Quantification and Discovery of Acyl-Acps by LC-MS/MS 3 Jeong-Won Nama,C, Lauren M

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Quantification and Discovery of Acyl-Acps by LC-MS/MS 3 Jeong-Won Nama,C, Lauren M bioRxiv preprint doi: https://doi.org/10.1101/870485; this version posted December 10, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. This article is a US Government work. It is not subject to copyright under 17 USC 105 and is also made available for use under a CC0 license. 1 BREAKTHROUGH REPORT 2 Quantification and Discovery of Acyl-ACPs by LC-MS/MS 3 Jeong-Won Nama,c, Lauren M. Jenkinsa,b,c, Jia Lia, Bradley S. Evansa,d, Jan G. 4 Jaworskia,d, and Doug K. Allena,b,d 5 a Donald Danforth Plant Science Center, 975 North Warson Road, St. Louis, Missouri 6 63132; 7 b USDA-ARS, Plant Genetics Research Unit, 975 North Warson Road, St. Louis, 8 Missouri 63132 (L.M.J, D.K.A) 9 c These authors contributed equally 10 d Corresponding Authors: doug.allen@ars.usda.gov, jaworskijg@gmail.com, 11 bevans@danforthcenter.org 12 Short Title: Acyl-ACP Discovery and Absolute Quantification 13 One-sentence Summary: Acyl-ACPs that enable fatty acid biosynthesis were uniquely 14 profiled and quantified resulting in discovery of novel ACP products with unknown 15 function and providing a method for rigorous comparisons. 16 The author(s) responsible for distribution of materials integral to the findings presented in this article in accordance with the policy described in the Instructions for Authors (www.plantcell.org) is (are): Doug K. Allen (doug.allen@usda.gov) and Bradley S. Evans (bevans@danforthcenter.org). bioRxiv preprint doi: https://doi.org/10.1101/870485; this version posted December 10, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. This article is a US Government work. It is not subject to copyright under 17 USC 105 and is also made available for use under a CC0 license. 17 ABSTRACT 18 Acyl carrier proteins (ACPs) are the scaffolds for fatty acid biosynthesis in living 19 systems, rendering them essential to a comprehensive understanding of lipid metabolism; 20 however, accurate quantitative methods to assess individual acyl-ACPs do not exist. A 21 robust method was developed to quantify acyl-ACPs at picogram levels. Acyl-ACP 22 elongation intermediates (3-hydroxyacyl-ACPs and 2, 3-trans-enoyl-ACPs), and 23 unexpected medium chain (C10:1, C14:1) and polyunsaturated long chain acyl-ACPs 24 (C16:3) were also identified, indicating the sensitivity of the method and that descriptions 25 of lipid metabolism and ACP function are incomplete. Such ACPs are likely important to 26 medium chain lipid production for fuels and highlight poorly understood lipid remodeling 27 events in the chloroplast. The approach is broadly applicable to Type II FAS systems 28 found in plants, bacteria, and mitochondria of animal and fungal systems because it uses 29 a strategy that capitalizes on a highly conserved Asp-Ser-Leu-Asp (DSLD) amino acid 30 sequence in ACPs to which acyl groups are attached. This allows for sensitive 31 quantification using LC-MS/MS with de novo generated standards and an isotopic 32 dilution strategy and will fill a gap in understanding, providing insights through 33 quantitative exploration of fatty acid biosynthesis processes for optimal biofuels, 34 renewable feed stocks, and medical studies in health and disease. 35 The author(s) responsible for distribution of materials integral to the findings presented in this article in accordance with the policy described in the Instructions for Authors (www.plantcell.org) is (are): Doug K. Allen (doug.allen@usda.gov) and Bradley S. Evans (bevans@danforthcenter.org). bioRxiv preprint doi: https://doi.org/10.1101/870485; this version posted December 10, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. This article is a US Government work. It is not subject to copyright under 17 USC 105 and is also made available for use under a CC0 license. 36 INTRODUCTION 37 The synthesis of fatty acyl chains is essential for the production of storage and 38 membrane lipids and functional molecules that modulate gene expression or contribute to 39 protein activity. Studies on acyl chains span from disease and nutrition to demands for 40 renewable fuels and feedstocks (Uauy et al., 2000; Peralta-Yahya et al., 2012; Zhu et al., 41 2016; Vanhercke et al., 2019). The lengths of up to 18 carbons are produced during fatty 42 acid biosynthesis (Li-Beisson et al., 2013) on an acyl carrier protein scaffold (Overath 43 and Stumpf, 1964) which is connected to the acyl chain through linkage of a 4’- 44 phosphopantetheine group to a serine residue of the protein (ACP; Fig. 1a). The acyl 45 chains are elongated by a series of four enzymatic steps that link ACP intermediates and 46 form a cycle that results in the addition of two carbons and four hydrogens. Two distinct 47 types of fatty acid synthase (FAS) complexes perform the repeated cycle of ketoacyl 48 synthesis, reduction, dehydration, and a second reduction producing long chain saturated 49 hydrocarbons (Fig. 1b). Yeast and mammals use a single large multifunctional FAS 50 protein (Type I) that includes multiple domains for the enzymatic steps and ACP region 51 (Bressler and Wakil, 1961; Hsu et al., 1965), whereas fatty acid synthesis in plants and 52 bacteria occur through the concerted action of individual proteins within a complex 53 including a distinct ACP that is 9-15 kDa in size (Alberts et al., 1963; White et al., 2005; 54 Cronan, 2014). 