Systems and methods for performing multiplexed targeted mass spectrometry

A method of performing multiplexed targeted mass spectrometry, comprising:

• acquiring, at a mass spectrometer by a plurality of targeted MS3 analyses during a plurality of acquisition segments, MS3 mass spectra of reporter ions dissociated from a plurality of isobaric tag-labeled target analytes included in a multiplex sample eluting from a column, wherein:
• the multiplex sample comprises a combination of a plurality of individual samples each including one or more of the isobaric tag-labeled target analytes,
• the one or more target analytes included in each individual sample are labeled with a distinct version of an isobaric tag comprising a reporter region from which the reporter ions are derived, and
• each targeted MS3 analysis is performed during an acquisition segment scheduled based on an expected retention time of a target analyte included in the plurality of target analytes;
• periodically acquiring, at the mass spectrometer by a plurality of MS2 analyses performed during the plurality of acquisition segments, MS2 mass spectra of product ions derived from the plurality of target analytes;
• and
• determining, by a computing device, a relative quantity of a target analyte included in the plurality of target analytes across the plurality of individual samples based on MS2 mass spectra acquired during an acquisition segment for the target analyte and MS3 mass spectra acquired during the acquisition segment for the target analyte.

A system for performing multiplexed targeted mass spectrometry, comprising:

• a mass spectrometer configured to receive components included in a multiplex sample and eluting from a chromatography column and mass analyze ions produced from the components, wherein:
• the components included in the multiplex sample include a plurality of target analytes;
• the multiplex sample comprises a combination of a plurality of individual samples each including one or more of the target analytes;
• and
• the one or more target analytes included in each individual sample are labeled with a distinct version of an isobaric tag comprising a reporter region;
• and
• a computing device configured to:
• acquire, by a plurality of targeted MS3 analyses during a plurality of acquisition segments, MS3 mass spectra of reporter ions dissociated from the plurality of target analytes included in the multiplex sample, wherein:
• each reporter ion is derived from the reporter region of an isobaric tag;
• and
• each targeted MS3 analysis is performed during an acquisition segment scheduled based on an expected retention time of a target analyte included in the plurality of target analytes;
• periodically acquire, by a plurality of MS2 analyses performed during the plurality of acquisition segments, MS2 mass spectra of product ions derived from the plurality of target analytes;
• and
• determine a relative quantity of a target analyte included in the plurality of target analytes across the plurality of individual samples based on MS2 mass spectra acquired during an acquisition segment for the target analyte and MS3 mass spectra acquired during the acquisition segment for the target analyte.