Protein Lipidation Types: Current Strategies for Enrichment and Characterization

Abstract

Post-translational modifications regulate diverse activities of a colossal number of proteins. For example, various types of lipids can be covalently linked to proteins enzymatically or non-enzymatically. Protein lipidation is perhaps not as extensively studied as protein phosphorylation, ubiquitination, or glycosylation although it is no less significant than these modifications. Evidence suggests that proteins can be attached by at least seven types of lipids, including fatty acids, lipoic acids, isoprenoids, sterols, phospholipids, glycosylphosphatidylinositol anchors, and lipid-derived electrophiles. In this review, we summarize types of protein lipidation and methods used for their detection, with an emphasis on the conjugation of proteins with polyunsaturated fatty acids (PUFAs). We discuss possible reasons for the scarcity of reports on PUFA-modified proteins, limitations in current methodology, and potential approaches in detecting PUFA modifications.

Keywords: mass spectrometry; polyunsaturated fatty acid; protein lipidation; proteomics.

Figure 1

Various analytical methods to identify and characterize protein lipidations. (A) Radioactive isotope-labeling. Any type of protein lipidation can be identified using this method if the corresponding isotope-labeled lipid is available; (B) Antibody affinity enrichment. In general, any type of protein lipidation can be detected if a suitable pan-antibody is available; (C–E) ABE and similar methods. These methods are used for detecting S-pamitoylation; (F) Click chemistry. Protein lipidations that can react with specific alkynyl/azide-lipid probes can be identified; (G) Biotin hydrazide affinity capture. Only proteins containing carbonyl or aldehyde groups are suitable for this method to detect the LDE modifications; (H) Lipid esterification. Some saturated or unsaturated fatty acid moieties derived from protein acylations can be identified if the process of esterification on dissociative lipid (usually hydrolysis) is available.

Figure 2

Methods to detect PUFA-modified proteins. (A) Flowchart of ABE and GC/LC-MS. Group A treats the supernatant from the acetone precipitation in cells; Group B treats the precipitation from the acetone precipitation in cells; Group C1 and D1 (+NH₂OH group) add NH₂OH and acetone to the above precipitation and further treats the second supernatant and precipitation as group C1 and D1; Group C2 and D2 (-NH₂OH group) add control and acetone to the above precipitation and further treats the second supernatant and precipitation as group C2 and D2; (B) The synthesis of the alkynyl-linoleic acid (alk-LA) probe. (C) Flowchart of the Click-chemistry method employed on total-protein or membrane-protein samples.