Membrane Proteome Analysis Research Articles

Proteomic and bioinformatic analysis of membrane proteome in type 2 diabetic mouse liver

Type 2 diabetes mellitus (T2DM) is the most prevalent and serious metabolic disease affecting people worldwide. T2DM results from insulin resistance of the liver, muscle, and adipose tissue. In this study, we used proteomic and bioinformatic methodologies to identify novel hepatic membrane proteins that are related to the development of hepatic insulin resistance, steatosis, and T2DM. Using FT-ICR MS, we identified 95 significantly differentially expressed proteins in the membrane fraction of normal and T2DM db/db mouse liver. These proteins are primarily involved in energy metabolism pathways, molecular transport, and cellular signaling, and many of them have not previously been reported in diabetic studies. Bioinformatic analysis revealed that 16 proteins may be related to the regulation of insulin signaling in the liver. In addition, six proteins are associated with energy stress-induced, nine proteins with inflammatory stress-induced, and 14 proteins with endoplasmic reticulum stress-induced hepatic insulin resistance. Moreover, we identified 19 proteins that may regulate hepatic insulin resistance in a c-Jun amino-terminal kinase-dependent manner. In addition, three proteins, 14-3-3 protein beta (YWHAB), Slc2a4 (GLUT4), and Dlg4 (PSD-95), are discovered by comprehensive bioinformatic analysis, which have correlations with several proteins identified by proteomics approach. The newly identified proteins in T2DM should provide additional insight into the development and pathophysiology of hepatic steatosis and insulin resistance, and they may serve as useful diagnostic markers and/or therapeutic targets for these diseases.

From Cells to Peptides: “One-Stop” Integrated Proteomic Processing Using Amphipols

In proteomics, detergents and chaotropes are indispensable for proteome analysis, not only for protein extraction, but also for protein digestion. To increase the protein extraction efficiency, detergents are usually added in the lysis buffer to extract membrane proteins out of membrane structure and to maintain protein in solutions. In general, these detergents need to be removed prior to protein digestion, usually by precipitation or ultrafiltration. Digestion often takes place in the presence of chaotropic reagents, such as urea, which often need to be removed prior to mass spectrometry. The addition and removal of detergents and chaotropes require multiple steps that are time-consuming and can cause sample losses. Amphipols (APols) are a different class of detergents that have physical and solubilization properties that are distinct from conventional detergents. They have primarily been used in protein structure analysis for membrane protein trapping and stabilization. Here, we demonstrate a simple and rapid protocol for total and membrane proteome preparation using APols. We demonstrate that APols added for cell lysis help maintain the proteome in solution, are compatible with protein digestion using trypsin, and can readily be removed prior to mass spectrometry by a one-step acidification and centrifugation. This protocol is much faster, can be performed in a single tube, and can readily replace the conventional detergent/chaotrope approaches for total and membrane proteome analysis.

A sample preparation method for micro-scale membrane proteome analysis

A sample preparation method for micro-scale membrane proteome analysis

Advancements in the analysis of the Arabidopsis plasma membrane proteome.

The plasma membrane (PM) regulates diverse processes essential to plant growth, development, and survival in an ever-changing environment. In addition to maintaining normal cellular homeostasis and plant nutrient status, PM proteins perceive and respond to a myriad of environmental cues. Here we review recent advances in the analysis of the plant PM proteome with a focus on the model plant Arabidopsis thaliana. Due to membrane heterogeneity, hydrophobicity, and low relative abundance, analysis of the PM proteome has been a special challenge. Various experimental techniques to enrich PM proteins and different protein and peptide separation strategies have facilitated the identification of thousands of integral and membrane-associated proteins. Numerous classes of proteins are present at the PM with diverse biological functions. PM microdomains have attracted much attention. However, it still remains a challenge to characterize these cell membrane compartments. Dynamic changes in the PM proteome in response to different biotic and abiotic stimuli are highlighted. Future prospects for PM proteomics research are also discussed.

Neuroproteomic profiling of human brain tissue using multidimensional separation techniques and selective enrichment of membrane proteins

Hydrophobic membrane proteins (MPs) occupy a unique niche in the brain proteome research due to their important physiological roles. Therefore, the extraction, separation, and identification of MPs are of great interest in proteomic analysis. We applied various proteomic techniques to enrich, separate, and analyze the human brain proteome, including membrane proteome. Temperature-induced phase fractionation with the nonionic surfactant Triton X-114 was used to simultaneously extract, separate, and concentrate low abundant hydrophobic and high abundant hydrophilic proteins from human brain tissue. The extracted and delipidated proteins were analyzed by two-dimensional gel electrophoresis (2DE). Approximately 600 spots were detected in the gels. In-solution digestion was performed on 3 kDa spin filters. Tryptic peptides were separated using RP nano-LC and analyzed using two different high performance mass spectrometers, linear ion trap-Fourier transform and a linear ion trap-Orbitrap to reveal the low abundant MPs. In total, 837 and 780 unique proteins were identified by using linear ion trap-Fourier transform and linear ion trap-Orbitrap mass spectrometers, respectively. More than 29% of the identified proteins were classified as MPs with significant biological functions such as ion channels and transporters. Our study establishes a simple and rapid shotgun approach for the characterization of the brain proteome, and allows comprehensive analysis of brain membrane proteomes.

