Advance Workflow in Lipoproteomics via Polymeric Ion Exchanger

Abstract

A workflow is designed for the analysis of lipoproteins, high density lipoproteins (HDL), apoproteins, and lipid fraction, employing an organic polymeric anion exchanger through the enrichment of lipoproteins/peptides from serum. Polymeric separation media are chemically stable over the wide pH range. Poly(GMA/DVB), poly(GMA/EGDMA), and poly(GPE/DVB) are synthesized by radical polymerization, derivatized as strong anion exchangers, and used for lipoproteins enrichment. Lipoprotein's surface is covered by phospholipids, having phosphate groups, therefore lipoproteins are enriched by the interaction of anion exchanger with the phosphate groups and eluted at the pH of 7.5. HDL are further isolated by precipitating the very low-density lipoproteins (VLDL) and low-density lipoproteins (LDL) with phosphotungstic acid as precipitating reagent, followed by delipidation via liquid/liquid extraction. Apolipoproteins profiling is done by MALDI-MS, and lipids are analyzed using gold nanoparticles in the LDI-MS process. This study introduces a lipoproteomics work flow in separation science which analyses the intact lipoproteins. Furthermore, solid phase extraction (SPE)-based methodology is reported for the first time in lipoproteomics. Use of organic polymers, high reproducibility, detailed analysis of lipoproteins, apoproteins/peptides, and lipids from the single serum sample are the distinctive features of this workflow. Being biomarkers of numerous diseases, lipoproteins have clinical significance, and this workflow can be used at diagnostic and therapeutic levels.

Enrichment of HDL proteome and phospholipidome from human serum via IMAC/MOAC affinity

“…Binding conditions are optimized by varying pH from 3 to 7.5 and then 8 in our previous work. At pH 3 and 8, phosphopeptides are also observed; however at 7.5 only lipo‐peptides are obtained (Javeed et al, ). IMAC affinity also shows high interaction for lipoproteins/peptides through the metal cations at set pH.…”

“…Later, gel filtration chromatography was employed for the extraction of HDLs from cardiovascular disease plasma samples; however, it lacks selectivity (Gordon, Deng, Lu, & Davidson, 2010). Furthermore, poly (GPE/dihydroxy benzoic acid [DHB])-, poly (GMA/EGDMA)-and poly(GMA/DVB)-based anion exchangers enrich lipoproteins (Javeed, Jabeen, Saeed, & Najam-ul-Haq, 2015). IMAC materials also enrich lipids (Lee, Venkataraman, Hwang, Han, & Hla, 2007) and HDLs (Collier, Jin, Topbas, & Bystrom, 2018).…”

In-Tip Lanthanum Oxide Monolith for the Enrichment of Phosphorylated Biomolecules

“…The digestion of β-casein, egg yolk, and human serum was carried out according to the reported protocol . Note that human serum samples were collected from the volunteers after taking their written consent and the approval was given by the ethical committee of the Institute.…”