O

endomembranes (dextran)

figure 21.1. The two-phase partitioning method (PEG/Dextran) to isolate a PM-enriched fraction. The PM vesicles are concentrated in the PEG upper phase; all the other endomem-branes are recovered in the dextran lower phase. The fraction enriched in PM will be pelleted by an ultracentrifugation (for details, see reference 8)

endomembranes are concentrated in the lower phase [7]. Almost all the PM proteomes reported so far showed the use of the two-phase partitioning method to purify PM fractions (Table 21.1). Two recent reviews discussed the advantages of that procedure and described its use for proteomics approaches [8, 9].

How to Assess Sample Purity? Historically, in order to estimate both the yield of PM protein recovery and the contamination from other subcellular compartments, enzymatic markers known from former biochemical studies as showing specific activities assigned to a membrane system were measured. Typically, P-type H+ ATPase activity was used as a specific marker of the plant PM [10], together with markers from other plant membrane system [reviewed in reference 11]. During the past decade, the number of well-characterized membrane proteins increased significantly, providing new tools for detecting individual proteins and consequently for characterizing a membrane fraction. Enrichment of immunoreactivity for known marker proteins of the target subcellular compartments, together with a decrease in the immunoreactivity for markers known to be part of other subcellular compartments, can be monitored. To date, complementary immunological tests are currently carried out on membrane fractions to assess both PM enrichment and PM purity (Table 21.1).

Still nowadays, endomembrane contaminants in the PM suspension cannot be avoided, because none of the available extraction/purification procedures allow the recovering of a 100% pure PM fraction so far. Among the advantages, the proteomics analyses themselves bring an overview of the contamination level, and not a view restricted to one, at most two proteins per endomembrane. Most of the reported PM proteomes point out as major contaminations (i) ribosomal proteins [12, 13] and (ii) proteins from other cell compartments, and especially the cytosol, that can be assessed by quantitative proteomics approaches [14]. Considering results combining both biochemical and enzymological tests on a same membrane fraction, contamination was assessed to be in the range of a few percent (Table 21.1 and references therein).

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