Background: Although total plasma lipoproteome consists of proteins that have shown promises as biomarkers that can identify Alzheimer's disease (AD), effect sizes are modest. The objective of this study is to provide initial proof-of-concept that the plasma lipoproteome more likely differ between AD cases and controls when measured in individual plasma lipoprotein fractions than when measured as total in immunodepleted plasma. Methods: We first developed a targeted proteomics method based on selected reaction monitoring (SRM) and liquid chromatography and tandem mass spectrometry for measurement of 120 tryptic peptides from 79 proteins that are commonly present in plasma lipoproteins. Then in a proof-of concept case-control study of 5 AD cases and 5 sex- and age-matched controls, we applied the targeted proteomic method and performed relatively quantification of 120 tryptic peptides in plasma lipoprotein fractions (fractionated by sequential gradient ultracentrifugation) and in immunodepleted plasma (of albumin and IgG). Unadjusted p values from two-sample t-tests and overall fold change was used to evaluate a peptide relative difference between AD cases and controls, with lower p values (< 0.05) or greater fold differences (> 1.05 or < 0.95) suggestive of greater peptide/protein differences. Results: Within-day and between-days technical precisions (mean %CV [SD] of all SRM transitions) of the targeted proteomic method were 3.95% (2.65) and 9.31% (5.59), respectively. Between-days technical precisions (mean % CV [SD]) of the entire plasma lipoproteomic workflow including plasma lipoprotein fractionation was 27.90% (14.61). Ten tryptic peptides that belonged to 5 proteins in plasma lipoproteins had unadjusted p values < 0.05, compared to no peptides in immunodepleted plasma. Furthermore, 27, 32, 17, and 20 tryptic peptides in VLDL, IDL, LDL and HDL, demonstrated overall peptide fold differences > 1.05 or < 0.95, compared to only 6 tryptic peptides in immunodepleted plasma. The overall comparisons, therefore, suggested greater peptide/protein differences in plasma lipoproteome when measured in individual plasma lipoproteins than as total in immunodepleted plasma. Specifically, protein complement C3's peptide IHWESASLLR, had unadjusted p values of 0.00007, 0.00012, and 0.0006 and overall 1.25, 1.17, 1.14-fold changes in VLDL, IDL, and LDL, respectively. After positive False Discovery Rate (pFDR) adjustment, the complement C3 peptide IHWESASLLR in VLDL remained statistically different (adjusted p value < 0.05). Discussion: The findings may warrant future studies to investigate plasma lipoproteome when measured in individual plasma lipoprotein fractions for AD diagnosis.
Bibliographical noteFunding Information:
We thank the FIT-AD Trials subjects and families for participating in the blood biomarker ancillary study. Our appreciation goes to Dr. Patric Clapshaw and Mr. Brian Wilbur from the Somolom Park (Burien, WA) who provided plasma samples for controls. Mass spectrometry was carried out in the Analytical Biochemistry Shared Resource of the Masonic Cancer Center, University of Minnesota, funded in part by Cancer Center Support Grant CA-077598. We thank Dr. Laura Parker’s lab at the University of Minnesota for reference peptide synthesis.
This work was supported by a grant from the Alzheimer’s Association (NIRG-15-362393). The FIT-AD Trial was supported by the National Institute on Aging of the National Institutes of Health Award Number 1R01AG043392-01A1. All subject interviews and data collections occurred at the CTSI that was supported by the National Institutes of Health National Center for Advancing Translational Sciences of the National Institutes of Health Award Number UL1TR000114. None of the sponsors had any role in study design, data analysis, or reporting of the results. The content is solely the responsibility of the authors and does not necessarily represent the official views of the Alzheimer’s Association and the National Institutes of Health. Dr. Li also receive research support from the NIH (R21 AG059068 and R01 AG059654).
- Alzheimer's disease
- Complement C3
- High-density lipoprotein (HDL)
- Intermediate density lipoprotein (IDL)
- Low density lipoprotein (LDL)
- Plasma lipoproteins
- Selected reaction monitoring (SRM)
- Targeted proteomics
- Very low-density lipoprotein (VLDL)