Free-Flow Electrophoresis of Plasma Membrane Vesicles Enriched by Two-Phase Partitioning Enhances the Quality of the Proteome from Arabidopsis Seedlings

Abstract

The plant plasma membrane is the interface between the cell and its environment undertaking a range of important functions related to transport, signaling, cell wall biosynthesis, and secretion. Multiple proteomic studies have attempted to capture the diversity of proteins in the plasma membrane using biochemical fractionation techniques. In this study, two-phase partitioning was combined with free-flow electrophoresis to produce a population of highly purified plasma membrane vesicles that were subsequently characterized by tandem mass spectroscopy. This combined high-quality plasma membrane isolation technique produced a reproducible proteomic library of over 1000 proteins with an extended dynamic range including plasma membrane-associated proteins. The approach enabled the detection of a number of putative plasma membrane proteins not previously identified by other studies, including peripheral membrane proteins. Utilizing multiple data sources, we developed a PM-confidence score to provide a value indicating association to the plasma membrane. This study highlights over 700 proteins that, while seemingly abundant at the plasma membrane, are mostly unstudied. To validate this data set, we selected 14 candidates and transiently localized 13 to the plasma membrane using a fluorescent tag. Given the importance of the plasma membrane, this data set provides a valuable tool to further investigate important proteins. The mass spectrometry data are available via ProteomeXchange, identifier PXD001795.

Stress-Related Mitogen-Activated Protein Kinases Stimulate the Accumulation of Small Molecules and Proteins in Arabidopsis thaliana Root Exudates

“…This could indicate an increase of signaling complexes at the cell periphery after MPK3/MPK6 activation. While some of the apoplastic proteins are normally classified as intracellular proteins, such as Varicose (AT3G13300) or NADP-malic enzyme 2 (AT5G11670), they have recently been found in extracellular/cell wall or plasma membrane proteomes (De Michele et al, 2016; Nguyen-Kim et al, 2016). In particular, these also include six members of the Histone 2A family (Table 1), which are typically part of the core nucleosomes (Mitra et al, 2009; Heard et al, 2015; Nguyen-Kim et al, 2016).…”

“…a The PubMed ID (PMID) of the publication reporting the plasma membrane (PM), extracellular or plastid proteomics data are indicated: Nguyen-Kim et al, 2016 (PMID:26572690); Mitra et al, 2009 (PMID:19334764); Heard et al, 2015 (PMID:25900983); De Michele et al, 2016 (PMID:26781341); Charmont et al, 2005 (PMID:15694452); Zhang and Peck, 2011 (PMID:21433285); Helm et al, 2014 (PMID:24361574); Melonek et al, 2016 (PMID:26987276) .…”

The plasma membrane proteome of Medicago truncatula roots as modified by arbuscular mycorrhizal symbiosis

“…However, none of these processes is specific to PM proteins. Consequently, in the absence of a consensus mechanism for incorporation of proteins into the PM, large-scale identification of plasmalemmic proteins requires subcellular fractionation procedures and proteomic analysis (de Michele et al 2016).…”

Integrating cell biology and proteomic approaches in plants

“…Accumulation of storage proteins in root MVBs was proved by immunolabeling of 2S albumin [74]. New proteins were also identified in purified Golgi [74] and plasma membrane vesicles [75] using aqueous two-phase partitioning in combination with freeflow electrophoresis. The subcellular localization of few of them was microscopically validated by using particle bombardment in onion epidermal cells with either C-or N-terminal yellow fluorescent protein (YFP) constructs [76].…”