| Endoplasmic reticulum (ER) takes part in the protein synthesis, glycogenolysis and biosynthesis of lipid, cholesterol. It also has a close relationship with membrane and secretory proteins. For liver tissue, ER plays a great role in the biotransformation of hydrophobic endogenous metabolites and xenobiotics. The disorder of ER function can cause cell dysfunction and diseases. To gain a better understanding of the critical role of ER function in liver, we present the ER proteome study on C57BL/6J mouse liver. In contrast to other proteome studies we focused on the multiple fractionation strategy to both make full use of sample and compare different subcellular fractions from single homogenate. At the same time, the thorough purity evaluation method was applied to obtain highly purified organelle.Subsequently, endoplasmic reticulum was profiled with a new three-dimensional gel-based large scale proteome profiling strategy which combined SDS-PAGE separation of intact proteins with liquid chromatography gas phase fractionation (GPF) tandem mass spectrometry analysis. The results showed that using the GPF strategy, the number of detected peptide and proteins increased by nearly 50 percent respectively. The capture of single charge peptide with low mass spectrometry signal which missed during full mass scan was amazingly enhanced in GPF scan with mass range of 980-1600Da. The results demonstrated that the 3-D strategy provides a good choice to accomplish a large scale proteome profiling.From 6,152 and 6,935 spectra, 921, 1065 different proteins with high confidence were separately identified in rER and sER. Gene Ontology annotation of these proteins delineated their relationships with the ER function, indicating an involvement of ER proteins in numerous cellular processes. Compared with the proteome of endoplasmic reticulum in rat liver, 662 proteins were shared by both, account 53.5% and 42.5% of rat and mouse ER proteome separately. We propose that these common proteins are stably expressed proteins which are essential for maintenance of ER normal function. GO annotation with hyper geometric proved this hypothesis. We found that structural molecular activity and cell homeostasis are highly enriched in common proteins, which indicates that these were constitutive expressed proteins. And the function of uniquely identified proteins in mouse is highly enriched in immune response, stimulus response, which indicates that these proteins are transit proteins. Spectral count has been widely accepted as proteomic semi-quantitative method. Here we use EM algorism optimized spectral count method to quantify our identified proteins. Cluster analysis with spearman correlation is applied. Closer analysis of selected clusters reveals proteins which co-cluster with sec61, calnexin, and aldehyde dehydrogenase. Swiss-Prot location shows that proteins in respectively organelle are most dominate; proteins with unknown location are also highly abundant, including 217 proteins only identified in transcriptome as indicated in Swiss-Prot database. Unexpectedly, we found some proteins previously reported in cytosol are highly enriched in ER, underrepresented in cytosol. Fluorescence location first validated Ifi35 is indeed ER localized. Global localization experiments which are daughter project of HLPP are in preparation.As part of HLPP, this study focused on the sample preservation and subcellular fractionation strategy. Based on systematic comparison and evaluation, we got highly enriched endoplasmic reticulum from human and mouse separately. Furthermore, gas phase fractionation as a three dimensional peptide fractionation is introduced into the SDS-LC-MS/MS protein identification strategy. And stringent data filter criteria is also applied, from 6,152 and 6,935 unique peptides, 921, 1065 proteins with over 95% confidence and two peptides match are identified separately in rER and sER. Based on protein semi-quantification and clustering, a total of 217 unknown proteins are co-clustered with marker proteins, which provide their localization reference. Unexpectedly, we also find that 4 cytosolic proteins are indeed ER localized. Fluorescence first validated the ER localization of Ifi35.
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