Proteomic Analysis Of Plasma Membranes From Human Fetal Liver And Human Red Blood Cell

The plasma membrane (PM) constitutes the interface between eukaryotic cells and their external environment. Consequently the functions of proteins embedded in this membrane include cell/cell and cell/extracellular matrix recognition, the reception and transduction of extracellular signals and the transport of solutes and water molecules into and out of the cell. Moreover, the plasma membrane has been extensively targeted for drug design because plasma membrane proteins account for 70% of all known drug targets. For these reasons, proteomic analysis of plasma membrane is an area of intense interest.The human fetal liver (HFL) during 16-24 weeks of gestation is a major site of fetal hematopoiesis and of liver differentiation and development. Therefore, proteomic analysis of HFL PM will be helpful to understand these physiological activities. PM of HFL was isolated by differential centrifugation and sucrose dense centrifugation, and the enrichment fold of PM is 16 evaluated by 5'-nucleotidase specific enzyme assay. The proteome of intact membrane and membrane treated by alkali (PMt) were analyzed respectively. We applied two alternative approaches to identify PM proteins: 1) SDS-PAGE-RPLC-ESI-Q-TOF MS and ion trap MS; 2) "shotgun", protein digestion-SCX-RPLC-ESI-MS/MS. These two approaches allowed us to identify 532 unique proteins and 82 protein groups, in which 126 proteins were clearly localized in plasma membrane and 41 were unknown. In addition, transmembrane analysis showed that 30.5% of all identified proteins contain transmembrane domain. 126 PM proteins covered all of functional modules on PM, including lipid raft, signal transduction, membrane skeleton, cell contact, membrane fusion, transporter, lipid metabolism enzyme and so on. To our knowledge, this is the most comprehensive proteomic analysis of plasma membrane proteins of normal human tissues to date.Intriguingly, some cytoplasmic proteins (such as HSP70, HSP27, GRP78 and protein disulfide isomerase), mitochondria proteins (HSP60, VDAC-1, ATPsynthetase) and nuclear proteins (such as histone proteins, hNRP, Lasp-1 ) were identified in HFL PM in high abundance, which were also reported by other studies to be localized in PM. This phenomenon is very interesting and might suggest that the proteins with the known localization in other organelles identified in PM are not simply considered as contamination, and should be due to physiological trans-localization.It is inevitable that the PM isolated from HFL is contaminated by intracellular membranes. We can't determine if other subcellular proteins identifided on PM are really localized on PM. Therefore, we analyzed proteome of human red blood cell (RBC) membranes and take it as in vivo eukaryotic plasma membrane reference proteome due to the absence of all organelles within RBC. To get integral membrane proteins and adequately remove cytoplasmic proteins, RBC membranes were treated by 1M KC1 and 0.1M Na2CC>3 4 times by turns. In total, 112 unique proteins were identified by SDS-PAGE-RPLC-ESI-MS/MS and "shotgun" strategies. Among them, there were 64 integral membrane proteins, accounting for 55% of all identified proteins, and 16 functional unknown proteins. Glycosylated proteins accounts for 24% of all proteins, and 88.5% of glycosylated proteins are integral membrane proteins. RBC membranes proteins predominately enrich on membrane skeleton, transporter, membrane traffic, blood group system (cell adhesion) and enzymes. Unexpectedly, 10% RBC membranes proteins are ras superfamily proteins, including 2 rap proteins and 9 rab proteins. They may be involved in special physiological activities. In addition, other subcellular proteins were identified on RBC membranes, including those involving in physiolgical activities such as redox, lipid metabolism, glycolysis, protein transport, protein degradation and DNA binding. And some of them were identified on PM of HFL. Therefore, It is very likely that these proteins are localized on PM.As reference proteome of PM, the proteome of RBC membranes appears similar features to that of HFL PM such as high ratio of transporter, membrane skeleton and metabolism enzymes. However, there are differences between them. The proportions of blood group proteins and membrane traffic proteins on RBC membranes are higher,...