização por Electrospray/métodos
18.
Mol Cell Proteomics ; 7(10): 1838-49, 2008 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-18445579

RESUMO

Cardiac troponin I (cTnI), the inhibitory subunit of the thin filament troponin-tropomyosin regulatory complex, is required for heart muscle relaxation during the cardiac cycle. Expressed only in cardiac muscle, cTnI is widely used in the clinic as a serum biomarker of cardiac injury. In vivo function of cTnI is influenced by phosphorylation and proteolysis; therefore analysis of post-translational modifications of the intact protein should greatly facilitate the understanding of cardiac regulatory mechanisms and may improve cTnI as a disease biomarker. cTnI (24 kDa, pI approximately 9.5) contains twelve serine, eight threonine, and three tyrosine residues, which presents a challenge for unequivocal identification of phosphorylation sites and quantification of positional isomers. In this study, we used top down electron capture dissociation and electron transfer dissociation MS to unravel the molecular complexity of cTnI purified from human heart tissue. High resolution MS spectra of human cTnI revealed a high degree of heterogeneity, corresponding to phosphorylation, acetylation, oxidation, and C-terminal proteolysis. Thirty-six molecular ions of cTnI were detected in a single ESI/FTMS spectrum despite running as a single sharp band on SDS-PAGE. Electron capture dissociation of monophosphorylated cTnI localized two major basal phosphorylation sites: a well known site at Ser(22) and a novel site at Ser(76)/Thr(77), each with partial occupancy (Ser(22): 53%; Ser(76)/Thr(77): 36%). Top down MS(3) analysis of diphosphorylated cTnI revealed occupancy of Ser(23) only in diphosphorylated species consistent with sequential (or ordered) phosphorylation/dephosphorylation of the Ser(22/23) pair. Top down MS of cTnI provides unique opportunities for unraveling its molecular complexity and for quantification of phosphorylated positional isomers thus allowing establishment of the relevance of such modifications to physiological functions and disease status.


Assuntos
Elétrons , Espectrometria de Massas/métodos , Miocárdio/metabolismo , Fosfoproteínas/química , Troponina I/química , Sequência de Aminoácidos , Humanos , Dados de Sequência Molecular , Peso Molecular , Fosfoproteínas/metabolismo , Fosforilação , Fosfosserina/metabolismo , Fosfotreonina/metabolismo , Isoformas de Proteínas/química , Isoformas de Proteínas/metabolismo , Troponina I/metabolismo
19.
Anal Chem ; 79(2): 477-85, 2007 Jan 15.
Artigo em Inglês | MEDLINE | ID: mdl-17222010

RESUMO

Electron-transfer dissociation (ETD) delivers the unique attributes of electron capture dissociation to mass spectrometers that utilize radio frequency trapping-type devices (e.g., quadrupole ion traps). The method has generated significant interest because of its compatibility with chromatography and its ability to: (1) preserve traditionally labile post-translational modifications (PTMs) and (2) randomly cleave the backbone bonds of highly charged peptide and protein precursor ions. ETD, however, has shown limited applicability to doubly protonated peptide precursors, [M + 2H]2+, the charge and type of peptide most frequently encountered in "bottom-up" proteomics. Here we describe a supplemental collisional activation (CAD) method that targets the nondissociated (intact) electron-transfer (ET) product species ([M + 2H]+*) to improve ETD efficiency for doubly protonated peptides (ETcaD). A systematic study of supplementary activation conditions revealed that low-energy CAD of the ET product population leads to the near-exclusive generation of c- and z-type fragment ions with relatively high efficiency (77 +/- 8%). Compared to those formed directly via ETD, the fragment ions were found to comprise increased relative amounts of the odd-electron c-type ions (c+*) and the even-electron z-type ions (z+). A large-scale analysis of 755 doubly charged tryptic peptides was conducted to compare the method (ETcaD) to ion trap CAD and ETD. ETcaD produced a median sequence coverage of 89%-a significant improvement over ETD (63%) and ion trap CAD (77%).


Assuntos
Espectrometria de Massas/métodos , Fragmentos de Peptídeos/análise , Proteínas/química , Proteômica/métodos , Sequência de Aminoácidos , Transtornos Dissociativos , Transporte de Elétrons , Dados de Sequência Molecular , Prótons , Tripsina/química
20.
Anal Chem ; 78(3): 718-25, 2006 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-16448044

RESUMO

A rectilinear ion trap (RIT) mass analyzer was incorporated into a mass spectrometer fitted with an electrospray ionization source and an atmospheric pressure interface. The RIT mass spectrometer, which was assembled in two different configurations, was used for the study of biological compounds, for which performance data are given. A variety of techniques, including the use of a balanced rf, elevated background gas pressure, automatic gain control, and resonance ejection waveforms with dynamically adjusted amplitude, were applied to enhance performance. The capabilities of the instrument were characterized using proteins, peptides, and pharmaceutical drugs. Unit resolution and an accuracy of better than m/z 0.2 was achieved for mass-to-charge (m/z) ratios up to 2000 Th at a scan rate of approximately 3000 amu/(charge.s) while reduced scan rates gave greater resolution and peak widths of less than m/z 0.5 over the same range. The mass discrimination in trapping externally generated ions was characterized over the range m/z 190-2000 and an optimized low mass cutoff value of m/z 120-140 was found to give equal trapping efficiencies over the entire range. The radial detection efficiency was measured as a function of m/z ratio and found to rise from 35% at low m/z values to more than 90% for ions of m/z 1800. The way in which the ion trapping capacity depends on the dc trapping potential was investigated by measuring the mass shift due to space charge effects, and it was shown that low trapping potentials minimize space charge effects by increasing the useful volume of the device. The collision-induced dissociation (CID) capabilities of the RIT instrument were evaluated by measuring isolation efficiency as a function of mass resolution as well as measuring peptide CID efficiencies. Overall CID efficiencies of more than 60% were easily reached, while isolation of an ion with unit resolution at m/z 524 was achieved with high rejection (>95%) of the adjacent ions. The overall analytical capabilities of the ESI-RIT instrument were demonstrated with the analysis of a mixture of pharmaceutical compounds using multiple-stage mass spectrometry.


Assuntos
Espectrometria de Massas por Ionização por Electrospray/métodos , Apoproteínas/análise , Pressão Atmosférica , Citocromos c/análise , Mioglobina/análise , Peptídeos/análise , Sensibilidade e Especificidade , Espectrometria de Massas por Ionização por Electrospray/instrumentação
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