Automatic Characterization of Lipids Using Charge Remote Fragmentation Ions and Peaks Characteristic of Fatty Acid Fragmentation From MALDI MS/MS Data
Introduction
MALDI mass spectrometry has been used for detecting phosphatidylcholines by direct tissue analysis (1-4). Recently, triacylglycerols and phosphatidylcholine in complex mixtures have been identified using MALDI MS/MS (5,6) wherein charge remote fragmentation (CRF) as well as other A-, B-, C-, G and J- ions are the determinants as described recently (7,8,9). The CRF ions facilitate localisation of double bonds and branching in fatty acid chains. However, the major challenge in MALDI MS/MS data analysis is the huge amount of data generated in the process. Annotation of the above mentioned signals requires time consuming manual spectral interpretation. We developed a software tool for characterizing lipids by MS/MS data which streamlines this type of data analysis.
Methods
Commercial standards and lipids from fats like olive oil were analysed using MALDI MS and MS/MS. Samples were prepared with DHB matrix in positive ion mode. MS/MS data was acquired using 1 keV collision energy and air as collision gas (5 x10-6 Torr). SimLipid® database has been created containing 36299 lipids and 1305386 structure-specific in-silico charge remote fragmentation (CRF) ions as well as other characteristic ions (7,8,9). SimLipid creates a list of candidate structures for each lipid MS/MS spectrum based on precursor m/z value and other information. For each candidate, in-silico fragment ions are matched against the experimental MS/MS data. A scoring mechanism was developed in order to differentiate isobaric candidates.
Preliminary Results/Abstract
The triacylglycerols (TAG) are all detected as singly charged sodiated molecular species. The MS/MS data of TAGs were subjected to batch analysis in SimLipid® software. All the structures are identified correctly by the program. Analysis of the MS/MS spectrum obtained for the TAG at m/z 577.4492 (known 30:0 fatty acid chain distribution with 3 fatty acids of the same lengths), results in 5 different candidate structures with the correct structure bearing common name '1,2,3-tricaprinoyl-glycerol' as the highest scored lipid followed by four different diacylglycerols that correspond to the 31:0 fatty acid chain distribution with 2 fatty acids of different lengths. CRF ions that are separated by mass difference of 14 Da indicate the loss of CH2 group, which are a result of the cleavages of C-C bonds within the fatty acid chains. CRF ions that show the losses of 12Da indicate double bonds and the cleavage of a C=C bond. For this data, due to the presence of similar fatty acids as well as closely related head group in the candidate structures, CRF ions alone could not distinguish the correct structure. The program distinguishes the TAG based characteristic ions. To verify the results of the prgram, we applied the technology on the analysis of lipids from different origins such as like olive oil brands, margarine and butter.
MALDI mass spectrometry has been used for detecting phosphatidylcholines by direct tissue analysis (1-4). Recently, triacylglycerols and phosphatidylcholine in complex mixtures have been identified using MALDI MS/MS (5,6) wherein charge remote fragmentation (CRF) as well as other A-, B-, C-, G and J- ions are the determinants as described recently (7,8,9). The CRF ions facilitate localisation of double bonds and branching in fatty acid chains. However, the major challenge in MALDI MS/MS data analysis is the huge amount of data generated in the process. Annotation of the above mentioned signals requires time consuming manual spectral interpretation. We developed a software tool for characterizing lipids by MS/MS data which streamlines this type of data analysis.
Methods
Commercial standards and lipids from fats like olive oil were analysed using MALDI MS and MS/MS. Samples were prepared with DHB matrix in positive ion mode. MS/MS data was acquired using 1 keV collision energy and air as collision gas (5 x10-6 Torr). SimLipid® database has been created containing 36299 lipids and 1305386 structure-specific in-silico charge remote fragmentation (CRF) ions as well as other characteristic ions (7,8,9). SimLipid creates a list of candidate structures for each lipid MS/MS spectrum based on precursor m/z value and other information. For each candidate, in-silico fragment ions are matched against the experimental MS/MS data. A scoring mechanism was developed in order to differentiate isobaric candidates.
Preliminary Results/Abstract
The triacylglycerols (TAG) are all detected as singly charged sodiated molecular species. The MS/MS data of TAGs were subjected to batch analysis in SimLipid® software. All the structures are identified correctly by the program. Analysis of the MS/MS spectrum obtained for the TAG at m/z 577.4492 (known 30:0 fatty acid chain distribution with 3 fatty acids of the same lengths), results in 5 different candidate structures with the correct structure bearing common name '1,2,3-tricaprinoyl-glycerol' as the highest scored lipid followed by four different diacylglycerols that correspond to the 31:0 fatty acid chain distribution with 2 fatty acids of different lengths. CRF ions that are separated by mass difference of 14 Da indicate the loss of CH2 group, which are a result of the cleavages of C-C bonds within the fatty acid chains. CRF ions that show the losses of 12Da indicate double bonds and the cleavage of a C=C bond. For this data, due to the presence of similar fatty acids as well as closely related head group in the candidate structures, CRF ions alone could not distinguish the correct structure. The program distinguishes the TAG based characteristic ions. To verify the results of the prgram, we applied the technology on the analysis of lipids from different origins such as like olive oil brands, margarine and butter.
