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1.
Rapid Commun Mass Spectrom ; 26(6): 621-30, 2012 Mar 30.
Artigo em Inglês | MEDLINE | ID: mdl-22328215

RESUMO

A combination of methodologies using the extremely high mass accuracy and resolution of 15-T Fourier transform ion cyclotron resonance (FT-ICR) mass spectrometry (MS) was introduced for the identification of intact cancer cell phospholipids. Lipids from a malignant glioma cell line were initially analyzed at a resolution of >200,000 and identified by setting the mass tolerance to ±1 mDa using matrix-assisted laser desorption/ionization (MALDI) 15-T FT-ICR MS in positive ion mode. In most cases, a database search of potential lipid candidates using the exact masses of the lipids yielded only one possible chemical composition. Extremely high mass accuracy (<0.1 ppm) was then attained by using previously identified lipids as internal standards. This, combined with an extremely high resolution (>800,000), yielded well-resolved isotopic fine structures allowing for the identification of lipids by MALDI 15-T FT-ICR MS without using tandem mass spectrometric (MS/MS) analysis. Using this method, a total of 38 unique lipids were successfully identified.


Assuntos
Lipídeos/análise , Neoplasias/química , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos , Linhagem Celular Tumoral , Ciclotrons , Bases de Dados Factuais , Análise de Fourier , Humanos , Fosfolipídeos/análise , Sensibilidade e Especificidade
2.
J Mass Spectrom ; 47(12): 1576-81, 2012 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-23280746

RESUMO

A peptide peak at m/z 1634 in the mass spectrum of tryptically digested cytochrome c has been ambiguously assigned to either a peptide IFVQKCAQCHTVEK or a peptide CAQCHTVEK combined with a heme group (CAQCHTVEK + heme (Fe(III))). A comprehensive investigation was performed to clearly identify the origin of the peak. Tryptic digests of cytochrome c were analyzed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS), liquid chromatography-tandem MS (LC-MS/MS), LC-ultraviolet (LC-UV), and MALDI Fourier transform-ion cyclotron resonance (FT-ICR) MS. The use of instruments with extremely high mass accuracy revealed the mass difference between the IFVQKCAQCHTVEK and the (CAQCHTVEK + heme (Fe(III))) ions. Fragmentation of the peptide associated with the unknown peak yielded a heme ion and other fragment ions originating from a (CAQCHTVEK + heme (Fe(III))) ion. Furthermore, an absorption peak at 395 nm confirmed the presence of a heme group in the unknown peptide. High mass accuracy analyses of MS and MS/MS spectra, in addition to three-dimensional UV contour mapping, showed that the peak at m/z 1634 is due to a (CAQCHTVEK + heme (Fe(III))) ion and not from protonated IFVQKCAQCHTVEK.


Assuntos
Citocromos c/química , Fragmentos de Peptídeos/química , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos , Sequência de Aminoácidos , Cromatografia Líquida de Alta Pressão/métodos , Citocromos c/metabolismo , Heme/química , Íons/química , Dados de Sequência Molecular , Fragmentos de Peptídeos/metabolismo , Tripsina/metabolismo
3.
Anal Biochem ; 414(1): 125-30, 2011 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-21352795

RESUMO

The effects of temperature on ultrasound-assisted tryptic protein digestion were comprehensively investigated using matrix-assisted laser desorption/ionization (MALDI) time-of-flight mass spectrometry. Three standard proteins, cytochrome c, myoglobin, and bovine serum albumin, were digested at 4°C (ice), room temperature (20-25), 37, and 55°C for 0 s, 30s, 1 min, and 5 min, in an ultrasonic bath. We found that the number of identified peptides generally increased with increasing temperature or digestion time. Compared with conventional overnight digestion at 37°C without ultrasonication, digestions performed under ultrasonication generally produced more peptides under most of the above listed conditions, mainly due to miscleaved peptides. Tryptic digestions were also performed under all the conditions evaluated without using ultrasound, where the most significant improvement with the application of ultrasound in terms of sequence coverage and the number of identified peptides was observed at 4°C, followed by room temperature, and 37°C, while no improvement was observed at 55°C with the application of ultrasound, which may be due to the fact that the current experiments were performed in an ultrasonic bath.


Assuntos
Citocromos c/metabolismo , Mioglobina/metabolismo , Fragmentos de Peptídeos/metabolismo , Soroalbumina Bovina/metabolismo , Tripsina/metabolismo , Ultrassom , Animais , Bovinos , Citocromos c/química , Cavalos , Mioglobina/química , Fragmentos de Peptídeos/química , Soroalbumina Bovina/química , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos , Temperatura
4.
Rapid Commun Mass Spectrom ; 25(1): 88-92, 2011 Jan 15.
Artigo em Inglês | MEDLINE | ID: mdl-21154656

