HPLC and mass spectrometry analysis of dolichol-phosphates at the cell culture scale

Abstract

Dolichols (Dol) are polyprenol lipids that are essential structural components of eukaryotic membranes. In addition, the phosphorylated derivatives of Dol function as lipid anchor of mono-and oligosaccharide precursors involved in protein glycosylation.The biological importance of Dol-phosphates (Dol-P) is illustrated by the severe outcome of human disorders linked to Dol biosynthetic defects, such as Dol-kinase deficiency. For characterization of inherited human diseases and evaluation of therapeutic trials, cultured cells often serve as a sole possible source for experimentation. Limited amounts of cell culture material render the quantitative analysis of Dol a challenging task. Here, we present HPLC and mass spectrometry based approaches to analyse and quantitate Dol-P from cultured human cells. The composition of naturally occurring Dol-P and the saturation state of the α-isoprene units was identified by negative ion electrospray ionization mass spectrometry. Furthermore, fluorescently labelled Dol-P were separated by HPLC and quantified by comparison to known amounts of the internal standard polyprenol-P. The effect of pravastatin, a 3-hydroxy-3-methyl-glutaryl coenzyme-A reductase inhibitor, on the formation of Dol-P in HeLa cells was investigated. As expected, this treatment led to a decrease of Dol-P down to 35% of normal levels. KeywordsDolichol-phosphate, mass spectrometry, HPLC, cholesterol pathway, statin, glycosylation 3 Introductory statement Dolichols (Dol) are essential components of eukaryotic membranes, where they contribute to the organisation and fluidity of the lipid bilayer and enhance vesicle fusion [1]. Their composition varies between the kingdoms of life in respect to the number of isoprene subunits, although all Dol are assembled in the identical trans-trans-polycis conformation. In contrast to prokaryotic polyprenols, Dol α-isoprene residues are saturated [2]. Starting from acetate, the pathway leading to the formation of Dol leads via mevalonate to farnesyl-pyrophosphate and is up to this point identical to the cholesterol and ubiquinone biosynthesis pathway [1]. The enzyme cis-prenyltransferase catalyzes the elongation of farnesyl-pyrophosphate by sequential condensation of isopentenyl-pyrophosphate [2]. Prior to the NADPH-dependent reduction of the α-isoprene unit, the entirely unsaturated polyprenol-pyrophosphate intermediates are dephosphorylated by mono-or pyrophosphate phosphatases. In a terminatory step, the To date, only few techniques have been described for the analysis and quantification of 12;13]. HPLC-based methods separate radio-labelled or fluorescently tagged polyprenol-P and are mainly restricted to tissue-derived samples [14; 15]. Whereas several mass spectrometry (MS) methods for the analysis of free Dol have been described [16; 17; 18; 19; 20; 21], no MS method has been established to analyse Dol-P from mammalian sources, despite the essential role of Dol-P in protein glycosylation [4].Solely, Griffiths and co-workers have configured their MS setup to s...

Glycoprotein Maturation and the UPR

“…Considering the low levels of Dol-P based structures in cells, a sensitive Dol-P detection can be achieved by labeling with the fluorescent compound 9-anthryldiazomethane. The procedure was originally described for tissue sample extraction (Elmberger et al, 1989) and we have adapted the method for cultured cells (Haeuptle et al, 2009). Detection of unlabeled Dol-P can also be achieved by negative ion electrospray mass spectrometry (ESI-MS), which, in combination to precursor ion fragmentation, provides detailed information on Dol-P composition and on the saturation state of the -isoprene unit.…”

“…Negative ion electrospray mass spectrometry of Dol-P Purified Dol-P can also be directly analyzed by ESI-MS (Haeuptle et al, 2009) without derivatization. The dried Dol-P sample is first dissolved in 90% acetonitrile, 10% nhexane, containing 0.01% diethylamine, and then vortexed and centrifuged to eliminate any particulate matter.…”

Improvement of Dolichol-linked Oligosaccharide Biosynthesis by the Squalene Synthase Inhibitor Zaragozic Acid

“…The impact of ZGA on Dol-P levels was first addressed using healthy control fibroblasts. After labeling with the fluorochrome 9-anthryldiazomethane, Dol-P levels were quantitated after HPLC separation (21). The resulting fluorescent HPLC profiles of untreated cells (Fig.…”

“…Prior to alkaline hydrolysis (20), 15 g of C 80 -polyprenyl-P standards was added. The extracted Dol and Dol-P were purified on a Sep-Pak C 18 column (Waters) and subsequently separated on a Sep-Pak silica column (Waters) (21). Dol-P was dimethylated using diazomethane generated in an Aldrich diazomethane generator system (Sigma) according to the manufacturer's instructions.…”

Liquid chromatography/tandem mass spectrometry of dolichols and polyprenols, lipid sugar carriers across evolution

“…Often, these unknowns are centered on those pathway steps involving the biosynthesis and assembly of dolichol or polyprenol, as well as their glycan-charged derivatives. Liquid chromatography (LC) coupled with electrospray ionization tandem mass spectrometry (ESI-MS/MS) has proven to be a highly sensitive and specific tool for studying dolichols and polyprenols in their alcohol or glycan-modified forms in members of all three domains of life [10–17]. …”