Membrane Proteome Profiling Research Articles

Metal organic layers enabled cell surface engineering coupling biomembrane fusion for dynamic membrane proteome profiling.

Systematically dissecting the highly dynamic and tightly communicating membrane proteome of living cells is essential for the system-level understanding of fundamental cellular processes and intricate relationship between membrane-bound organelles constructed through membrane traffic. While extensive efforts have been made to enrich membrane proteins, their comprehensive analysis with high selectivity and deep coverage remains a challenge, especially at the living cell state. To address this problem, we developed the cell surface engineering coupling biomembrane fusion method to map the whole membrane proteome from the plasma membrane to various organelle membranes taking advantage of the exquisite interaction between two-dimensional metal-organic layers and phospholipid bilayers on the membrane. This approach, which bypassed conventional biochemical fractionation and ultracentrifugation, facilitated the enrichment of membrane proteins in their native phospholipid bilayer environment, helping to map the membrane proteome with a specificity of 77% and realizing the deep coverage of the HeLa membrane proteome (5087 membrane proteins). Furthermore, membrane N-phosphoproteome was profiled by integrating the N-phosphoproteome analysis strategy, and the dynamic membrane proteome during apoptosis was deciphered in combination with quantitative proteomics. The features of membrane protein N-phosphorylation modifications and many differential proteins during apoptosis associated with mitochondrial dynamics and ER homeostasis were found. The method provided a simple and robust strategy for efficient analysis of membrane proteome, offered a reliable platform for research on membrane-related cell dynamic events and expanded the application of metal-organic layers.

Membrane Proteomic Profiling of Soybean Leaf and Root Tissues Uncovers Salt-Stress-Responsive Membrane Proteins.

Cultivated soybean (Glycine max (L.)), the world's most important legume crop, has high-to-moderate salt sensitivity. Being the frontier for sensing and controlling solute transport, membrane proteins could be involved in cell signaling, osmoregulation, and stress-sensing mechanisms, but their roles in abiotic stresses are still largely unknown. By analyzing salt-induced membrane proteomic changes in the roots and leaves of salt-sensitive soybean cultivar (C08) seedlings germinated under NaCl, we detected 972 membrane proteins, with those present in both leaves and roots annotated as receptor kinases, calcium-sensing proteins, abscisic acid receptors, cation and anion channel proteins, proton pumps, amide and peptide transporters, and vesicle transport-related proteins etc. Endocytosis, linoleic acid metabolism, and fatty acid biosynthesis pathway-related proteins were enriched in roots whereas phagosome, spliceosome and soluble NSF attachment protein receptor (SNARE) interaction-related proteins were enriched in leaves. Using label-free quantitation, 129 differentially expressed membrane proteins were found in both tissues upon NaCl treatment. Additionally, the 140 NaCl-induced proteins identified in roots and 57 in leaves are vesicle-, mitochondrial-, and chloroplast-associated membrane proteins and those with functions related to ion transport, protein transport, ATP hydrolysis, protein folding, and receptor kinases, etc. Our proteomic results were verified against corresponding gene expression patterns from published C08 RNA-seq data, demonstrating the importance of solute transport and sensing in salt stress responses.

Membrane proteomic profiling enhances drug target detection

AbstractMembrane proteins are an important category of drug targets in human diseases. However, membrane protein characterization is challenging due to their hydrophobicity, low abundance, and poor protease accessibility of the proteins. To facilitate reproducible and sensitive profiling of the druggable membrane proteome, we reported a strategy combining membrane protein extraction and data‐independent acquisition mass spectrometry (DIA‐MS). By a single‐shot DIA analysis for a model lung cancer cell line, the results show good enrichment of 2,740 confidently identified membrane proteins, which accounts for 71% proteins identified from the membrane‐enriched sample and includes 202 FDA‐approved drug targets and 462 uniquely identified membrane proteins compared to the result from whole‐cell lysate. Furthermore, the membrane fraction enrichment significantly enhanced protein abundance of 1,245 membrane proteins, and 22 proteins were identified in the non‐small‐cell lung cancer pathway, including 6 lung cancer drug targets. The approach demonstrated better identification in higher intensities and signal‐to‐noise ratios of peptide ions. The drug targets increased 3.3–25.9‐fold (median 15.5‐fold) in peptide abundance and 1.0–12.0% in (median 5.3%) protein coverage. The membrane proteomic profiling strategy will advance the applications of quantitative proteomics in drug target discovery.

