Maximum entropy deconvolution in electrospray mass spectrometry

Ferrige, Anthony G.; Seddon, M. J.; Jarvis, S.; Skilling, John; Aplin, Robert

doi:10.1002/rcm.1290050810

Cited by 152 publications

(111 citation statements)

References 1 publication

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“…Conventional mass spectra were recorded at a resolution set to give a peak width at half-height of 0.4 m/z units for a monoisotopic peak of a singly charged ion. Protein spectra were accumulated for 5 min under the control of MassLynx software (Micromass) and were processed using the maximum entropy approach (38). Relative proportions of protein constituents of mixtures were estimated from peak areas in the maximum entropy-processed spectra.…”

Section: Methodsmentioning

confidence: 99%

Evidence for the Location of Bicyclomycin Binding to theEscherichia coli Transcription Termination Factor Rho

Riba¹,

Gaskell²,

Cho

et al. 1998

Journal of Biological Chemistry

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The commercial antibiotic bicyclomycin (Bcm) has been shown to target the essential transcription termination factor Rho in Escherichia coli. Little is known about the Bcm binding domain in Rho. A recent structure-activity relationship study led us to evaluate the reductive amination probe, 5a-(3-formylanilino)dihydrobicyclomycin (FD-Bcm). Biochemical studies showed that FD-Bcm possessed inhibitory activities comparable to Bcm in Rho-dependent ATPase and transcription termination assays. Incubation of Rho with FD-Bcm, ATP, and poly(C) followed by NaBH 4 reduction and dialysis led to an appreciable loss of ATPase activity. Inclusion of Bcm with FD-Bcm in the reductive amination reaction protected Rho, indicating that Bcm and FD-Bcm competed for the same binding site in Rho. Incubation of Rho with FD-Bcm and poly(C) followed by NaBH 4 reduction provided a sample with residual ATPase activity (12%). Mass spectrometric analysis indicated the presence of two proteins in an approximate 1.2:1 ratio, whose masses corresponded to wild-type Rho (47,010 Da) and lysine-modified Rho (47,417 Da), respectively. Trypsin digestion of the Rho sample followed by high performance liquid chromatography separation and tandem mass spectrometry analysis identified the site of modification as Lys 181 within the combined tryptic fragment, Gly-Leu-Ile-Val-Ala-Pro-Pro-Lys-Ala-Gly-Lys (residues 174 -184). Similar analysis of a lesser modified sample (following incubation with inclusion of ATP) showed that addition had again occurred at Lys 181 . These findings provide the first structural information concerning the site of Bcm binding in Rho.Bicyclomycin (Bcm) 1 is a commercial antibiotic of novel structure (1-8). The primary site of action in Escherichia coli is unique and has been identified as the transcription termination factor, Rho (9). Rho is composed of six identical 47-kDa proteins of 419 amino acids (10) and exists in a planar, hexagonal (11-13) arrangement of proposed C 6 (14) or D 3 (15) geometry. Rho-dependent transcription termination sites occur at the ends of transcription units, at regulatory points before or between genes, and within genes (16). Transcription termination begins with the recognition and binding of Rho to discrete regions (rut sites) within the newly synthesized RNA chains (17). Binding is governed by a general requirement for a cytosine-rich sequence, and it extends across all six subunits of the functional protein, encompassing approximately 80 nucleotides (17)(18)(19)(20). Rho then extends its interaction toward the 3Ј end of the RNA polymerase elongation complex in a process coupled to ATP hydrolysis. The Rho-mediated release of RNA from the transcription bubble results from a destabilization of the complex, believed to be caused, in part, by the ATPase-dependent 5Ј 3 3Ј RNA-DNA helicase action of the protein (21).Details of the Bcm-Rho interaction have emerged in recent investigations (9,(22)(23)(24). Chemical studies have shown that nucleophilic amino acids readily react with Bcm, consistent with sever...

