Method and apparatus for identifying proteins in mixtures

Abstract

Protein identification in a complex sample begins by selecting a database having proteins likely to be in the sample. In-silico digestion is performed and a target peptide is selected from produced peptides. The masses of the Y- and B-ion fragments of the target peptide are determined. These masses are used to search previously obtained low- and high-energy AMRTs obtained from LC/MS analysis of the complex sample for masses on the list. Any mass observed in the data within a detection threshold are considered a hit. If enough hits accumulate in a given retention time, the target peptide is identified as being in the sample. The list of peptides identified in the complex sample can be used to identify the proteins present in the sample, track the chromatographic retention times of peptides between samples, and quantitate the peptides and proteins present in complex samples.

1 - 25 . (canceled) 
     
     
         26 . A method for identifying proteins in a mixture comprising:
 selecting a target peptide having a target precursor;   determining masses associated with the target precursor and corresponding Y and B fragment ions of the target precursor;   performing an experiment using the mixture, said performing an experiment including:
 applying a low-energy mode in a mass spectrometer and obtaining first mass information and first chromatographic retention time information primarily associated with precursors; and 
 applying an elevated energy mode in the mass spectrometer and obtaining second mass information and second chromatographic retention time information primarily associated with fragments of the precursors; 
   comparing the associated masses of the target precursor and the corresponding Y and B fragment ions of the target precursor to the first mass information and the second mass information to determine mass matches;   determining chromatographic retention times of the mass matches; and   identifying, based on said mass matches and the chromatographic retention times of the mass matches, whether the target peptide is present in the mixture.   
     
     
         27 . The method of  claim 26 , wherein the low energy mode is applied by applying a low voltage in a collision cell of the mass spectrometer and the elevated energy mode is applied by applying a high voltage in the collision cell. 
     
     
         28 . The method of  claim 26 , further comprising switching between the low energy mode and the elevated energy mode in accordance with a protocol that alternates application of the low energy mode and the elevated energy mode with a sufficient frequency whereby each of the low energy mode and the elevated energy mode is applied a plurality of times during a chromatographic peak width. 
     
     
         29 . The method of  claim 26 , further comprising:
 digesting the mixture to obtain a peptide mixture; and   separating the peptide mixture by performing liquid chromatographic separation, wherein the separated peptide mixture is further processed using the mass spectrometer in said applying a low-energy mode and said applying an elevated energy mode.   
     
     
         30 . The method of  claim 26 , wherein a mass match is determined if any of said target precursor and said corresponding Y and B fragment ions of the target precursor have a corresponding mass in any of the first mass information and the second mass information. 
     
     
         31 . The method of  claim 30 , further comprising creating a detection chromatogram identifying a number of ions within each of a plurality of chromatographic retention time intervals. 
     
     
         32 . The method of  claim 31 , wherein the detection chromatogram is derived using Gaussian shaped peaks and each mass match is represented by a Gaussian shaped peak in the detection chromatogram. 
     
     
         33 . The method of  claim 31 , wherein the detection chromatogram is a histogram comprising a series of bins, a center of each of said bins corresponding to a chromatographic retention time and a width of said each bin corresponding to a chromatographic retention time interval, and wherein each mass match has a corresponding chromatographic retention time included in one of said bins having an associated count that is incremented by one for said each mass match. 
     
     
         34 . The method of  claim 26 , wherein the target peptide is a theoretical peptide selected from a database and the mass matches are determined in accordance with a search tolerance. 
     
     
         35 . The method of  claim 34 , wherein the search tolerance is automatically determined from data using statistics, or is user-specified. 
     
     
         36 . The method of  claim 34 , wherein said database is a specialized database having proteins likely to be found in the mixture. 
     
     
         37 . The method of  claim 26 , further comprising:
 determining a detection threshold; and   identifying whether a peptide is present based on whether a count of mass matches in a particular chromatographic retention time interval exceeds the detection threshold.   
     
     
         38 . The method of  claim 37 , further comprising:
 estimating a noise floor associated with the detection threshold; and   analyzing a significance of a peptide identification based on the count and the estimate of the noise floor.   
     
     
         39 . The method of  claim 37 , wherein said detection threshold varies with a complexity of the mixture. 
     
     
         40 . The method of  claim 37 , wherein the target peptide is identified as being in the mixture in accordance with a count based on mass matches to said corresponding Y and B fragment ions of the target precursor without a mass match to said target precursor. 
     
     
         41 . The method of  claim 26 , wherein all chromatographic peaks related to the target precursor have the same peak shape and peak width. 
     
     
         42 . The method of  claim 41 , wherein peak shape includes analysis of a peak's apex, up-slope inflection point and down-slope inflection point. 
     
     
         43 . A method for identifying proteins in a mixture comprising:
 selecting a target peptide having a target precursor;   determining masses associated with the target precursor and corresponding Y and B fragment ions of the target precursor;   performing an experiment using the mixture, said performing an experiment including:
 applying a low-energy mode in a mass spectrometer and obtaining first mass information and first chromatographic retention time information primarily associated with precursors; and 
 applying an elevated energy mode in the mass spectrometer and obtaining second mass information and second chromatographic retention time information primarily associated with fragments of the precursors; 
   searching, in accordance with a search tolerance, the first mass information and the second mass information to determine mass matches with any of the associated masses of the target precursor and the corresponding Y and B fragment ions of the target precursor;   determining chromatographic retention times of the mass matches; and   identifying, based on said mass matches and the chromatographic retention times of the mass matches, whether the target peptide is present in the mixture.   
     
     
         44 . The method of  claim 43 , further comprising switching between the low energy mode and the elevated energy mode in accordance with a protocol that alternates application of the low energy mode and the elevated energy mode with a sufficient frequency whereby each of the low energy mode and the elevated energy mode is applied a plurality of times during a chromatographic peak width. 
     