Spatially resolved cell tagging and surfaceome labeling via targeted photocatalytic decaging

<h2>Summary</h2> Spatially resolved <i>in situ</i> tagging of the cell of interest is crucial for in-depth mechanistic dissection of multicellular architectures or processes. With continuing interest in bioorthogonal photocatalytic decaging chemistry, we herein report the extracellular-targeted photocatalytic decaging system (CAT-Ex) for spatially resolved cell tagging and surface proteome profiling under living conditions. An antibody-conjugated photocatalysis system was established and extensively validated, enabling photocatalytic decaging of biotin precursors and proximal quinone methide probes on target cells. Visible-light-controlled selective cell tagging in cell mixture as well as in primary cells from tumor xenografts were demonstrated. Spatially resolved membrane proteome profiling was further achieved by coupling quinone methide decaging chemistry with CAT-Ex, revealing a potential microdomain protein cluster surrounding the endogenous HER2 receptor. Finally, we expanded our strategy to photocatalytic prodrug decaging for selective tumor cell killing, establishing CAT-Ex as a general platform for diverse photo-controlled molecular manipulations on targeted cells with spatial-temporal precision.

Combined Transcriptome and Proteome Profiling for Role of pfEMP1 in Antimalarial Mechanism of Action of Dihydroartemisinin.

ABSTRACTMalaria parasites induce morphological and biochemical changes in the membranes of parasite-infected red blood cells (iRBCs) for propagation. Artemisinin combination therapies are the first-line antiplasmodials in countries of endemicity. However, the mechanism of action of artemisinin is unclear, and drug resistance decreases long-term efficacy. To understand whether artemisinin targets or interacts with iRBC membrane proteins, this study investigated the molecular changes caused by dihydroartemisinin (DHA), an artemisinin derivative, in Plasmodium falciparum 3D7 using a combined transcriptomic and membrane proteomic profiling approach. Optical microscopy and scanning electron microscopy showed that DHA can cause morphological variation in the iRBC membrane. We identified 125 differentially expressed membrane proteins, and functional analysis indicated structural molecule activity and protein export as key biological functions of the two omics studies. DHA treatment decreased the expression of var gene variants PF3D7_0415700 and PF3D7_0900100 dose-dependently. Western blotting and immunofluorescence analysis showed that DHA treatment downregulates the var gene encoding P. falciparum erythrocyte membrane protein-1 (pfEMP1). pfEMP1 knockout significantly increased artemisinin sensitivity. Results showed that pfEMP1 might be involved in the antimalarial mechanism of action of DHA and pfEMP1 or its regulated factors may be further exploited in antiparasitic drug design. The findings are beneficial for elucidating the potential effects of DHA on iRBC membrane proteins and developing new drugs targeting iRBC membrane.IMPORTANCE Malaria parasites induce morphological and biochemical changes in the membranes of parasite-infected red blood cells (iRBCs) for propagation, with artemisinin combination therapies as the first-line treatments. To understand whether artemisinin targets or interacts with iRBC membrane proteins, this study investigated the molecular changes caused by dihydroartemisinin (DHA), an artemisinin derivative, in Plasmodium falciparum 3D7 using a combined transcriptomic and membrane proteomic profiling approach. We found that DHA can cause morphological changes of iRBC membrane. Structural molecule activity and protein export are considered to be the key biological functions based on the two omics studies. pfEMP1 might be involved in the DHA mechanism of action. pfEMP1 or its regulated factors may be further exploited in antiparasitic drug design. The findings are beneficial for elucidating the potential effects of DHA on iRBC membrane proteins and developing new antimalarial drugs targeting iRBC membrane.