55 In plants, the production of acyl chains take place predominantly in the 56 chloroplast, though also to a lesser extent in the mitochondria, using acyl-ACP 57 intermediates in both instances (Ohlrogge et al., 1979; Chuman and Brody, 1989). The 58 chain elongation process is terminated by thioesterase reactions that release a non- 59 esterified fatty acid from the ACP and regenerate the ACP substrate pool for further fatty 60 acid biosynthetic reactions. The released fatty acid can then be activated to an acyl-CoA 61 to make glycerolipids for storage oil production, waxes, surface lipids or membrane 62 biogenesis (Li-Beisson et al., 2013). 63 Given the central role of acyl-ACPs in fatty acid biosynthesis, their quantitative 64 analysis can help our understanding of lipid metabolism; however, the acyl group is a 65 small percentage of the total acyl-ACP weight (usually significantly less than 3%) and 66 current methods are confined by biochemical approaches that analyze intact proteins or The author(s) responsible for distribution of materials integral to the findings presented in this article in accordance with the policy described in the Instructions for Authors (www.plantcell.org) is (are): Doug K. Allen (doug.allen@usda.gov) and Bradley S. Evans (bevans@danforthcenter.org). bioRxiv preprint doi: https://doi.org/10.1101/870485; this version posted December 10, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. This article is a US Government work. It is not subject to copyright under 17 USC 105 and is also made available for use under a CC0 license. 67 their subunits. After ACP was demonstrated to be the acyl carrier for fatty acid synthesis 4 bioRxiv preprint doi: https://doi.org/10.1101/870485; this version posted December 10, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. This article is a US Government work. It is not subject to copyright under 17 USC 105 and is also made available for use under a CC0 license. 68 (Goldman et al., 1963) studies examined expression (Hloušek-Radojčić et al., 1992; 69 Bonaventure and Ohlrogge, 2002), subcellular roles (Brody et al., 1997; Wada et al., 70 1997; Cronan et al., 2005; Witkowski et al., 2007) and the impact of sequence on 71 function and growth (De Lay and Cronan, 2007; Zhu et al., 2019); however studies 72 quantifying the amount of acyl-ACPs are uncommon. 73 Levels of acyl-ACPs were measured based on urea polyacrylamide gel separation 74 and immunodetection (Post-Beittenmiller et al., 1991); however multiple isoforms could 75 not be resolved (Ohlrogge and Kuo, 1985) and only a subset of all acyl-ACPs were 76 identified by antibodies and gel gradients, limiting the extent of quantification. Focusing 77 more specifically on the acyl component, Kopka and coworkers (Kopka et al., 1995) 78 developed an assay for individual acyl-ACP levels with gas chromatography linked to 79 mass spectrometry (GC-MS) by generating butylamide derivatives. Levels of spinach leaf 80 acyl-ACPs qualitatively matched those inferred from densitometry of urea-based gels; 81 however, since the chemical derivatization was not specific to acyl-ACP thioester bonds, 82 acyl-CoAs and other thioesters present at greater levels must be meticulously removed by 83 anion exchange chromatography prior to derivatization. Moreover, short chain ACPs 84 cannot be detected through this strategy. 85 We report a novel method to quantitatively analyze acyl-ACPs with tandem mass 86 spectrometry based on a highly conserved contiguous amino acid region that flanks the 87 attachment of the acyl chain. Enzymatic cleavage resulted in an acyl chain connected to a 88 phosphopantetheinyl group and a tripeptide that was sensitively quantified by LC- 89 MS/MS. 15N-labeled ACP standards were enzymatically synthesized de novo for absolute 90 quantification of acyl-ACP levels using an isotopic dilution strategy. The method 91 identified 3-hydroxyacyl-ACP and 2, 3-trans-enoyl-ACP, two of the three acyl-ACP 92 elongation intermediates for each 2-carbon acyl-ACP product of the fatty acid 93 biosynthetic cycle and unanticipated medium chain unsaturated acyl-ACPs that to date 94 have no known function in plant metabolism.
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