Quantification and Discovery of Acyl-ACPs by LC-MS/MS

Acyl carrier proteins (ACPs) are the scaffolds for fatty acid biosynthesis in living systems, rendering them essential to a comprehensive understanding of lipid metabolism; however, accurate quantitative methods to assess individual acyl-ACPs do not exist. A robust method was developed to quantify acyl-ACPs at picogram levels. Acyl-ACP elongation intermediates (3-hydroxyacyl-ACPs and 2, 3- trans -enoyl-ACPs), and unexpected medium chain (C10:1, C14:1) and polyunsaturated long chain acyl-ACPs (C16:3) were also identified, indicating the sensitivity of the method and that descriptions of lipid metabolism and ACP function are incomplete. Such ACPs are likely important to medium chain lipid production for fuels and highlight poorly understood lipid remodeling events in the chloroplast. The approach is broadly applicable to Type II FAS systems found in plants, bacteria, and mitochondria of animal and fungal systems because it uses a strategy that capitalizes on a highly conserved Asp-Ser-Leu-Asp (DSLD) amino acid sequence in ACPs to which acyl groups are attached. This allows for sensitive quantification using LC-MS/MS with de novo generated standards and an isotopic dilution strategy and will fill a gap in understanding, providing insights through quantitative exploration of fatty acid biosynthesis processes for optimal biofuels, renewable feed stocks, and medical studies in health and disease.