On-line cysteine modification for protein analysis: new probes for electrochemical tagging nanospray mass spectrometry

A series of electrogenerated selective electrophiles based on substituted benzoquinones has been characterized as tags for l-cysteine and cysteine residues in proteins. The electrophiles are generated electrochemically from the corresponding hydroquinones. It is shown from mass spectrometry analysis that the electrogenerated benzoquinone can tag the biomolecules. The rate constants pertaining to the addition of l-cysteine onto the electrogenerated benzoquinones have been determined using electrochemical techniques. The substitution patterns have been unraveled leading to the assessment of site-specific rate constants. It is shown that the rate constants are primarily dependent on the electronic nature of the substituents as expressed by the Hammett substitution constant. The apparent tagging yields observed for l-cysteine in nanospray mass spectrometry experiments do not correspond to the yields expected from the electrochemical study, as the ionisation efficiencies are highly dependent on the tag. Finally, the on-line tagging has been tested using β-lactoglobulin A and myoglobin. Based on these results, it is concluded that the tagging reaction is selective towards cysteine when it takes place in the nanospray interface. The results show that the methodology presented can be used for a rapid characterization and identification of reactive sites in biomolecules.     

Selective Electroanalytical Assay for Cysteine at a Boron Doped Diamond Electrode

The electrochemical oxidation of the cysteine‐quinone adduct has been examined as a means of providing an electroanalytical cysteine specific detection protocol. The appliance of square‐wave voltammetry allowed 0.5 μM as a limit of detection. The effects of various biologically relevant interferences including other thiols were studied and found to present no change in the voltammetric profile. The practical applicability and efficiency of the methodology was probed through the determination of cysteine concentration in growth tissue medium.     

Cystine and cysteine analysis as the same phenylthiocarbamyl derivatives by HPLC in protein hydrolyzates

Summary A new approach is described, and a novel explanation presented, for the high performance liquid chromatographic analysis of cystine and cysteine as their phenylthiocarbamyl derivatives. PTC cystine and cysteine have been eluted with the same retention times and molar responses, most probably due to electrophilic attack of phenylisothiocyanate on cystine resulting in the scission of the disulfide bond yielding two moles of cysteine. Further, total PTC cystine and cysteine have been measured both in model solutions and in standard protein hydrolyzates (lysozyme, bovine albumin, ribonuclease) with the same linearity as the other ineteen amino acids. The reproducibility of the measurements, at the 250–750 pmole level, proved to be 4.1% (Relative Standard Deviation %) or less.     

Ormosil Encapsulated Pyrroloquinoline Quinone‐Modified Electrochemical Sensor for Thiols

An organically modified sol‐gel glass (ORMOSIL) encapsulating pyrroloquinoline quinone (PQQ)‐modified electrode for the rapid, sensitive and simple determination of thiol‐containing compounds such as cysteine and glutathione is reported. The effect of applied potential, nature of thiol compound and pH on the response of the sensor was examined and optimum conditions were determined. The electrochemical responses and detection limits were found to be sensitive to the nature of thiols and pH. The electrochemical responses for cysteine and glutathione at an applied potential of ?0.2 V (vs. Ag/AgCl) were found to be linear with detection limits of 18 nM for cysteine and 36 nM for glutathione at pH 3.5, whereas the detection limits at pH 8.5 were 0.5 μM for cysteine and 1 μM for glutathione. The electrode retained 95% of the original response for 7 days when stored at 4 °C. The ORMOSIL‐encapsulated PQQ was also characterized by spectrophotometry. The absorbance measurement using 5,5′‐dithiobis(2‐nitrobenzoic acid) at 412 nm justify the PQQ‐mediated oxidation of glutathione whereas fluorescence measurements (excitation wavelength=380 nm; emission wavelength=480 nm) justify the successful encapsulation of PQQ in ORMOSIL matrix.     

Synthesis of a homotrifunctional conjugation reagent based on maleimide chemistry

A novel homotrifunctional conjugation reagent, 1,3,5-tris-(N-maleimidomethyl)benzene has been synthesized in high yield with minimum purification. The reactivity of this compound was examined by using l-cysteine as a nucleophile.     

饱和锌的粘土矿物吸附半胱氨酸的机理

用红外光谱、X射线衍射（XRD）和热分析研究了饱和过渡金属锌的粘土矿物吸附半胱氨酸的机理；XRD结果显示吸附了半胱氨酸后的粘土矿物的d(001)方向的距离明显增大，说明被吸附的半胱氨酸进入了粘土的层状结构之中；饱和了锌的粘土矿物吸附半胱氨酸有两种方式，其一为弱相互作用，另一种强相互作用；后一种方式吸附的半胱氨酸与粘土的过渡金属之间形成了配合结构，结合力比较强，在热作用下，它们一直到395℃左右才分解；强相互作用吸附的半胱氨酸主要是以去质子的两性离子和阴离子形式存在于粘土矿物上，与过渡金属形成螯合的六元环结构。     

Comparison of pulsed amperometric detection and integrated voltammetric detection for inorganic sulfur compounds in liquid chromatography

A Variety of potential–time waveforms are useful in pulsed electrochemical detection (PED) when applied for the amperometric detection of numerous polar organic compounds following their separation by liquid chromatography (LC). Here, we compare the waveforms for pulsed amperometric detection (PAD) and integrated voltammetric detection (IVD) applied for detection of organosulfur compounds at Au electrodes in acidic media. In PAD waveforms, electrodes response is measured at a constant detection potentials. In IVD waveforms, electrodes current is integrated throughout a fast cyclic scan of the detection potential. As a consequence of this difference in detection strategy, the background signal for IVD is significantly smaller for PAD in the detection of organosulfur compounds whose response mechanisms require the concomitant formation of surface oxides on Au electrodes. Furthermore, in comparison to Pad, IVD has a larger sensitivity and a diminished system peak from 0₂ dissolved in the sample. Use of a preadsorption step increases detection sensitivity in both PAD and IVD. The limit of detection (S/N=3)for cysteine in LC-IVD is ca. 6 nM for a 50-μl injection (i.e., 300 fmol) using a detection waveform that includes a 1000-ms preadsorption period.