Deep Membrane Proteome Profiling of Rat Hippocampus in Simulated Complex Space Environment by SWATH

Despite the development and great progress in the field of space biology, the astronauts are still facing many challenges in space. The space environment in which astronauts stay includes microgravity, noise, circadian rhythms disorder, and confinement, which has deep effect both on the physiology and psychology of astronauts. It was reported that long-term flight could cause the astronauts’ anxiety and depression. However, the underlying mechanism is not yet fully understood. Therefore, in the present study, the rat tail suspension model with noise, circadian rhythms, and confinement was employed to simulate complex space environment. We found that the rats exhibited the depressive-like behavior by the sucrose preference, forced swimming, and open-field tests. The membrane proteome of the rat hippocampus was investigated by “SWATH quantitation” technology both in control and simulated complex space environment (SCSE) groups. Out of 4520 quantified proteins, 244 differentially expressed membrane proteins were obtained between the SCSE and control rats, which were functionally enriched in a series of biological processes, such as translation, protein phosphorylation, brain development, endocytosis, nervous system development, axonogenesis, and vesicle-mediated transport. We found a reduction level of neurexin-2, the light, medium, heavy polypeptide of neurofilament, rab 18, synaptogyrin 1, and syntaxin-1A and an increase level of neuroligin-1, munc18, snapin, synaptotagmin XII, complexin-1, etc., which may play a key part in the development of depression. Furthermore, GSK-3 β protein was upregulated in mass spectrometry, which was further validated by western blotting. The results of the study do the favor in designing the effective countermeasures for the astronauts in the future long-term spaceflight.

Membrane proteomic profiling of the heart: past, present, and future.

Cardiovascular diseases remain the most rapidly rising contributing factor of all-cause mortality and the leading cause of inpatient hospitalization worldwide, with costs exceeding $30 billion annually in North America. Cell surface and membrane-associated proteins play an important role in cardiomyocyte biology and are involved in the pathogenesis of many human heart diseases. In cardiomyocytes, membrane proteins serve as critical signaling receptors, Ca2+ cycling regulators, and electrical propagation regulators, all functioning in concert to maintain spontaneous and synchronous contractions of cardiomyocytes. Membrane proteins are excellent pharmaceutical targets due to their uniquely exposed position within the cell. Perturbations in cardiac membrane protein localization and function have been implicated in the progression and pathogenesis of many heart diseases. However, previous attempts at profiling the cardiac membrane proteome have yielded limited results due to poor technological developments for isolating hydrophobic, low-abundance membrane proteins. Comprehensive mapping and characterization of the cardiac membrane proteome thereby remains incomplete. This review will focus on recent advances in mapping the cardiac membrane proteome and the role of novel cardiac membrane proteins in the healthy and the diseased heart.

Membrane proteome profiling of Mentha arvensis leaves in response to Alternaria alternata infection identifies crucial candidates for defense response

