Probing the oligomeric structure of an enzyme by electrospray ionization time-of-flight mass spectrometry.
نویسندگان
چکیده
Electrospray ionization time-of-flight (ESI-TOF) mass spectrometry was used to study the quaternary structure of 4-oxalocrotonate tautomerase (EC 5.3.2; 4OT), and four analogues prepared by total chemical synthesis. Wild-type 4OT is a hexamer of 62 amino acid subunits and contains no cysteine residues. The analogues were: (desPro1)4OT, a truncated construct in which Pro1 was deleted; (Cpc1)4OT in which Pro1 was replaced with cyclopentane carboxylate; a derivative [Met(O)45]4OT in which Met45 was oxidized to the sulfoxide; and an analogue (Nle45)4OT in which Met45 was replaced with norleucine. ESI of (Nle45)4OT, (Cpc1)4OT, and 4OT from solution conditions under which the native enzyme was fully active (5 mM ammonium bicarbonate buffer, pH 7.5) gave the intact hexamer as the major species detected by TOF mass spectrometry. In contrast, analysis of [Met(O)45]4OT and (desPro1)4OT under similar conditions yielded predominantly monomer ions. The ESI-TOF measurements were consistent with structural data obtained from circular dichroism spectroscopy. In the context of kinetic data collected for 4OT and these analogues, ESI-TOF mass spectrometry also provided important evidence for the structural and mechanistic significance of the catalytically important Pro1 residue in 4OT.
منابع مشابه
Design, Construction and Calibration of a Laser Ionization Time-of-Flight Mass Spectrometer
A time-of-flight mass spectrometer (TOF-MS) developed in our laboratory at Isfahan University of Technology is described here. The TOF-MS instrument uses laser as the ionization source which provides an opportunity to investigate the ions formed in laser ablation or desorption. The TOF-MS has an ionization chamber containing an accelerator and an ion lens to focus the ions into a one meter line...
متن کاملA Rapid, Simple, Liquid Chromatographic-Electrospray Ionization, Ion Trap Mass Spectrometry Method for the Determination of Finasteride in Human Plasma and its Application to Pharmacokinetic Study
A fast, accurate, sensitive, selective and reliable method using reversed-phase high performance liquid chromatography coupled to electrospray ionization ion trap mass spectrometry was developed and validated for the determination of finasteride in human plasma. After protein precipitation with perchloric acid, satisfactory separation was achieved on a Zorbax Eclipse® C8 analytical column u...
متن کاملA Rapid, Simple, Liquid Chromatographic-Electrospray Ionization, Ion Trap Mass Spectrometry Method for the Determination of Finasteride in Human Plasma and its Application to Pharmacokinetic Study
A fast, accurate, sensitive, selective and reliable method using reversed-phase high performance liquid chromatography coupled to electrospray ionization ion trap mass spectrometry was developed and validated for the determination of finasteride in human plasma. After protein precipitation with perchloric acid, satisfactory separation was achieved on a Zorbax Eclipse® C8 analytical column u...
متن کاملRapidly switchable matrix-assisted laser desorption/ionization and electrospray quadrupole-time-of-flight mass spectrometry for protein identification.
We describe a new interface for a prototype quadrupole-quadrupole-time-of-flight (TOF) mass spectrometer (Centaur, Sciex) that allows rapid switching between electrospray ionization (ESI) and matrix-assisted laser desorption/ionization (MALDI) modes of operation. Instrument performance in both modes is comparable (i.e., resolution approximately 10,000 FWHM, mass accuracy <10 ppm, sensitivity ap...
متن کاملQuantification of Melittin in Iranian Honey Bee (Apis mellifera meda) Venom by Liquid Chromatography-electrospray Ionization-ion Trap Tandem Mass Spectrometry (LC-ESI-IT-MS/MS)
The current research aimed to quantify melittin (MEL) in Iranian honey bee (Apis mellifera meda) venom. To this end, a liquid chromatography-electrospray ionization-ion trap tandem mass spectrometry (LC-ESI-IT-MS/MS) approach was employed. Melittin is the main toxic peptide of honey bee venom with various biological and pharmacological activities. It was extracted with...
متن کاملذخیره در منابع من
با ذخیره ی این منبع در منابع من، دسترسی به آن را برای استفاده های بعدی آسان تر کنید
برای دانلود متن کامل این مقاله و بیش از 32 میلیون مقاله دیگر ابتدا ثبت نام کنید
ثبت ناماگر عضو سایت هستید لطفا وارد حساب کاربری خود شوید
ورودعنوان ژورنال:
- Proceedings of the National Academy of Sciences of the United States of America
دوره 93 14 شماره
صفحات -
تاریخ انتشار 1996