Mass spectrometry based systems and methods for implementing multistage MS/MS analysis for identification of experimental glycolipid samples
Mass spectrometry (MS) based systems and methods of implementing multi-stage MS/MS analysis for identification of experimental glycolipid samples. A processor communicatively coupled to a memory and a reference spectral database (1) implements a first stage analysis of glycan fragment data including determining, based on a matching reference glycan of a candidate reference glycolipid, a glycan structure of a glycan portion of the experimental glycolipid. The processor further (2) implements a second stage analysis of lipid/glycolipid fragment data including determining a lipid structure of a lipid portion of the experimental glycolipid by performing a spectral comparison of one or more MS/MS spectral values of a shifted experimental lipid fragment data with one or more MS/MS spectral values of matching reference lipid/glycolipid fragment data of the candidate reference glycolipid. A combination of the determined glycan structure and lipid structure gives the structure of each specific glycolipid in the experimental glycolipid sample.
1 . A mass spectrometry (MS) based system configured to implement multi-stage MS/MS analysis for identification of experimental glycolipid samples, the MS based system comprising:
a mass spectrometer configured to receive and analyze an experimental glycolipid sample to determine experimental MS/MS spectral data of the experimental glycolipid sample;
a reference spectral database configured to store a plurality of reference glycolipid datasets, each of the reference glycolipid datasets corresponding to a reference glycolipid and comprising at least (a) reference glycan fragment data defining MS/MS spectra of a glycan portion of the reference glycolipid, and (b) reference lipid and/or glycolipid fragment data defining MS/MS spectra of a lipid portion of the reference glycolipid;
a memory storing program instructions; and
a processor communicatively coupled to the memory and the reference spectral database,
wherein the processor is configured to execute the program instructions to cause the processor to:
(1) implement a first stage analysis of glycan fragment data comprising:
determining, from the experimental MS/MS spectral data, experimental glycan fragment data defining MS/MS spectra of a glycan portion of the experimental glycolipid sample,
identifying a matching reference glycan of a candidate reference glycolipid selected from the reference spectral database, wherein fragment data of the matching reference glycan comprises one or more MS/MS spectral values that correspond to one or more MS/MS spectral values of the experimental glycan fragment data,
determining, based on the matching reference glycan of the candidate reference glycolipid, a glycan structure of the glycan portion of the experimental glycolipid, and
(2) implement a second stage analysis of lipid and/or glycolipid fragment data comprising:
determining, from the experimental MS/MS spectral data, experimental lipid and/or glycolipid fragment data defining MS/MS spectra of a lipid portion of the experimental glycolipid sample,
rule-based shifting, by the processor, one or more mass values of the reference lipid and/or glycolipid fragment data of the candidate reference glycolipid, while maintaining, during the rule-based shifting, the experimental glycan fragment data identified during the first stage analysis, to generate shifted reference lipid and/or glycolipid fragment data,
performing a spectral comparison of one or more MS/MS spectral values of the shifted reference lipid and/or glycolipid fragment data with one or more MS/MS spectral values of the experimental lipid and/or glycolipid fragment data, and
determining, based on reference lipid and/or glycolipid fragment data of the candidate reference glycolipid, a lipid structure of the lipid portion of the experimental glycolipid,
wherein, based on the spectral comparison, the experimental glycolipid sample is determined to be of a same glycolipid species as the candidate reference glycolipid, the lipid structure of the lipid portion of the experimental glycolipid being different from a lipid structure of the candidate reference glycolipid as characterized by a difference between the experimental lipid and/or glycolipid fragment data and the reference lipid and/or glycolipid fragment data.
2 . The MS based system of claim 1 , wherein the experimental glycolipid sample is a human or animal bodily fluid or tissue based glycolipid sample.
3 . The MS based system of claim 1 , wherein the experimental glycolipid sample includes one or more unidentified glycolipids as one or more corresponding biomarkers.
4 . The MS based system of claim 1 , wherein the one or more MS/MS spectral values of the matching reference glycan fragment data correspond to one or more MS/MS spectral values of the experimental glycan fragment data by at least a glycan based relative intensity threshold value; or
the one or more MS/MS spectral values of the experimental lipid and/or glycolipid fragment data correspond to one or more MS/MS spectral values of the reference lipid and/or glycolipid fragment data or the shifted reference lipid and/or glycolipid fragment data by at least a lipid and/or glycolipid based relative intensity threshold value.
5 . The MS based system of claim 1 further comprising generating a matching score for the experimental glycolipid sample based on a comparison of MS/MS spectra of (1) the reference glycan fragment data and either the shifted reference lipid and/or glycolipid fragment data or the reference lipid and/or glycolipid fragment data, with (2) the experimental glycan fragment data and the experimental lipid and/or glycolipid fragment data.
6 . The MS based system of claim 1 , wherein the reference spectral database is updated with a new glycolipid dataset corresponding to the experimental glycolipid sample, the new glycolipid dataset stored in the reference spectral database, and the new glycolipid dataset comprising at least (a) the experimental glycan fragment data, and (b) the experimental lipid and/or glycolipid fragment data.
7 . The MS based system of claim 1 , wherein the processor is communicatively coupled to the mass spectrometer, and where the processor is further configured to receive the MS/MS spectral data of the experimental glycolipid sample from the mass spectrometer.