ABSTRACTThe leaf spot disease of Mentha arvensis, caused by Alternaria alternata, is a devastating foliar disease worldwide and leads to considerable economic losses. In this investigation, 2-dimensional gel electrophoresis (2-DE) was used to identify the membrane proteins potentially involved in M. arvensis - A. alternata interaction. Membrane proteins, isolated from leaves of control and infected plants, were analyzed by 2-DE and identified using mass spectrometry (MALDI TOF–TOF MS/MS). Our analysis identified 21 differentially expressed membrane proteins including several interesting receptors and channel proteins. Of these identified proteins, 34% were found to be involved in plant defense responses. Leucine-rich repeat family protein/ protein kinase family protein which plays critical role in stress response and nucleotide-binding site–leucine-rich repeat (NBS-LRR) which is involved in detecting the advent of pathogen on plant surface were identified to be up-regulated in our study. Interestingly, AKT1-like potassium channel protein which is known to play a crucial role in maintaining ion homeostasis within the cell was also upregulated in the infected sample. In addition, ADP ribolysation factor (ARF)-GTPase activating domain containing protein, a membrane trafficking protein, was also up-regulated in the current study. Protein-protein interaction network analysis followed by functional enrichment revealed that transmembrane ion transport-related proteins represented a major class in this network followed by nucleic acid binding proteins and proteins with kinase activities respectively. Together, our investigation identified several key defense-related proteins which are crucial sensors for detecting pathogen invasion and can serve as a potential resource to understand disease resistance mechanism in mint.

A robust mass spectrometry method for rapid profiling of erythrocyte ghost membrane proteomes

BackgroundRed blood cell (RBC) physiology is directly linked to many human disorders associated with low tissue oxygen levels or anemia including chronic obstructive pulmonary disease, congenital heart disease, sleep apnea and sickle cell anemia. Parasites such as Plasmodium spp. and phylum Apicomplexa directly target RBCs, and surface molecules within the RBC membrane are critical for pathogen interactions. Proteomics of RBC membrane ‘ghost’ fractions has therefore been of considerable interest, but protocols described to date are either suboptimal or too extensive to be applicable to a larger set of clinical cohorts.MethodsHere, we describe an optimised erythrocyte isolation protocol from blood, tested for various storage conditions and explored using different fractionation conditions for isolating ghost RBC membranes. Liquid chromatography mass spectrometry (LC–MS) analysis on a Q-Exactive Orbitrap instrument was used to profile proteins isolated from the comparative conditions. Data analysis was run on the MASCOT and MaxQuant platforms to assess their scope and diversity.ResultsThe results obtained demonstrate a robust method for membrane enrichment enabling consistent MS based characterisation of > 900 RBC membrane proteins in single LC–MS/MS analyses. Non-detergent based membrane solubilisation methods using the tissue and supernatant fractions of isolated ghost membranes are shown to offer effective haemoglobin removal as well as diverse recovery including erythrocyte membrane proteins of high and low abundance.ConclusionsThe methods described in this manuscript propose a medium to high throughput framework for membrane proteome profiling by LC–MS of potential applicability to larger clinical cohorts in a variety of disease contexts.

Membrane Proteomics of Impaired Energetics and Cytoskeletal Disorganization in Elderly Diet-Induced Diabetic Mice.

Diabetic cardiomyopathy is a well-recognized complication of diabetes, but its pathophysiology is unclear. We aimed to investigate the mechanisms underlying cardiac dysfunction in an elderly type 2 diabetic (T2DM) mouse model, using membrane proteomic analyses. Elderly mice were fed a high fat diet for 12 weeks to induce T2DM, and myocardial structure and function were assessed by echocardiography. Cardiomyocytes were isolated by Langendorff perfusion and subjected to iTRAQ-based quantitative membrane proteomic profiling, immunoblotting, and real-time quantitative reverse-transcriptase polymerase chain reaction. Compared to controls, elderly T2DM mice showed worse systolic function, more myocardial fibrosis and up-regulation of several heart failure markers (all p < 0.05). Cardiomyocyte membrane proteomic profiling revealed that 417 proteins had differential expressions related to perturbations in several biological processes in T2DM mice compared with the control. The most up-regulated proteins were involved in oxidative phosphorylation, whereas many down-regulated proteins were involved in cytoskeletal regulation. Differential protein expression correlated with myocardial systolic velocity by tissue Doppler. In addition, cardiomyocyte immunofluorescence staining showed greater disorganization of thick/parallel F-actin stress fibers and marked reduction in F-to-G-actin ratio in T2DM vs control (p < 0.05), which paralleled worsened myocardial systolic velocity. We concluded that cardiac contractile dysfunction in elderly T2DM mice was associated with impaired energetics and cytoskeletal disorganization.

