Mass spectrometric analysis of recombinant human α-2 interferon

Padrón, Gabrìel; Besada, Vladimir; Agraz, Alberto; Quiñones, Y.; Herrera, Luís; Shimonishi, Yasutsugu; Takao, Toshifumi

doi:10.1016/s0003-2670(00)84101-x

Cited by 12 publications

(6 citation statements)

References 8 publications

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“…The re-sulting proteins are identical to the natural human ones. hrIFNa2 contains the two disulfide bonds correctly positioned, as vcrified by MS [19]. Samples were dissolved in 0.1 M ammonium acetate, pH 5.5 or 0.05 M glycine/NaOH, pH 9.4. Carboxymethylation of the protein was performed according to a modification of the published procedure [20].…”

Section: Protein Preparationmentioning

confidence: 99%

Time‐resolved fluorescence study of human recombinant interferon α₂

Vincent

Sierra

Berberan‐Santos

et al. 1992

European Journal of Biochemistry

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Human recombinant interferon t12 belongs a to family of proteins active against a wide range of viruses. It contains two tryptophan residues located at positions 77 and 141 in the peptide sequence. The fluorescence cmission spectrum of these tryptophan residues displays a maximum at 335 nm. The fluorescence intensity decay is described by one broad excited-state-lifetime population centered around a value of 1.7 ns (full width at half maximum, 1.5 ns). These observations suggest that in the native protein, both tryptophan residues emit from similar environments, not directly exposed to the surrounding solvent. The anisotropy decay is essentially biexponential. The correlation-time value characterizing the Brownian rotation of the protein varies linearly with the viscosity/temperature ratio. The calculated hydrodynamic volumes are compatible with the existence of a dimer and a tetramer, at pH 5.5 and 9.4, respectively. Addition of urea at pH 5.5 disrupts the dimer and modifies to some extent the excited-state-lifetime distribution which becomes more heterogeneous. Disulfidebond reduction also dissociates the dimer and leads to a highly heterogeneous fluorescence-intensity decay with four excited-state-lifetime populations. An opening of the local structure in the Trp region of the protein is likely to occur in these conditions. The fast-anisotropy-decay components can be due to either fast rotation or energy transfer between the indoles. Close proximity of the two Trp residues (less than 1 nm) is suggested from steady-state and time-resolved fluorescence-anisotropy measurements in vitrified medium [95% (by mass) glycerol at -38 "C]. This suggestion is in agreement with the recently published three-dimensional structure of the homologous protein murine interferon j3 [Senda, T., Shimazu, T., Matsuda. S. Kawano, G., Shimizu, H., Nakamura, K. T. & Mitsui, Y.(1992) EMBO J . 11,3193-32011.Human (h) recombinant (r) interferon a2 (IFNaJ is a 19-kDa protein of 165 amino acids belonging to the cytokine family and is very active in conferring protection against a wide range of viruses [1, 21. This is therefore an extremely important protein in pathological agression. Unlike that of INFy [3], its three-dimensional structure, as well as important physicochemical properties, remain unknown, despite attempts to obtain crystals of sufficient quality for X-ray analysis [4]. Several models of protein folding have been proposed [5][6][7][8][9] on the basis of sequence similarity with interleukin 2 [lo], and on the relative percentage of x-helical structure deduced from the CD spectrum [11]. These @-helices are assumed to fold to preserve an hydrophobic core [6,7,9]. The recently published crystalline structure of the closely related protein murine (m) IF",' [12, 131 provides some support for these models. One of the bcst-characterized structural feature of the IFNa family of proteins [12], but which is absent in IFNB, is thc disulfide bond occurring between Cys29 and Cys139. A second disulfide bridge is also present between Cysl an...

