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

Abstract

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.

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

“…Full scan MS spectra obtained by ESI showed signals from multiply charged ions of rHuINF ␣-2b and their adducts with one or more ions of trifluoroacetic acid from mobile phase (deconvoluted spectrum at m/z 19 265.73, 19 380.66, 19 493.52, etc.). Observed mass difference of 114 and 113 in deconvoluted spectra can be used as indication of good instrument calibration and accurate molecular mass determination [15]. Almost the same mass accuracy (m/z 19 265.63) was obtained by separate analysis performed on MALDI-TOF instrument with internal calibration.…”

“…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].…”

“…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].…”

Accelerated on‐column lysine derivatization and cysteine methylation by imidazole reaction in a deuterated environment for enhanced product ion analysis

“…To find the optimal conditions for lysine on‐column tagging two models of tryptic or Lys‐C digest were chosen: recombinant human interferon α ‐2b ( rHu INF α ‐2b)15 and recombinant human erythropoietin ( rHu EPO) 16. Protein rHu INF α ‐2b after tryptic or Lys‐C digestion produces very unstable peptides that can be easily oxidized.…”