Dissolving capability difference based sequential extraction: A versatile tool for in-depth membrane proteome analysis

Profiling membrane proteins would facilitate revealing disease mechanism and discovering new drug targets as they play essential roles in cellular signaling, substrate transport, and cell adhesion. However, the analysis of membrane proteins still remains a challenge due to their high hydrophobicity, as well as the suppression effect of high abundant soluble proteins. In this work, to achieve a membrane proteome profiling, a sample preparation strategy based on sequential extraction at the protein level assisted by a range of extraction reagents with different dissolving capabilities, followed by nano-RPLC-ESI-MS/MS analysis was developed and applied for HeLa cell line analysis. It was found that with progressively harsher extraction reagents (i.e., 2 M NaCl, 4 M urea, 0.1 M Na2CO3, and 10% 1-dodecyl-3- methyl-imidazolium chloride (C12ImCl) performed, much more high hydrophobic proteins and low abundant proteins were identified. With our developed strategy, 5553 of the identified proteins (4419 gene products) were annotated to be membrane proteins and 2573 proteins (2183 gene products) have at least one transmembrane domain, to our best knowledge, which is the most comprehensive membrane proteome dataset for HeLa cell line. Notably, 110 of the identified membrane proteins were discovered in the “missing proteins” list referred to those in the neXtProt database. All above results indicated that our strategy has great potential to tackle the difficult but relevant task of identifying and profiling membrane proteins.

Glycan Moieties as Bait to Fish Plasma Membrane Proteins.

Plasma membrane proteome analysis is of significance for screening candidate biomarkers and drug targets. However, due to their low abundance and lack of specific groups that can enable their capture, the plasma membrane proteins (PMPs) are under-represented. On the basis of the fact that PMPs are embedded in or anchored to the phospholipid bilayer of the plasma membrane and the glycan moieties of proteins and lipids located on the plasma membrane are exposed outside of the cell surface, we proposed a strategy to capture PMPs, termed as glycan moieties-directed PMPs enrichment (GMDPE). With the glycan moieties exposed outside of the cells as bait to ensure the selectivity and the phospholipid bilayer as raft to provide the sensitivity, we applied this strategy into the plasma membrane proteome analysis of HeLa cells, and in total, 772 PMPs were identified, increased by 4.5 times compared to those identified by the reported cell surface biotinylation method. Notably, among them, 86 CD antigens and 16 ion channel proteins were confidently identified. All these results demonstrated that our proposed approach has great potential in the large scale plasma membrane proteome profiling.

Rapid High-pH Reverse Phase StageTip for Sensitive Small-Scale Membrane Proteomic Profiling.

Membrane proteins are crucial targets for cancer biomarker discovery and drug development. However, in addition to the inherent challenges of hydrophobicity and low abundance, complete membrane proteome coverage of clinical specimen is usually hindered by the requirement of large amount of starting materials. Toward comprehensive membrane proteomic profiling for small amounts of samples (10 μg), we developed high-pH reverse phase (Hp-RP) combined with stop-and-go extraction tip (StageTip) technique, as a fast (∼15 min.), sensitive, reproducible, high-resolution and multiplexed fractionation method suitable for accurate quantification of the membrane proteome. This approach provided almost 2-fold enhanced detection of peptides encompassing transmembrane helix (TMH) domain, as compared with strong anion exchange (SAX) and strong cation exchange (SCX) StageTip techniques. Almost 5000 proteins (∼60% membrane proteins) can be identified in only 10 μg of membrane protein digests, showing the superior sensitivity of the Hp-RP StageTip approach. The method allowed up to 9- and 6-fold increase in the identification of unique hydrophobic and hydrophilic peptides, respectively. The Hp-RP StageTip method enabled in-depth membrane proteome profiling of 11 lung cancer cell lines harboring different EGFR mutation status, which resulted in the identification of 3983 annotated membrane proteins. This provides the largest collection of reference peptide spectral data for lung cancer membrane subproteome. Finally, relative quantification of membrane proteins between Gefitinib-resistant and -sensitive lung cancer cell lines revealed several up-regulated membrane proteins with key roles in lung cancer progression.

