Analysis of proteolytic processing by mass spectrometry
AssigneeKoninklijke Philips Electronics N.V.
InventorRalf Hoffman
Life of this patent
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Abstract
The present invention relates to the simultaneous analysis of samples for determining differential proteolytic processing based on isotopic labelling of N-terminal peptides, wherein the isotopic labelling is achieved by incorporation of 18O during enzymatic proteolysis.
Claims16
An in vitro method for investigation differences in proteolytic processing between two or more different samples comprising the steps of:
- a) modifying the amine of the N-terminus and of Lysine of the proteins in said samples,
- b) cleaving the modified proteins into peptides and simultaneously labelling each of the samples with either O or 18O,
- c) isolating N-terminal peptides,
- d) pooling of the labelled samples after step (b) or of the isolated N-terminal peptides of step (c),
- e) subjecting the N-terminal peptides to MS,
- f) selecting relevant peptide fractions for further analysis, and
- g) identifying peptides which are generated by proteolytic processing.
A kit of reagents comprising a set of two or more isobaric labelling reagents and H218O.
A device (100′) for analysis of two protein samples using isotopic labelling comprising two sample sources (101), a protein modification unit (103′) with a source of modifying reagent (104′), a labelling and protein cleavage unit (105) and corresponding label sources (107), a N-terminal peptide isolation unit (106), a separation unit (108), a mass spectrometer unit (109) and a data analysis unit (110).
A device (100) for multiplex analysis of protein samples using double labelling comprising at least two sample sources (101), a labelling unit (103) with at least two sources of labelling reagent (104), a labelling and protein cleavage unit (105) and corresponding label sources (107), a N-terminal peptide isolation unit (106) a separation unit (108), a mass spectrometer unit (109) and a data analysis unit (110).
Dependencies are inferred from the claim text.