Quantification of Dolichyl Phosphates Using Phosphate Methylation and Reverse-Phase Liquid Chromatography–High Resolution Mass Spectrometry

Abstract

Dolichyl monophosphates (DolPs) are essential lipids in glycosylation pathways that are highly conserved across almost all domains of life. The availability of DolP is critical for all glycosylation processes, as these lipids serve as membrane-anchored building blocks used by various types of glycosyltransferases to generate complex post-translational modifications of proteins and lipids. The analysis of DolP species by reverse-phase liquid chromatography-mass spectrometry (RPLC–MS) remains a challenge due to their very low abundance and wide range of lipophilicities. Until now, a method for the simultaneous qualitative and quantitative assessment of DolP species from biological membranes has been lacking. Here, we describe a novel approach based on simple sample preparation, rapid and efficient trimethylsilyl diazomethane-dependent phosphate methylation, and RPLC-MS analysis for quantification of DolP species with different isoprene chain lengths. We used this workflow to selectively quantify DolP species from lipid extracts derived of Saccharomyces cerevisiae, HeLa, and human skin fibroblasts from steroid 5-α-reductase 3- congenital disorders of glycosylation (SRD5A3-CDG) patients and healthy controls. Integration of this workflow with global lipidomics analyses will be a powerful tool to expand our understanding of the role of DolPs in pathophysiological alterations of metabolic pathways downstream of HMG-CoA reductase, associated with CDGs, hypercholesterolemia, neurodegeneration, and cancer.

A pseudoautosomal glycosylation disorder prompts the revision of dolichol biosynthesis

“…Consistent with these observations, previous studies reported unchanged dolichol levels with an increase in the polyprenol/dolichol ratio in fibroblasts from patients with SRD5A3-CDG. 31 , 32 This indicated that high polyprenol levels allow cells to bypass the requirement for DHRSX and, as previously postulated, for SRD5A3 in a cell-type-dependent manner. 4 , 31 This compensation could explain the relatively mild glycosylation deficiency in certain SRD5A3-CDG patients 49 , 63 and in DHRSX-CDG patients 3 and 4 in our study.…”

“…This context-dependent behavior resembles what has previously been described in SRD5A3-CDG patient cells and might be due to compensatory changes. 4 , 31 , 32 …”

“…The dimethylation of dolichol phosphate and polyprenol phosphate was performed as described in Kale et al. 32 The organic fraction containing isoprenoid species was completely dried and resuspended in 200 μL of dichloromethane:methanol (6.5:5.2, v/v). 10 μL of 2 mol/L TMSD (in hexane) was added and incubated for 40 min at room temperature.…”

The N-glycosylation defect in Lec5 and Lec9 CHO cells is caused by absence of the DHRSX gene

“…Dimethylation of isoprenoid phosphates using trimethylsilyl diazomethane (TMSD) The dimethylation of dolichol phosphate and polyprenol phosphate was performed as described in Kale et al, (2023) (38): The organic fraction containing isoprenoid species was completely dried and resuspended in 200 μL of dichloromethane:methanol (6.5:5.2, v/v). 10 μL of 2 mol/L TMSD was added and incubated for 40 min at room temperature.…”

Blood-Derived Lipid and Metabolite Biomarkers in Cardiovascular Research from Clinical Studies: A Recent Update

“…The analysis of such low-abundant metabolites needs targeted analysis, and may sometimes require additional selective sample enrichment/purification steps during the sample preparation procedures prior to MS analysis. In recent years, for the analysis of low-abundant metabolites, several new MS-based analytical methods and techniques (e.g., [54,55]) have been developed. In the future, such analytical methods could be used to comprehensively characterize the homeostasis of these low-abundance analytes under health and disease conditions.…”