Ion detector of time-of-flight mass spectrometer with registration of leading and trailing edges.

The accuracy of the ion flight time measurement in the time-of-flight mass spectrometer is critical to achieving high resolution. The pulse amplitude variation of the detector pulses leads to the registration time spread at a given pulse detection threshold. This time spread can be eliminated by determining the position of the pulse apex. To determine the position of the pulse apex, the output of the ion detector is fed simultaneously to the two channels of the time-to-digital converter. In this case, the first channel is set to register the leading edge, and the second channel is set to register the trailing edge of the pulse. Using a simple processing of the received data, the position of the pulse tip is determined. Thus, the dependence of the temporal position of the peak on the pulse amplitude is largely eliminated. Examples are given, and the efficiency of using this algorithm to increase the resolution of time-of-flight mass spectral peak registration is demonstrated.

Note: Isolated high-voltage thermo-compensated DC power supply for mass spectrometry.

The design of a highly stable isolated high voltage power supply is proposed and developed. The power supply is characterized by good temperature stability (<15 ppm/°C) and very low ripple level (<7 ppm) which were reduced by using a sinusoidal voltage in the transformer conversion. We anticipate that the power supplies of this type can find their utility in modern high-resolution mass spectrometers, particularly in time-of-flight and Orbitrap-type instruments providing a high resolution of ∼100 000 FWHM and high accuracy of m/z determination.

Hetero- and auto-activation of recombinant glutamyl endopeptidase from Bacillus intermedius.

Glutamyl endopeptidase from Bacillus intermedius (BIGEP) is a secretory serine proteinase specifically hydrolyzing peptide bonds involving alpha-carboxyl groups of glutamic and aspartic acids. In this work, different BIGEP forms (full-length precursor, precursor without signal peptide and mature part) were expressed in Escherichia coli and the process of enzyme maturation was studied in vitro. BIGEP precursor renaturation leads to autocatalytic hydrolysis of the propeptide at Glu(-16). At the same time, the enzyme activation requires the complete removal of the prosequence by other proteinases. The mature part of BIGEP cannot be activated, which indicates that the propeptide is required for the active protein formation. The data obtained allowed us to apply directed mutagenesis of the processing site to obtain a BIGEP form that matured autocatalytically. This approach makes it possible to produce the enzyme without extrinsic proteinases, which is a prerequisite for using it in limited hydrolysis of proteins and peptides.

Electrospray mass spectrometric study of melittin trypsinolysis by a kinetic approach.

The kinetics of tryptic digestion of melittin was studied by combined electrospray ionization time-of-flight mass spectrometry and high-performance liquid chromatography. The ratios of the kinetic constants for cleavage of the peptide bonds that are susceptible to trypsin action were determined. It is shown that trypsin does not manifest affinity for the hydrolysis of the peptide bonds inside the Arg,Lys cluster series as efficiently as it cleaves the peptide at the separately localized Lys residue. This feature demonstrates clearly the advantage of the kinetic approach to tryptic mapping of proteins. The kinetic approach allows the determination of not only discrete structural segments in protein structure but also their relative locations and their amino acid sequences. Using the melittin digests and some artificially prepared amino acids and dipeptides mixtures as models, it is shown that the presence and nature of basic amino acids predetermines the charge states of the molecules analyzed by electrospray but not the yields of their ions. The aliphatic parts of the molecules seem to be more important in determining the actual ion yields.