Abstract 1607: Quantitative membrane proteome profiling to discover therapeutic targets for adult T-cell leukemia (ATL)

Abstract Human T-cell leukemia virus type 1 (HTLV-1) is one of retroviruses known as the causative agent of adult T-cell leukemia (ATL), which is infected within 20 million people in the world. ATL develops at the prevalence of only 5% among HTLV-1-infected individuals after a long latency period over 30 years. However, the outcomes of conventional treatment for ATL are not satisfactory, exhibiting overall 5-year survival rate of 20%. To discover novel therapeutic targets for ATL, we performed label-free quantitative membrane proteome analysis for CD4+ T-cell subpopulation in which HTLV-1 infected cells are enriched. Tryptic digests of CD4+ T-cells were prepared from 14 normal donors (ND), 21 asymptomatic carriers (AC), and 13 ATL patients. To focus on the membrane derived peptides, glycosylated peptides were enriched by lectin column chromatography (Mol Cell Proteomics. 2010,9(9),1819) since most of membrane proteins are glycosylated. The eluates were analyzed by LC/MS/MS and 8,658 N-glycosylated peptides derived from 1,134 N-glycosylated proteins were identified. The therapeutic targets were subsequently extracted by a couple of criteria as follows. The all-or-nothing analysis explored peptides detected in none or 1 case of ND+AC group, resulting in identification of 31 peptides derived from 19 proteins. Additionally Welch's t-test (ATL vs. ND+AC) identified 33 peptides derived from 14 proteins (p &amp;lt; 0.05 and fold change &amp;gt; 2.0). The identified therapeutic target molecules above included cell adhesion molecule 1 (CADM1 or TSLC1) which overexpresses in ATL cells and induces organ infiltration of tumor cells. Programmed cell death 1 ligand 1 (PD1L1) was also found in our therapeutic target list, which was known to be up-regulated in ATL cells and involved in induction of immune tolerance. These facts strongly emphasize the credibility of our quantitative profiling results. We would like to show the results of functional inhibitory experiments for novel therapeutic targets and discuss the uncovered molecular mechanisms of ATL progression. Note: This abstract was not presented at the meeting. Citation Format: Makoto Ishihara, Natsumi Araya, Tomoo Sato, Risa Fujii, Ayako Tatsuguchi, Naomi Saichi, Hidewaki Nakagawa, Yoshihisa Yamano, Koji Ueda. Quantitative membrane proteome profiling to discover therapeutic targets for adult T-cell leukemia (ATL). [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1607. doi:10.1158/1538-7445.AM2014-1607

1-Dodecyl-3-methylimidazolium chloride-assisted sample preparation method for efficient integral membrane proteome analysis.

Due to their extremely hydrophobic nature, the analysis of integral membrane proteins (IMPs) is of great challenge. Although various additives have been applied to improve the solubility of IMPs, they still suffer from low solubilization efficiency, incompatibility with trypsin digestion, or interference with MS detection. Herein, the systematic study on the effect of ionic liquid structure on membrane protein solubilization and trypsin biocompatibility was performed, based on which 1-dodecyl-3-methylimidazolium chloride (C12Im-Cl) was selected for the sample preparation of IMPs. Compared with other commonly used additives, such as sodium dodecyl sulfate (SDS), Rapigest, and methanol, C12Im-Cl showed the best performance. In addition, with a strong cation exchange trap column, it could be easily removed after trypsin digestion, which not only was beneficial to avoid protein precipitation during digestion but also had no adverse effect on LC-MS-based separation and detection. Such a C12Im-Cl-assisted sample preparation method was further applied to the membrane proteome analysis of rat brain. Compared with the SDS-assisted method, 1.4 and 3.5 times improvement on the identified IMP and hydrophobic peptide number were achieved (251 vs 178, and 982 vs 279). All these results demonstrated that the C12Im-Cl-assisted sample preparation method is of great promise to promote the large-scale membrane proteome profiling.

