Patent DE-102008043241-B3 (DE)
Title: Method for determining relative quantity of cysteine- and/or methionine-containing peptides, comprises providing a peptide isolate obtained from microorganisms and carrying out a fusion of peptide isolate with reference peptide isolate
Inventor: SCHMIDT FRANK
IPC: G01N33/68
Filed: 2008-10-28
Granted: 2010-04-15
Abstract: The method comprises providing a peptide isolate obtained from microorganisms that are cultivated from a 3>6>sulfur-containing sulfur source as renewable sulfur source, carrying out a fusion of peptide isolate with same protein quantity of a reference peptide isolates obtained from the microorganisms, and degrading the peptide compound of peptides from the fusion of the peptide isolates so that specific, reproducible cysteine- and/or methionine-containing peptides consist of peptide-fragments that optionally consist of sulfur isotopes ( 3>2>S or 3>6>S). The method comprises providing a peptide isolate obtained from microorganisms that are cultivated from a 3>6>sulfur-containing sulfur source as renewable sulfur source, carrying out a fusion of peptide isolate with same protein quantity of a reference peptide isolate obtained from the microorganisms, degrading the peptide compound of peptides from the fusion of the peptide isolates so that specific, reproducible cysteine- and/or methionine-containing peptides consist of peptide-fragments that optionally consist of sulfur isotopes ( 3>2>S or 3>6>S), mass spectrometrically measuring the degraded peptide fragment and determining the ratio of the signal intensities for a 3>2>S/ 3>6>S-peptide fragment pair, where the signal of the 3>6>S-peptide fragment is displaced per cysteine content and per methionine respectively to 4 u in the measured area as the corresponding sequence identical of 3>2>S-peptide fragment. A mass spectrometric determination of 3>6>S-peptide fragment is assigned a determined peptide with partially known peptide sequence in an additional step using peptide mass finger print (PMF) alogarithms. A selection of peptide fragments is carried out between the splitting and the mass spectrometric measurement. Only one peptide of the peptide isolate is subjected to degradation and further treatment. The microorganism is Pseudomonas putida, Mycobacteriumand/or Corynebacterium. The mass spectrometric measurement is matrix-assisted laser desorption/ionization (MALDI)-, MALDI/time-of-flight secondary ion mass spectrometer (TOF)-, MALDI/TOF/TOF-, electrospray ionization (ESI)-iron trap-, liquid chromatography (LC)-ESI-quadrupole-TOF-, Q-TRAP-MS(RTM: Hybrid linear ion trap mass spectrometer) or MS/MS measurement. In the production of peptide isolates or the reference peptide isolate, a protein reconditioning method selected from size exclusion chromatography, fractioned ammonium sulfate precipitation, fractioned extraction by phenol, ion exchange xhromatography and/or reversed-phase chromatography. The degradation is an enzyme-mediated degradation. The ratio of the signal intensities are determined for 3>2>S/ 3>6>S-peptide fragment pair that consists of cysteine- and/or methionine. The 3>6>sulfur-containing sulfur source is calcium sulfate (Ca 3>6>SO 4), magnesium sulfate (Mg 3>6>SO 4) and/or ferrous sulfate (Fe 3>6>SO 4).