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Section: Protein Preparationmentioning

confidence: 99%

Time‐resolved fluorescence study of human recombinant interferon α₂

Vincent

Sierra

Berberan‐Santos

et al. 1992

European Journal of Biochemistry

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“…Digestion with trypsin did not produce such an extensive oxidation. According to the previous studies, oxidation of methionine-containing peptide fragments as a result of proteolysis was not reported [10,11,15]. Under mild oxidizing conditions (0.05% hydrogen peroxide, v/v) in less then 1 h, rHuINF ␣-2b forms monomethionine sulfoxide variant at position Met111 [1,27].…”

Section: Very Low Intensity Signalsmentioning

confidence: 99%

“…Different ionization techniques were used in previous rHuINF ␣-2b studies, e.g. fast atom bombardment [10], 252 Cf plasma desorption, Cs + liquid secondary ion mass spectrometry [11] and thermospray [12]. However, mild ionization gained by matrix-assisted laser desorption/ionization [13,14] and ESI (electrospray ionization) and TOF analyzers (time-of-flight analyzers) enabled protein analysis with high mass accuracy at the femtomole level [15].…”

Section: Introductionmentioning

confidence: 99%

Evaluation of recombinant human interferon α-2b structure and stability by in-gel tryptic digestion, H/D exchange and mass spectrometry

Cindrić¹,

Galić²,

Vuletić³

et al. 2006

Journal of Pharmaceutical and Biomedical Analysis

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“…In each LC-MS analysis, the observed m/z value of interferon a-2b and TFA adducts differed by up to 1 amu from the calculated average m/z value ( Figure 1). The absence of a disulphide bridge would be observed through the mass value obtained in the experiment, and any incorrect disulphide bridges in protein would be observed through the shift in chromatographic retention time ( Figure 2) [4]. In MALDI-TOF MS analysis (Figure 3), the obtained mass value was inaccurate up to 5 amu due to the instrument's poor resolution when operating in linear mode [7].…”

Section: Maldi-tof Ms Conditionsmentioning

confidence: 99%

“…Peptide mapping of interferon a-2b can confirm the primary structure and provide more information about stability and conformational changes in the protein [4]. Estimation of the exact mass of the protein and each peptide fragment after trypsin digestion plays an important role in protein characterisation [5 -7].…”

Section: Introductionmentioning

confidence: 99%

Characterisation of interferon α‐2b by liquid chromatography and mass spectrometry techniques

Cindrić¹,

Vuletić

2003

J of Separation Science

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Characterisation of interferon a-2b by liquid chromatography and mass spectrometry techniquesInterferon a-2b produced by Escherichia coli consists of 165 amino acids and contains two disulphide bonds; its purity was confirmed by LC-UV (DAD)-FLD and LC-MS techniques. A C 4 column was used with UV detection at 214 nm; diode array detector (DAD) spectra were recorded from 200 -400 nm and fluorescence detection was performed at specific wavelengths of trypthophan emission and excitation. Peptide mapping was performed with trypsin. Peptides produced by trypsin digestion were analysed by LC-UV (DAD)-FLD, LC-MS, and LC-MS/MS using a C 18 column. Amino acid sequence coverage was about 95%. UV spectra in the range from 200 nm to 400 nm, emission (Em) and excitation (Ex) spectra of each separated peptide were additionally compared with spectra of the same peptide produced by digestion of European Pharmacopaeia interferon a-2b standard (spectral matching). The chromatogram of any interferon a-2b (drug substance or certificated standard) sample produced in the same manner with the same amino acid composition should be similar to the chromatogram obtained by the method described in this paper. Molecular masses of peptides were obtained from MS experiments and MS/MS experiments gave additional structural information. The molecular mass of interferon a-2b was obtained by MALDI-TOF MS analysis in linear mode, with an accuracy comparable to the theoretical average mass l 5 atomic mass units. The molecular mass was obtained from the deconvoluted ESI mass spectrum.

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Mass spectrometric analysis of recombinant human α-2 interferon

Cited by 12 publications

References 8 publications

Time‐resolved fluorescence study of human recombinant interferon α₂

Time‐resolved fluorescence study of human recombinant interferon α₂

Evaluation of recombinant human interferon α-2b structure and stability by in-gel tryptic digestion, H/D exchange and mass spectrometry

Characterisation of interferon α‐2b by liquid chromatography and mass spectrometry techniques

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Mass spectrometric analysis of recombinant human α-2 interferon

Cited by 12 publications

References 8 publications

Time‐resolved fluorescence study of human recombinant interferon α2

Time‐resolved fluorescence study of human recombinant interferon α2

Evaluation of recombinant human interferon α-2b structure and stability by in-gel tryptic digestion, H/D exchange and mass spectrometry

Characterisation of interferon α‐2b by liquid chromatography and mass spectrometry techniques

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Time‐resolved fluorescence study of human recombinant interferon α₂

Time‐resolved fluorescence study of human recombinant interferon α₂