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US 2004/0002118 A1Published application
Method for determining mass altering moiety in peptides
OwnerZeev Smilanskyinventor
Life of this patent
Estimating the term…
Abstract
A method is described for determining the mass of a mass altering moiety, and for identifying a cleavage altering sequence, wherein the mass altering moiety or a cleavage altering sequence is present in an assayed peptide and is absent from a corresponding database peptide, or is present in a database peptide and is absent from an assayed peptide.
Claims37
A method for determining the mass of a mass altering moiety, which is present in an assayed peptide and is absent from a corresponding database peptide, or is present in a database peptide and is absent from an assayed peptide, the method comprising:
- (i) treating the assayed peptide with a first digestion agent to obtain a first digestion product comprising a plurality of first assayed fragments;
- and determining the mass spectrum of the digestion product to obtain one or more mass values of the individual first assayed fragments Mia;
- (ii) treating the assayed peptide with a further digestion agent to obtain a further digestion product comprising a plurality of further assayed fragments;
- and determining the mass spectrum of the further digestion product to obtain one or more mass values of the individual further assayed fragments Mja;
- (iii) optionally repeating step (ii) according to the number of different further digestion agents, obtaining mass values of the individual further assayed fragments Mka, Mla, Mma, etc.;
- (iv) optionally identifying the assayed peptide, in case of a peptide not identified earlier, by a suitable protein identification method;
- (v) obtaining masses Mit of the individual theoretical fragments of the database peptide corresponding to the assayed peptide, which fragments are obtained by the theoretical digestion of said database peptide with said first digestion agent;
- (vi) obtaining masses Mjt of the individual theoretical fragments of the database peptide corresponding to the assayed peptide, which fragments are obtained by the theoretical digestion of said database peptide with said further digestion agent;
- (vii) comparing each of Mia with each database value Mit, to obtain a plurality of differences Di=Mia−Mit and discarding all Di values lower than a predetermined threshold value to give a plurality of selected differences Di′;
- (viii) comparing each of Mja with each database value Mjt, to obtain a plurality of differences Dj=Mja−Mjt and discarding all Dj values lower than a predetermined threshold value to give a plurality of selected differences Dj′;
- (ix) comparing selected differences Di′ and Dj′, preferably comprising overlapping theoretical fragments, and identifying those which are essentially identical;
- and optionally repeating steps (vi) to (ix), according to the number of different further digestion agents, obtaining selected differences Dk′/Dl′/Dm′, etc. The required mass of said mass altering moiety is thereby defined by said essentially identical Di′/Dj′ values.
9 more elements
A method for identifying a cleavage altering sequence which is present in an assayed peptide and is absent from a corresponding database peptide, or is present in a database peptide and is absent from an assayed peptide, wherein said cleavage altering sequence alters a cleavage site for at least one digestion agent used in the assay, the method comprising the steps of:
- (i) treating the assayed peptide with a first digestion agent to obtain a first digestion product comprising a plurality of first assayed fragments;
- and determining the mass spectrum of the digestion product to obtain one or more mass values of the individual first assayed fragments Mia;
- (ii) treating the assayed peptide with a further digestion agent to obtain a further digestion product comprising a plurality of further assayed fragments;
- and determining the mass spectrum of the further digestion product to obtain one or more mass values of the individual further assayed fragments Mja;
- (iii) optionally repeating step (ii) according to the number of different further digestion agents, obtaining mass values of the individual further assayed fragments Mka, Mla, Mma, etc.;
- (iv) optionally identifying the assayed peptide, in case of a peptide not identified earlier, by a suitable protein identification method;
- (v) obtaining masses Mit of the individual theoretical fragments of the database peptide corresponding to the assayed peptide, which fragments are obtained by the theoretical digestion of said database peptide with said first digestion agent;
- (vi) obtaining masses Mjt of the individual theoretical fragments of the database peptide corresponding to the assayed peptide, which fragments are obtained by the theoretical digestion of said peptide with said further digestion agent;
- (vii) optionally repeating step (vi) according to the number of different further digestion agents, obtaining masses Mkt , Mlt, Mmt, etc., of the individual theoretical fragments.
- (viii) comparing each of Mia with each database value Mit, to obtain a plurality of differences Di=Mia−Mit;
- discarding all Mia and Mit for which at least one of the Di values is lower than a predetermined threshold value;
- and thus identifying orphan Mia that have no corresponding Mit, and orphan Mit that have no corresponding Mia;
- (ix) comparing each of Mja with each database value Mjt, to obtain a plurality of differences Dj=Mja−Mjt;
- discarding all Mja and Mjt for which at least one of the Dj values is lower than a predetermined threshold value;
- and thus identifying orphan Mja that have no corresponding Mjt, and orphan Mjt that have no corresponding Mja;
- (x) optionally repeating step (ix) according to the number of different further digestion agents, and thus identifying orphan Mka, Mia, Mma, etc., that have no corresponding Mkt, Mlt, Mmt, etc., and identifying orphan Mkt, Mlt, Mmt, etc. that have no corresponding Mka, Mla, Mma, etc;
- (xi) defining a first orphan region as the subset of the amino acid sequences of the database peptide which includes all the theoretical fragments corresponding to orphan Mit for said first digestion agent;
- defining a further orphan region as the subset of the amino acid sequences of the database peptide which includes all the theoretical fragments corresponding to orphan Mjt for said further digestion agent;
- optionally repeating this for further digestion agents Mkt etc.;
- and finally defining a peptide orphan region as the intersection of the first orphan region with all further orphan regions, thus consisting of a subset of sequences of the peptide that were not identified by any of the digestion agents;
- (xii) theoretically altering the amino acid sequence of said peptide orphan region, by adding, deleting or changing one or more amino acids thereof, to obtain altered database fragments;
- and calculating a set of theoretical values of masses Malt of said altered fragments;
- (xiii) comparing each Malt with an orphan Mia;
- orphan Mja, orphan Mka, etc. and selecting those Malt of which the difference from an orphan Mia, Mja, Mka etc. is smaller than a predetermined threshold value. Malt representing the correct change is selected based on a predetermined criterion, for example, confirmation by the largest number of different digestion agents;
- and thus identifying the amino acid sequence which is present only in the assayed peptide or in the database peptide as the altered database fragment contributing to said Malt.
22 more elements
A kit for determining a mass altering moiety and/or cleavage altering sequence of a peptide for use with mass spectroscopy, comprising two or more digestion agents, means for digesting peptides with the agents, and an instruction manual.
Dependencies are inferred from the claim text.