With the intention of enhancing existing knowledge on endomembrane proteomics in plants, Heard et al. (2015) devised an affinity purification workflow for mass spectrometric analysis in Arabidopsis thaliana seedlings.1 Their results give an initial overview of proteins involved in endosomal and secretory pathways, allowing researchers to predict localization within plants.
The research team grew A. thaliana plants from seeds, harvesting them for protein extraction and affinity purification at eight days. They created a modified affinity enrichment workflow for seedlings stably expressing a range of fluorescence-tagged markers that would validate isolation from endosomal compartments. Heard et al. chose seven subcellular proteins—RABD2a/ARA5, RABF2b/ARA7, RABF1/ARA6 and RABG3f—as endomembrane markers, in addition to GOT1, CLC2 and VAMP711 for subcellular localization. Using these seven bait proteins, the researchers could demonstrate that the enrichment strategy worked, in addition to providing cellular localization data for proteins co-identified.
Following protein extraction and purification, Heard et al. separated the samples using 4–20% Tris-Glycine gel electrophoresis, and then excised relevant bands for in-gel trypsin digestion. They analyzed the digests by liquid chromatography–tandem mass spectrometry (LC-MS/MS) using an Orbitrap Fusion or LTQ Orbitrap XL mass spectrometer (both Thermo Scientific). The team inspected the results with Proteome Discoverer, v1.2, (Thermo Scientific) for raw spectral data analysis.
The scientists also conducted transient expression studies by microprojectile bombardment of four- to five-week-old A. thaliana leaves. They studied the results with confocal microscopy to identify cellular location with live cell imaging.
Heard et al. found that the modified affinity enrichment protocol gave reliable results for proteome isolation from the endosomal compartment. They confirmed the findings by Western immunoblotting to show enrichment. From these sample preparations, the team identified 433 proteins by LC-MS/MS analysis showing good enrichment with one or more bait proteins. They found overlaps in proteins identified between bait protein pulldowns, but also some unique identities for individual markers. Five proteins showed good enrichment for all seven bait proteins, whereas four enriched with six out of the seven.
Further structural analysis for indication of cellular localization and possible origin showed that 34% of the proteins identified contained one or more transmembrane domains (n = 64 contained one transmembrane domain; n = 58 contained multiple domains). This left 66% of the initial 433 proteins identified from LC-MS/MS analysis as putative cargo proteins.
Heard et al. are confident that the results obtained are valuable for use by other research teams as markers to predict cellular protein localization in plants. They describe the results as a starting point for research into endomembrane regulatory proteins in plants, suggesting that further validation in future studies is necessary.
1. Heard, W., et al. (2015) “Identification of regulatory and cargo proteins of endosomal and secretory pathways in Arabidopsis thaliana by proteomic dissection,” Molecular and Cellular Proteomics, 14(7) (pp. 1796–1813), doi: 10.1074/mcp.M115.050286.