Reprogramming- and pluripotency-associated membrane proteins in mouse stem cells revealed by label-free quantitative proteomics

Induced pluripotent stem cells (iPSCs), derived from somatic cells and functionally very similar to embryonic stem cells (ESCs), are at the center stage of intense research in regenerative medicine. We carried out the first membrane proteomic profiling of mouse iPSCs, in comparison with ESCs and adult mouse tail tip fibroblasts (TTFs) from which iPSCs were generated. Using a proteomic workflow combining membrane fractionation, SDS-PAGE separation and nanoUPLC-MS(E) technology, we identified 673, 679 and 682 non-redundant proteins from mouse iPSC, ESC and TTF membrane fractions, respectively. Label-free quantitation revealed 155 reprogramming-associated and 128 pluripotency-associated transmembrane proteins. Furthermore, a small group of 23 membrane proteins mainly involved in amino acid/glucose/ion transport, membrane fusion and vesicular trafficking were found potentially regulated between miPSCs and mESCs. Expression changes of selected proteins were verified by qPCR, western blot and/or immunofluorescence analyses in a wider array of cell types. Notably, epithelial cell adhesion molecules, glucose transporters 1 and 3, transferrin receptor and several nuclear membrane-associated components were highly expressed in both iPSCs and ESCs, relative to TTFs. Moreover, knock-down of glucose transporter 3 in ESCs impaired the beating function of ESC-derived cardiomyocytes, suggesting its potential role in mediating stem cell differentiation. This study constitutes a membrane proteomic resource for murine iPSCs and ESCs, and offers a comparison between pluripotent stem cells and fibroblasts in the proteomic landscape. An integrated proteomics platform combining technologies of membrane fractionation, LC-MS(E) analysis and label-free quantitation was developed to identify membrane proteins with their abundances related to reprogramming of fibroblasts or maintenance of stem cell pluripotency. The high similarity in the membrane proteomic patterns between iPSCs and ESCs strengthens the usefulness of iPSCs in biomedical research and therapeutic application. Moreover, we found a small subset of membrane proteins potentially regulated between miPSCs and mESCs. This membrane proteomic resource of pluripotent stem cells would be expected to inspire further investigations leading to discovery of new regulatory factors or membrane markers for reprogramming and pluripotency.

Coupling Formic Acid Assisted Solubilization and Online Immobilized Pepsin Digestion with Strong Cation Exchange and Microflow Reversed-Phase Liquid Chromatography with Electrospray Ionization Tandem Mass Spectrometry for Integral Membrane Proteome Analysis

In this study, a facile system for membrane proteome profiling was established, in which membrane proteins were solubilized by formic acid, online digested by a pepsin-based immobilized enzyme reactor (pepsin-IMER), and analyzed by strong cation exchange and microflow reversed-phase liquid chromatography with electrospray ionization tandem mass spectrometry (SCX-μRPLC-ESI-MS/MS). Under optimized conditions, such a system showed excellent compatibility between all crucial steps and was successfully applied for analyzing integral membrane proteins extracted from rat liver microsomes. Out of the 235 unique proteins positively identified, 39% (91/235) were annotated as membrane proteins with one or more transmembrane domains (TMDs). It is anticipated that the efficient sample treatment and the relevant online analytical system might provide a promising tool for automated and comprehensive profiling of membrane proteomes.