8 . The MS based system of claim 1 , wherein the mass spectrometer comprises collision-induced dissociation (CID).
9 . The MS based system of claim 1 , wherein the one of more mass values of the reference lipid and/or glycolipid fragment data are shifted by 14n, 2n′, or a combination thereof.
10 . The MS based system of claim 1 , wherein the one or more mass values of the reference lipid and/or glycolipid fragment data are shifted by the mass of an isotope label, a chemical modification, a chemical tag, or a combination thereof.
11 . A mass spectrometry (MS) based method of implementing multi-stage MS/MS analysis for identification of experimental glycolipid samples, the MS based method comprising:
(1) implementing, by a processor communicatively coupled to a memory and a reference spectral database, a first stage analysis of glycan fragment data comprising:
determining, from experimental MS/MS spectral data of an experimental glycolipid sample as analyzed by a mass spectrometer, experimental glycan fragment data defining MS/MS spectra of a glycan portion of the experimental glycolipid sample,
identifying a matching reference glycan of a candidate reference glycolipid selected from the reference spectral database, wherein the spectral database stores a plurality of reference glycolipid datasets, each of the reference glycolipid datasets corresponding to a reference glycolipid and comprising at least (a) reference glycan fragment data defining MS/MS spectra of a glycan portion of the reference glycolipid, and (b) reference lipid and/or glycolipid fragment data defining MS/MS spectra of a lipid portion of the reference glycolipid, and wherein fragment data of the matching reference glycan comprises one or more MS/MS spectral values that correspond to one or more MS/MS spectral values of the experimental glycan fragment data, and
determining, based on the matching reference glycan of the candidate reference glycolipid, a glycan structure of the glycan portion of the experimental glycolipid, and
(2) implementing, by the processor, a second stage analysis of lipid and/or glycolipid fragment data comprising:
determining, from the experimental MS/MS spectral data, experimental lipid and/or glycolipid fragment data defining MS/MS spectra of a lipid portion of the experimental glycolipid sample,
rule-based shifting, by the processor, one or more mass values of the reference lipid and/or glycolipid fragment data of the candidate reference glycolipid, while maintaining, during the rule-based shifting, the experimental glycan fragment data identified during the first stage analysis, to generate shifted reference lipid and/or glycolipid fragment data,
performing a spectral comparison of one or more MS/MS spectral values of the shifted reference lipid and/or glycolipid fragment data with one or more MS/MS spectral values of the experimental lipid and/or glycolipid fragment data, and
determining, based on reference lipid and/or glycolipid fragment data of the candidate reference glycolipid, a lipid structure of the lipid portion of the experimental glycolipid,
wherein, based on the spectral comparison, the experimental glycolipid sample is determined to be of a same glycolipid species as the candidate reference glycolipid, the lipid structure of the lipid portion of the experimental glycolipid being different from a lipid structure of the candidate reference glycolipid as characterized by a difference between the experimental lipid and/or glycolipid fragment data and the reference lipid and/or glycolipid fragment data.
12 . The MS based method of claim 11 , wherein the experimental glycolipid sample is a human or animal bodily fluid or tissue based glycolipid sample.
13 . The MS based method of claim 11 , wherein the experimental glycolipid sample includes one or more unidentified glycolipids as one or more corresponding biomarkers.
14 . The MS based method of claim 11 , wherein the one or more MS/MS spectral values of the matching reference glycan fragment data correspond to one or more MS/MS spectral values of the experimental glycan fragment data by at least a glycan based relative intensity threshold value; or
the one or more MS/MS spectral values of the experimental lipid and/or glycolipid fragment data correspond to one or more MS/MS spectral values of the reference lipid and/or glycolipid fragment data or the shifted reference lipid and/or glycolipid fragment data by at least a lipid and/or glycolipid based relative intensity threshold value.
15 . The MS based method of claim 11 , wherein a matching score is generated, by the processor, for the experimental glycolipid sample based on a comparison of MS/MS spectra of (1) the reference glycan fragment data and either the shifted reference lipid and/or glycolipid fragment data or the reference lipid and/or glycolipid fragment data, with (2) the experimental glycan fragment data and the experimental lipid and/or glycolipid fragment data.
16 . The MS based method of claim 11 , wherein the reference spectral database is updated with a new glycolipid dataset corresponding to the experimental glycolipid sample, the new glycolipid dataset stored in the reference spectral database, and the new glycolipid dataset comprising at least (a) the experimental glycan fragment data, and (b) the experimental lipid and/or glycolipid fragment data.
17 . The MS based method of claim 11 , wherein the processor is communicatively coupled to the mass spectrometer, and where the processor is further configured to receive the MS/MS spectral data of the experimental glycolipid sample from the mass spectrometer.
18 . The MS based method of claim 11 , wherein the mass spectrometer comprises collision-induced dissociation (CID).
19 . The MS based method of claim 11 , wherein the one or more mass values of the reference lipid and/or glycolipid fragment data are shifted by 14n, 2n′, or both.
20 . The MS based method of claim 11 , wherein the one or more mass values of the reference lipid and/or glycolipid fragment data are shifted by the mass of an isotope label, a chemical modification, a chemical tag, or a combination thereof.