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Section: Methodsmentioning

confidence: 99%

Evidence for the Location of Bicyclomycin Binding to theEscherichia coli Transcription Termination Factor Rho

Riba¹,

Gaskell²,

Cho

et al. 1998

Journal of Biological Chemistry

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“…Molecular masses are based on the following atomic weights of the elements: C ϭ 12.011, H ϭ 1.00794, n ϭ 14.00674, O ϭ 15.9994 and S ϭ 32.066 (25). The raw ESI-MS spectra were processed using a maximum entropy (MaxEnt) based approach (26,27) employing the MemSys5 program (MaxEnt Solutions Ltd., Cambridge, UK) incorporated as part of the VG Mass Lynx software suite on a 60 MHz Pentium PC supplied with the spectrometer.…”

Section: Methodsmentioning

confidence: 99%

The Multi-hemoglobin System of the Hydrothermal Vent Tube Worm Riftia pachyptila

Zal

Lallier

Green³

et al. 1996

Journal of Biological Chemistry

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The deep-sea tube worm Riftia pachyptila Jones possesses a complex of three extracellular Hbs: two in the vascular compartment, V1 (ϳ3500 kDa) and V2 (ϳ400 kDa), and one in the coelomic cavity, C1 (ϳ400 kDa). These native Hbs, their dissociation products and derivatives were subjected to electrospray ionization mass spectrometry (ESI-MS). The data were analyzed by the maximum entropy deconvolution system. We identified three groups of peaks for V1 Hb, at ϳ16, 23-27, and 30 kDa, corresponding to ( Several models have been proposed for the quaternary structure of annelid hexagonal bilayer hemoglobins (Hbs) 1 (1-5) and vestimentifera Hbs (6) but no definitive agreement exists. However, it is well established that HBL Hbs have a hexagonal symmetry and consist of two types of chains, globin-chains (ϳ17,000 Da) accounting for approximately 70% of total mass and heme-deficient linker chains (ϳ24,000 -32,000 Da) necessary for assemblage into the HBL structure (7). A detailed understanding of this molecular structure requires the knowledge of: (i) the molecular mass of the native molecule; (ii) the number and proportions of all constitutive polypeptide chains; (iii) the number and relation between subunits; and (iv) the determination of linker proportions. In a companion study (8) we have confirmed that Riftia pachyptila (Jones), a vestimentiferan living around deep-sea hydrothermal vents (9 -11), possesses three hemoglobins, two of them dissolved in the vascular blood (V1 and V2), and one in the coelomic fluid (C1). Their molecular weights have been determined by scanning transmission electron microscopy mass mapping (STEM) and by multi-angle laser light scattering (MALLS). Both methods yielded approximately the same molecular weights with mass significantly higher than the literature data for V1. V1, V2, and C1 had M r of 3396 Ϯ 540 ϫ 10 3

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“…1 ml of a sample at 1 mg ml À1 in 0.1%(v/v) formic acid was fractioned by reverse-phase chromatography using a gradient from 3 to 70%(v/v) acetonitrile with 0.1%(v/v) formic acid on a BEH C4 column (1.7 mm, 100 Â 100 mm). The acquired MS data were processed using a maximum-entropy technique (MaxEnt) to obtain a deconvoluted spectrum (Ferrige et al, 1991).…”

Section: Purification Of Ftl Sds-page and Esi Massspectrometric Analmentioning

confidence: 99%

Crystallization and preliminary X-ray diffraction studies of frutalin, an α-D-galactose-specific lectin fromArtocarpus incisaseeds

Monteiro-Moreira

Pereira²,

Neto

et al. 2015

Acta Cryst Sect F

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Frutalin is an -d-galactose-specific carbohydrate-binding glycoprotein with antitumour properties and is a powerful tool for tumour biomarker discovery. The crystallization and preliminary X-ray diffraction analysis of this lectin, which was isolated from Artocarpus incisa seeds, are reported here. Frutalin was purified and submitted to mass-spectrometric analysis. Diverse masses at approximately 16 kDa were observed in the deconvoluted spectra, which support the presence of isoforms. The best frutalin crystals were grown within a week in 0.1 M citric acid pH 3.5 which contained 25% PEG 3350 as a precipitant at 293 K, and diffracted to a maximum resolution of 1.81 Å . The monoclinic crystals belonged to space group I2, with unit-cell parameters a = 76.17, b = 74.56, c = 118.98 Å , = 96.56 . A molecular-replacement solution was obtained which indicated the presence of four monomers per asymmetric unit. Crystallographic refinement of the structure is in progress.

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Maximum entropy deconvolution in electrospray mass spectrometry

Cited by 152 publications

References 1 publication

Evidence for the Location of Bicyclomycin Binding to theEscherichia coli Transcription Termination Factor Rho

Evidence for the Location of Bicyclomycin Binding to theEscherichia coli Transcription Termination Factor Rho

The Multi-hemoglobin System of the Hydrothermal Vent Tube Worm Riftia pachyptila

Crystallization and preliminary X-ray diffraction studies of frutalin, an α-D-galactose-specific lectin fromArtocarpus incisaseeds

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