Isobaric mass tagging and triple quadrupole mass spectrometry to determine lipid biomarker candidates for Alzheimer's disease

Abstract

The isobaric tagging method widely used in proteomic and lipidomic fields, with the multiple reaction monitoring (MRM) approach using a triple quadrupole mass spectrometer, was applied to identify biomarker candidates from plasma samples for Alzheimer’s disease (AD). We focused on the following phospholipids that have amino groups as the functional group: phosphatidylethanolamine (PE), Lyso-PE, phosphatidylserine, and Lyso-phosphatidylserine. We also investigated fatty acids that have a carboxy group. A sixplex tandem mass tag (TMT) was used for the isobaric tagging method in this study. The TMT reaction had high reproducibility in human plasma. A total of 196 human plasma samples from three AD cohorts were used for the study, and compared to pooled plasma quality control (QC) samples. The described method required only 40 MRM measurements, including the pooled QC samples, for a full comparison of the data. We found that the content of free fatty acids increased in AD samples in all the three cohorts, alkenyl PEs (ePEs) decreased over a one-year interval in AD patients, and ePEs weakly correlated with amyloid peptide (a-beta) 1–42 in cerebrospinal fluid. In conclusion, total free fatty acids in plasma are a risk factor for AD, and ePEs monitor candidates for AD. Therefore, TMT-lipidomics is a powerful approach for the determination of plasma biomarkers because of the high sample throughput.

Limitations of deuterium‐labeled internal standards for quantitative electrospray ionization mass spectrometry analysis of fatty acid metabolites

“…As a result, target compounds labeled with stable isotopes have been used as ISs for a long time 8–11 . Furthermore, the IS method has been applied to many targets in fields such as proteomics, 12,13 metabolomics 14,15 and lipidomics 16,17 . However, stable‐isotope‐labeled compounds are expensive, and, sometimes, ISs labeled with stable isotopes are not commercially available.…”

Development of Tandem Mass Tag Labeling Method for Lipid Molecules Containing Carboxy and Phosphate Groups, and Their Stability in Human Serum

“…Representative spectra obtained by TMT reporter ion-specific precursor ion scanning analysis and product ion scan spectra of derivatized PA, Lyso-PA(LPA), arachidonic acid and eicosanoid standards are shown in Figure 3B,C. for the TMT labeling of free fatty acids, which we have already reported [22], no unreacted PAs or fatty acids were detected, and the progress of TMT formation based on PAs and free fatty acids was observed. When there was no EDC reagent and only DMTMM was used, the TMT derivatization reaction proceeded to free fatty acids, but it did not react with PA. Figure 2 shows these chemical reaction schemes for phosphor-lipids and carboxy-lipids.…”

“…Since this reaction also proceeded for the carboxy group, unreacted free fatty acids were observed when the fatty acids and PA reacted simultaneously. When EDC was added to the one-pot reaction solution containing 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (DMTMM) for the TMT labeling of free fatty acids, which we have already reported [ 22 ], no unreacted PAs or fatty acids were detected, and the progress of TMT formation based on PAs and free fatty acids was observed. When there was no EDC reagent and only DMTMM was used, the TMT derivatization reaction proceeded to free fatty acids, but it did not react with PA. Figure 2 shows these chemical reaction schemes for phosphor-lipids and carboxy-lipids.…”

“…SRM analysis was performed using the positive ion mode ESI, with a collision energy of 46 eV for TMT-derivatized compounds. For the sixplex aminoxyTMT sample, Fatty acid species possessing carbon chains 12 to 24 carbons in length were targeted [22], 132 eicosanoids or other fatty acid derivatives [36], five LPA species (16 → 153.1, respectively, in negative ion mode. The SRM settings for non-labeled eicosanoids have been described previously [36].…”

“…Many papers in the field of proteomics have demonstrated the good reliability of relative quantification. Isobaric tag approaches in lipidomics [ 19 ] and metabolomics [ 20 ] for the compounds with amino groups have been used, the TMT labeling method for carboxy groups has been developed as a functional group other than amino groups [ 21 ], and we have previously conducted the quantitative analysis of free fatty acids using approximately 200 human plasma samples [ 22 ]. To take advantage of relative quantification by TMT labelling, we propose a strategy for using a common quality control (QC) sample each time for one of six sample in sixplex measurement, thereby allowing for comparisons of large number of sample sets as well as different datasets.…”