     
         45 . A system comprising:
 a mass spectrometer;   a database from which a target peptide is selected;   a computer coupled, to the database and an output of the mass spectrometer, said computer having code executing thereon which, when executed, causes the computer to perform a method, the method comprising:
 selecting the target peptide having a target precursor; 
 determining masses associated with the target precursor and corresponding Y and B fragment ions of the target precursor; 
 obtaining first mass information and first chromatographic retention time information primarily associated with precursors whereby the first mass information and the first chromatographic retention time information are acquired from data collected in accordance with applying a low energy mode in the mass spectrometer; 
 obtaining second mass information and second chromatographic retention time information primarily associated with fragments of the precursors whereby the second mass information and the second chromatographic retention time information are acquired from data collected in accordance with applying an elevated energy mode in the mass spectrometer; 
 comparing the associated masses of the target precursor and the corresponding Y and B fragment ions of the target precursor to the first mass information and the second mass information to determine mass matches; 
 determining chromatographic retention times of the mass matches; and 
 identifying, based on said mass matches and the chromatographic retention times of the mass matches, whether the target peptide is present in the mixture. 
   
     
     
         46 . The system of  claim 45 , wherein the low energy mode is applied by applying a low voltage in a collision cell of the mass spectrometer and the elevated energy mode is applied by applying a high voltage in the collision cell. 
     
     
         47 . The system of  claim 45 , wherein the mass spectrometer is switched between the low energy mode and the elevated energy mode in accordance with a protocol that alternates application of the low energy mode and the elevated energy mode with a sufficient frequency whereby each of the low energy mode and the elevated energy mode is applied a plurality of times during a chromatographic peak width. 
     
     
         48 . A system comprising:
 a mass spectrometer;   a database from which a target peptide is selected;   a computer coupled to the database and an output of the mass spectrometer, said computer having code executing thereon which, when executed, causes the computer to perform a method, the method comprising:
 selecting the target peptide having a target precursor; 
 determining masses associated with the target precursor and corresponding Y and B fragment ions of the target precursor; 
 obtaining first mass information and first chromatographic retention time information primarily associated with precursors whereby the first mass information and first chromatographic retention time information are acquired from data collected in accordance with applying a low energy mode in a mass spectrometer; 
 obtaining second mass information and second chromatographic retention time information primarily associated with fragments of the precursors whereby the second mass information and second chromatographic retention time information are acquired from data collected in accordance with applying an elevated energy mode in the mass spectrometer; 
 searching, in accordance with a search tolerance, the first mass information and the second mass information to determine mass matches with any of the associated masses of the target precursor and the corresponding Y and B fragment ions of the target precursor; 
 determining chromatographic retention times of the mass matches; and 
 identifying, based on said mass matches and the chromatographic retention times of the mass matches, whether the target peptide is present in the mixture. 
   
     
     
         49 . The system of  claim 48 , wherein the mass spectrometer is switched between, the low energy mode and the elevated energy mode in accordance with a protocol that alternates application of the low energy mode and the elevated energy mode with a sufficient frequency whereby each of the low energy mode and the elevated energy mode is applied a plurality of times during a chromatographic peak width. 
     
     
         50 . The system of  claim 49 , wherein the low energy mode is applied by applying a low voltage in a collision cell of the mass spectrometer and the elevated energy mode is applied by applying a high voltage in the collision cell. 
     
     
         51 . A method for identifying proteins in a mixture comprising:
 selecting a target peptide having a target precursor;   determining masses associated with the target precursor and corresponding Y and B fragment ions of the target precursor;   performing an experiment using the mixture, said performing an experiment including:
 applying a first energy mode in a mass spectrometer causing at least some fragmentation of one or more precursors; 
   obtaining first mass information and first chromatographic retention time information acquired from data collected in accordance with said first energy mode;   comparing the associated masses of the target precursor and the corresponding Y and B fragment ions of the target precursor to the first mass information to determine mass matches;   determining chromatographic retention times of the mass matches; and   identifying, based on said mass matches and the chromatographic retention times of the mass matches, whether the target peptide is present in the mixture.   
     
     
         52 . The method of  claim 51 , wherein the first energy mode is any of a low-energy mode used to obtain mass and chromatographic retention time information primarily associated with precursors, a high energy mode used to obtain mass and chromatographic retention time information primarily associated with fragments of the precursors, and a fixed energy mode using a voltage that is an intermediate value between a first voltage used in the low-energy mode and a second voltage used in the high energy mode. 
     
     
         53 . A system comprising:
 a mass spectrometer;   a database from which a target peptide is selected;   a computer coupled to the database and an output of the mass spectrometer, said computer having code executing thereon which, when executed, causes the computer to perform a method, the method comprising:
 selecting the target peptide having a target precursor; 
 determining masses associated with the target precursor and corresponding Y and B fragment ions of the target precursor; 
 obtaining first mass information and first chromatographic retention time information acquired from data collected in accordance with a first energy mode applied in the mass spectrometer causing at least some fragmentation of one or more precursors; 
 comparing the associated masses of the target precursor and the corresponding Y and B fragment ions of the target precursor to the first mass information to determine mass matches; 
 determining chromatographic retention times of the mass matches; and 
 identifying, based on said mass matches and the chromatographic retention times of the mass matches, whether the target peptide is present in the mixture. 
   
     
     
         54 . The system of  claim 51 , wherein the first energy mode is any of a low-energy mode used to obtain mass and chromatographic retention time information primarily associated with precursors, a high energy mode used to obtain mass and chromatographic retention time information primarily associated with fragments of the precursors, and a fixed energy mode using a voltage that is an intermediate value between a first voltage used in the low-energy mode and a second voltage used in the high energy mode.