US 5,885,841 A · System and methods for qualitatively and quantitatively comparing complex admixtures using single ion chromatograms derived from spectroscopic analysis of such admixtures

Abstract

System for analyzing a protein sample, comprising a reactor vessel, a chromatographic column, a mass spectrometer, and a computer system. The reactor vessel comprises an enzyme activity capable of digesting the protein sample in order to provide a plurality of peptide digests, an inlet port for receiving the protein to be digested, and an exit port for discharging the peptide digests. The chromatographic column comprises a chromatographic medium capable of chromatographically fractionating the peptide digests as the peptide digests are eluted through the column, wherein the chromatographic column comprises an inlet port for receiving the peptide digests, said inlet port being in flow communication with the exit port of the reactor vessel, and wherein the chromatographic column comprises an exit port for discharging an effluent comprising the chromatographically fractionated peptide digests. The mass spectrometer is capable of generating a mass spectroscopic data set comprising data from which a first plurality of selective ion chromatograms for the fractionated peptide digests can be generated. The mass spectrometer has an inlet port for receiving the chromatographically fractionated peptide digests. The inlet port is in flow communication with the exit port of the chromatographic column. The computer system is operationally coupled to the mass spectrometer such that the computer system is capable of analyzing the mass spectroscopic data set. The computer system comprises programming enabling the computer system to analyze protein samples using selective ion chromatograms derived from the mass spectroscopic data set.

Claims55

Dependent claims

An apparatus for analyzing a protein sample, comprising:

(a) a reactor vessel comprising an enzyme activity capable of digesting the protein sample in order to provide a plurality of peptide digests, an inlet port for receiving the protein to be digested, and an exit port for discharging the peptide digests;
(b) a chromatographic column comprising a chromatographic medium capable of chromatographically fractionating the peptide digests as the peptide digests are eluted through the column, wherein the chromatographic column comprises an inlet port for receiving the peptide digests, said inlet port being in flow communication with the exit port of the reactor vessel, and wherein the chromatographic column comprises an exit port for discharging an effluent comprising the chromatographically fractionated peptide digests;
(c) a mass spectrometer capable of generating a mass spectroscopic data set, wherein said mass spectrometer has an inlet port for receiving the chromatographically fractionated peptide digests, said inlet port being in flow communication with the exit port of the chromatographic column;
(d) a computer system operationally coupled to the mass spectrometer such that the computer system is capable of analyzing said mass spectroscopic data set, wherein the computer system comprises a program comprising:
(i) instructions enabling said computer system to compute a plurality of selective ion chromatograms, wherein said selective ion chromatograms are derived from said mass spectroscopic data set;
(ii) instructions enabling said computer system to compute a plurality of peak areas of said selective ion chromatograms;
and
(iii) instructions enabling said computer to calculate at least one value indicative of the abundance of at least one of the peptide digests, wherein said value is derived from information comprising at least two peak areas selected from a peak area of an intended form of a peptide digest, a peak area of a first variant of said intended form of a peptide digest, a peak area of a second variant of said intended form of a peptide digest, and a peak area of a reference peptide digest.

5 more elements

An apparatus for comparing a first protein sample to a second protein sample, comprising:

(a) a reactor vessel comprising an enzyme activity capable of digesting each protein sample in order to provide a plurality of peptide digests corresponding to each of the samples, an inlet port for receiving the protein sample to be digested, and an exit port for discharging the corresponding peptide digests;
(b) a chromatographic column comprising a chromatographic medium capable of chromatographically fractionating the peptide digests resulting from the digestion of each protein sample, wherein the chromatographic column comprises an inlet port for receiving the peptide digests, said inlet port being in flow communication with the exit port of the reactor vessel, and wherein the chromatographic column comprises an exit port for discharging an effluent comprising the chromatographically fractionated peptide digests;
(c) a mass spectrometer capable of generating a mass spectroscopic data set, wherein said mass spectrometer has an inlet port for receiving the chromatographically fractionated peptide digests, said inlet port being in flow communication with the exit port of the chromatographic column;
(d) a computer system operationally coupled to the mass spectrometer such that the computer system is capable of analyzing said mass spectroscopic data set generated for each plurality of peptide digests, wherein the computer system comprises a program comprising:
(i) instructions enabling the computer system to compute a first plurality of selective ion chromatograms corresponding to the plurality of digests of the first protein sample and a second plurality of selective ion chromatograms corresponding to the plurality of digests of the second protein sample, wherein the first plurality of selective ion chromatograms is derived from the mass spectroscopic data set generated for the peptide digests of the first protein sample, and the second plurality of selective ion chromatograms is derived from the mass spectroscopic data set generated for the peptide digests of the second protein sample;
(ii) peak picking instructions enabling the computer system to automatically locate chromatographic peaks of the first and second pluralities of selective ion chromatograms;
(iii) instructions enabling the computer system to automatically match peaks between the first and second pluralities of selective ion chromatograms in order to identify matching peak pairs of the first and second pluralities of selective ion chromatograms;
and
(iv) instructions enabling the computer system to output results of said peak matching.

