This study demonstrates the use of Nanoxis LPI™ FlowCell in conjunction with LC-MS/MS to profile the protein content of a plasma membrane preparation derived from Arabidopsis thaliana, with respect to identity, membrane association types and subcellular locations.
Arabidopsis plasma membranes were isolated using an aqueous two-phase system1. LPI™ Maxi FlowCells were used to immobilize plasma membrane vesicles which were then digested with trypsin to produce peptide samples suitable for LC-MS/MS analysis. Plasma membranes were isolated and donated by the laboratory of Plant Physiology of the University of Groningen, the Netherlands.
Peptide matching using Mascot searching against the Swiss-Prot Arabidopsis thaliana subset resulted in identification of 249 proteins based on 843 unique peptides.
Figure 1. Membrane association class distribution
To assess the membrane purity of the sample a membrane association analysis was performed. 138 proteins (55%) were classed as membrane associated. A further 22% were noted as ribosomal, which can be classified as both soluble and membrane bound, and the remaining 23% were classed as soluble. Within the membrane protein group, proteins anchored to the membrane by a transmembrane domain or lipid anchor dominated (94), compared to peripheral (44), as seen in Figure 1.
Subcellular localization analysis of the membrane proteins revealed that the dominant membrane compartment represented was the plasma membrane, indicating that the aqueous two-phase enrichment process used for the sample preparation was efficient. 88 membrane proteins originated from the plasma membrane, with smaller contributions from ER/Golgi, vacuole, chloroplast and cytoskeleton (Figure 2). Note that some proteins have dual location and are therefore counted twice in this graph.
1. Larsson, C., Widell, S., Kjellbom, P. (1987) Methods Enzymol. 148, 558-568