Increased coronin-1C expression is related to hepatocellular carcinoma invasion and metastasis

To search for hepatocellular carcinoma (HCC) invasion related biomarkers using the cell membrane proteomics approaches, and to validate the markers using experimental and clinical specimens. The HCCLM9 and MHCC97L cells with a similar genetic background and remarkably different metastasis behaviors were used for comparative membrane proteome profiling using sodium dodecyl sulfate polyacrylamide gel electrophoresis and electrospray ionization mass spectrometry technologies. Candidate protein makers were further validated by western blot on cells, immunohistochemistry (IHC) on animal tumor tissues, and tissue micro-array on clinical specimens. The membrane proteins of MHCC97L and HCCLM9 cells were compared by sodium dodecyl sulfate polyacrylamide gel electrophoresis analyses. 14 proteins were identified by ESI-MS/MS among the differential bands. Coronin-1C was overexpressed in HCCLM9 (7.31+/-0.73) versus MHCC97L (2.84+/-0.99) validated by western blot. Elevated coronin-1C expression was observed in liver cancer tissues of HCCLM9 nude mice. IHC study in 115 human HCC specimens demonstrated that patients with higher coronin-1C expression had more advanced stage. The study suggests that coronin-1C could be a potential molecule to predict HCC invasive behavior.

Separation and identification of mouse liver membrane proteins using a gel-based approach in combination with 2DnanoLC-Q-TOF-MS/MS

In this work, we present results of membrane proteome profiling from mouse liver tissues using a gel-based approach in combination with 2DnanoLC-Q-TOF-MS/MS. Following purification of the membrane fraction, SDS-PAGE was carried out as a useful separation step. After staining, gels with protein bands were cut, reduced, alkylated and trypsin-digested. The peptide mixtures extracted from each gel slice were fractionated by two-dimensional nano liquid chromatography (2DnanoLC) coupled online with tandem mass spectrometry analysis (NanoESI-Q-TOF-MS/MS). The proteins were identified by MASCOT search against a mouse protein database using a peptide and fragment mass tolerance of ±0.5 Da. Protein identification was carried out using a Mowse scoring algorithm with a confidence level of 95% and processed by MSQuant v1.5 software for further validation. In total, 318 verified membrane proteins from mouse liver tissues were identified; 66.67% of them (212 proteins) contained at least one or more transmembrane domains predicted by the SOSUI program and 43 were found to be unique microsome membranes. Furthermore, GRAVY values of membrane proteins varied in the range -1.1276 to 0.9016 and only 31 (9.76%) membrane proteins had positive values. The functions and subcellular locations of the identified proteins were categorized as well, according to universal GO annotations.

Selenium binding protein 1 in ovarian cancer

Selenium binding protein 1 (SELENBP1) was identified to be the most significantly down-regulated protein in ovarian cancer cells by a membrane proteome profiling analysis. SELENBP1 expression levels in 4 normal ovaries, 8 benign ovarian tumors, 12 borderline ovarian tumors and 141 invasive ovarian cancers were analyzed with immunohistochemical assay. SELENBP1 expression was reduced in 87% cases of invasive ovarian cancer (122/141) and was significantly reduced in borderline tumors and invasive cancers (p<0.001). Cox multivariate analysis within the 141 invasive cancer tissues showed that SELENBP1 expression score was a potential prognostic indicator for unfavorable prognosis of ovarian cancer (hazard ratio [HR], 2.18; 95% CI=1.22-3.90; p=0.009). Selenium can disrupt the androgen pathway, which has been implicated in modulating SELENBP1 expression. We investigated the effects of selenium and androgen on normal human ovarian surface epithelial (HOSE) cells and cancer cells. Interestingly, SELENBP1 mRNA and protein levels were reduced by androgen and elevated by selenium treatment in the normal HOSE cells, whereas reversed responses were observed in the ovarian cancer cell lines. These results suggest that changes of SELENBP1 expression in malignant ovarian cancer are an indicator of aberration of selenium/androgen pathways and may reveal prognostic information of ovarian cancer.