RESUMO

The effect of vortex-induced vibration during tryptic digestion was investigated by applying different vibrational speeds (0, 600, 1200, or 2500 rpm) to digestion solutions for varying durations (10, 20, 30, 40, or 60 min) at two different incubation temperatures (25°C or 37°C). The most rapid digestion was observed with the highest vibrational speed and temperature. With the application of 2500 rpm at 37°C, the tryptic digestion of each of three standard proteins (cytochrome c, myoglobin, or bovine serum albumin) provided complete disappearance of the protein within 60 min, as determined by matrix-assisted laser desorption/ionization mass spectrometry. Compared to conventional overnight digestion, 60-min vortex-assisted tryptic digestion generated longer peptides, due primarily to the limited digestion time and provided better sequence coverages (89% vs. 78% for cytochrome c, 100% vs. 87% for myoglobin, and 38% vs. 26% for BSA). The longer peptides should be advantageous to analytical methods such as the middle-down approach that benefit from increased sequence coverage of proteins. Vortex-assisted tryptic digestion is expected to be a useful method for rapid tryptic digestion of proteins.


Assuntos
Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/metabolismo , Tripsina/química , Tripsina/metabolismo , Animais , Bovinos , Citocromos c/química , Citocromos c/metabolismo , Mioglobina/química , Mioglobina/metabolismo , Soroalbumina Bovina/química , Soroalbumina Bovina/metabolismo , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Fatores de Tempo , Vibração
5.
J Am Soc Mass Spectrom ; 21(12): 2000-4, 2010 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-20850343

RESUMO

Peaks originating from unknown compounds on stainless steel plates used in matrix-assisted laser desorption/ionization (MALDI) mass spectrometers are observed around m/z 304.3, 332.3, 360.4, and 388.4 regardless of the matrix and/or solvent, and are even observed with bare plates. These peaks were characterized using three different types of MALDI-MS instrumentation: MALDI-TOF MS, MALDI-TOF/TOF MS, and MALDI-FTMS. The fragmentation data from MALDI-TOF/TOF MS and accurate mass determination by MALDI-FTMS enabled identification of the chemical formulae and structures. The unknown compounds are, in fact, likely benzylalkylmethylammonium salts, as confirmed by closely matching fragmentation patterns with a commercially available benzalkonium chloride.

6.
Rapid Commun Mass Spectrom ; 24(7): 901-8, 2010 Apr 15.
Artigo em Inglês | MEDLINE | ID: mdl-20196188

RESUMO

A simple and effective digestion method was developed using a syringe. A 3 mL syringe was used to apply a pressure of 6 atm to expedite tryptic digestion. Application of a pressure of 6 atm during digestion resulted in better digestion efficiency than digestion under atmospheric pressure. The protein peaks in the matrix-assisted laser desorption/ionization mass spectra of three model proteins (cytochrome c, horse heart myoglobin, and bovine serum albumin (BSA)) completely disappeared within 30 min at 37 degrees C under a pressure of 6 atm, with greater numbers of peptides observed in 30 min pressure-assisted digestion than in overnight atmospheric pressure digestion. This is mostly due to the miscleaved peptides. Similar sequence coverages were obtained for 30 min pressure-assisted digestion and overnight atmospheric pressure digestion of the three model proteins (92% vs. 88% for cytochrome c, 100% vs. 97% for horse heart myoglobin, and 53% vs. 53% for BSA).


Assuntos
Fragmentos de Peptídeos/química , Mapeamento de Peptídeos/instrumentação , Proteínas/química , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/instrumentação , Tripsina/química , Acetonitrilas/química , Sequência de Aminoácidos , Animais , Bovinos , Citocromos c/química , Citocromos c/metabolismo , Cavalos , Dados de Sequência Molecular , Mioglobina/química , Mioglobina/metabolismo , Fragmentos de Peptídeos/metabolismo , Mapeamento de Peptídeos/métodos , Pressão , Proteínas/metabolismo , Soroalbumina Bovina/química , Soroalbumina Bovina/metabolismo , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos , Seringas , Tripsina/metabolismo
7.
Rapid Commun Mass Spectrom ; 22(16): 2561-4, 2008 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-18655002

RESUMO

Magnetic iron(II, III) oxide (magnetite, Fe(3)O(4)) nanoparticles were used to selectively enrich phosphopeptides from tryptic digests of bovine beta-casein and from tryptic digest mixtures containing bovine beta-casein, cytochrome c, bovine serum albumin, and horse heart myoglobin. The magnetic property of the particles permits an easy and speedy enrichment process. No enrichment of phosphopeptides was observed from ferric magnetic iron(III) oxide (Fe(2)O(3)) nanoparticles. These data collectively demonstrate that the enrichment of phosphopeptides using magnetic iron(II, III) oxide nanoparticles is a practical method for the selective analysis of phosphopeptides and could be helpful in isolating and analyzing phosphorylated peptides from complex biological samples.


Assuntos
Óxido Ferroso-Férrico/química , Magnetismo , Nanopartículas Metálicas/química , Fosfopeptídeos/química , Animais , Caseínas/química , Bovinos , Citocromos c/química , Cavalos , Miocárdio/química , Mioglobina/química , Soroalbumina Bovina/química , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos
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