6 more elements

A method of comparing a protein sample to a reference protein, comprising the steps of:

(a) providing a predefined list of a plurality of peptides resulting from digestion of the reference protein by an enzyme activity, wherein said predefined list comprises at least two peptide digests selected from an intended form of a peptide digest, a first variant of said intended form of a peptide digest, a second variant of said intended form of a peptide digest, and a reference peptide digest, and wherein each of said peptides is characterized by a mass interval and a retention time interval for which each peptide is expected to be detected by a mass spectrometer;
(b) characterizing the reference protein sufficiently such that the relative abundance of one or more of said plurality of peptides relative to one or more other peptides of the reference protein can be determined;
(c) digesting the protein sample into a plurality of peptides using the enzyme activity;
(d) fractionating the plurality of peptides resulting from digestion of the protein sample;
(e) subjecting the fractionated peptides to mass spectroscopic analysis using a mass spectrometer in order to provide a mass spectroscopic data set from which a plurality of selective ion chromatograms for the fractionated peptide digests can be computed;
(f) computing a plurality of selective ion chromatograms wherein the selective ion chromatograms are derived from the mass spectroscopic data set and wherein each of the selective ion chromatograms is computed for a mass interval corresponding to a mass interval of one of the plurality of listed peptides resulting from digestion of the reference protein;
(g) calculating a plurality of peak areas of the selective ion chromatograms, wherein each peak area is calculated over a retention time interval corresponding to a retention time interval of one of the plurality of listed peptides resulting from digestion of the reference protein;
(h) calculating a value indicative of the relative abundance of at least one fractionated peptide to the extent such fractionated peptide is detected by the mass spectrometer, wherein said relative abundance value is derived from information comprising a plurality of said peak areas of the selective ion chromatograms;
and
(i) comparing said value of said at least one peptide to the corresponding relative abundance value of the corresponding at least one peptide of the reference protein.

7 more elements

An apparatus for comparing a first admixture of organic species to a second admixture of organic species, comprising:

(a) a chromatographic column comprising a chromatographic medium capable of chromatographically fractionating the organic species of each admixture, wherein the chromatographic column comprises an inlet port for receiving the admixtures, and wherein the chromatographic column comprises an exit port for discharging an effluent comprising the chromatographically fractionated organic species of each admixture;
(b) a mass spectrometer capable of generating a mass spectroscopic data set comprising data from which plurality of selective ion chromatograms for each fractionated admixture of organic species can be generated, wherein said mass spectrometer has an inlet port for receiving the chromatographically fractionated admixtures, said inlet port being in flow communication with the exit port of the chromatographic column;
(c) a computer system operationally coupled to the mass spectrometer such that the computer system is capable of analyzing said mass spectroscopic data set generated for each fractionated admixture, wherein the computer system comprises a program comprising:
(i) instructions enabling the computer system to compute a first plurality of selective ion chromatograms corresponding to the first organic admixture and a second plurality of selective ion chromatograms corresponding to the second admixture, wherein the first plurality of selective ion chromatograms is derived from the mass spectroscopic data set generated for the first admixture, and the second plurality of selective ion chromatograms is derived from the mass spectroscopic data set generated for the second admixture;
(ii) peak picking instructions enabling the computer system to automatically locate chromatographic peaks of the first and second pluralities of selective ion chromatograms;
(iii) instructions enabling the computer system to automatically match peaks between the first and second pluralities of selective ion chromatograms in order to identify matching peak pairs of the first and second pluralities of selective ion chromatograms;
and
(iv) instructions enabling the computer system to output results of said peak matching.

5 more elements

A method of comparing a sample admixture comprising a plurality of organic species to a reference admixture, comprising the steps of:

(a) providing a list of a plurality of organic species present in the reference admixture, wherein each of said organic species is characterized by a mass interval and a retention time interval for which each organic species is expected to be detected by a mass spectrometer;
(b) characterizing the reference admixture sufficiently such that the relative abundance of one or more of said organic species relative to one or more other organic species of the reference admixture can be determined;
(c) fractionating the organic species of the admixture sample;
(d) subjecting the fractionated organic species to mass spectroscopic analysis using a mass spectrometer in order to provide a mass spectroscopic data set from which a plurality of selective ion chromatograms for the fractionated organic species can be computed;
(e) computing a plurality of selective ion chromatograms, wherein the selective ion chromatograms are derived from the mass spectroscopic data set and wherein each of the selective ion chromatograms is computed for a mass interval corresponding to a mass interval of one of the plurality of listed organic species of the reference admixture;
(f) calculating a plurality of peak areas of the selective ion chromatograms, wherein each peak area is calculated over a retention time interval corresponding to a retention time interval of one of the organic species of the reference admixture;
(g) calculating a value indicative of the relative abundance of at least one fractionated organic species of the admixture sample to the extent such fractionated organic species is detected by the mass spectrometer, wherein said relative abundance value is derived from information comprising a plurality of said peak areas of the selective ion chromatograms;
and
(h) comparing said value of said at least one organic species of the admixture sample to the corresponding relative abundance value of the corresponding at least one organic species of the reference admixture.

6 more elements

Dependencies are inferred from the claim text.

System and methods for qualitatively and quantitatively comparing complex admixtures using single ion chromatograms derived from spectroscopic analysis of such admixtures

Life of this patent

System and methods for qualitatively and quantitatively comparing complex admixtures using single ion chromatograms derived from spectroscopic analysis of such admixtures

Life of this patent

Abstract

Claims55