Improved proteomic profiling of the cell surface of culture-expanded human bone marrow multipotent stromal cells

A comprehensive analysis of the membrane proteome is essential to explain the biology of multipotent stromal cells and identify reliable protein biomarkers for the isolation as well as tracking of cells during differentiation and maturation. However, proteomic analysis of membrane proteins is challenging and they are noticeably under-represented in numerous proteomic studies. Here we introduce new approach, which includes high pressure-assisted membrane protein extraction, protein fractionation by gel-eluted liquid fraction entrapment electrophoresis (GELFREE), and combined use of liquid chromatography MALDI and ESI tandem mass spectrometry. This report presents the first comprehensive proteomic analysis of membrane proteome of undifferentiated and culture-expanded human bone marrow multipotent stromal cells (hBM-MSC) obtained from different human donors. Gene ontology mapping using the Ingenuity Pathway Analysis and DAVID programs revealed the largest membrane proteomic dataset for hBM-MSC reported to date. Collectively, the new workflow enabled us to identify at least two-fold more membrane proteins compared to published results on hBM-MSC. A total of 84 CDs were identified including 14 CDs identified for the first time. This dataset can serve as a basis for further exploration of self-renewal, differentiation and characterization of hBM-MSC.

GOFAST: An Integrated Approach for Efficient and Comprehensive Membrane Proteome Analysis

Membrane proteomics, the large-scale analysis of membrane proteins, is often constrained by the difficulties of achieving fully resolvable separation and resistance to proteolysis, both of which could lead to low recovery and low identification rates of membrane proteins. Here, we introduce a novel integrated approach, GELFrEE Optimized FASP Technology (GOFAST) for large-scale and comprehensive membrane proteins analysis. Using an array of sample preparation techniques including gel-eluted liquid fraction entrapment electrophoresis (GELFrEE), filter-aided sample preparation (FASP), and microwave-assisted on-filter enzymatic digestion, we identified 2 090 proteins from the membrane fraction of a leukemia cell line (K562). Of these, 37% are annotated as membrane proteins according to gene ontology analysis, resulting in the largest membrane proteome of leukemia cells reported to date. Our approach combines the advantages of GELFrEE high-loading capacity, gel-free separation, efficient depletion of detergents, and microwave-assisted on-filter digestion, minimizing sample losses and maximizing MS-detectable sequence coverage of individual proteins. In addition, this approach also shows great potential for the identification of alternative splicing products.

Shotgun analysis of membrane proteomes by an improved SDS-assisted sample preparation method coupled with liquid chromatography–tandem mass spectrometry

Analysis of the membrane proteins, particularly the integral membrane proteins, is limited by the inherent membrane hydrophobicity. Sodium dodecyl sulfate (SDS) is one of the most efficient reagents used for the extraction of membrane proteins, but its presence in samples interferes with LC–MS-based proteomic analyses because it affects RP-LC separations and electrospray ionization. In this paper, we present an improved sample preparation strategy based on SDS-assisted digestion and peptide-level SDS-removal using an optimized potassium dodecyl sulfate (KDS) precipitation method (SSDP method) for shotgun analysis of the membrane proteome. This method utilizes a high concentration of SDS (1.0%) to lyse the membranes and to solubilize the hydrophobic membrane proteins, resulting in a more complete protein digestion in the diluted SDS buffer (0.1% SDS), and a high efficiency of SDS removal and peptide recovery by the optimized KDS precipitation for protein identification. The SSDP method provides evidence that proteins can be efficiently digested, and the SDS can be decreased to <0.01% allowing >95% peptide recovery. Compared to other sample preparation methods commonly used in shotgun membrane proteomics, the newly developed method not only increased the identified number of the total proteins, membrane proteins and integral membrane proteins by an average of 33.1%, 37.2% and 40.5%, respectively, but also leading to the identification of highest number of matching peptides. All the results showed that the method yielded better recovery and reliability in the identification of the proteins especially the highly hydrophobic integral membrane proteins, and thus providing a promising tool for the shotgun analysis of membrane proteome.

Development and evaluation of an entirely solution-based combinative sample preparation method for membrane proteomics

The hydrophobic nature of many membrane proteins, especially integral membrane proteins, brings great difficulties to their analysis. To improve the analysis of membrane proteins, an entirely solution-based combinative sample preparation (CSP) method was developed and its application to the shotgun analysis of rat liver membrane proteomes was evaluated in this study. This CSP method comprehensively uses the strong ability of sodium dodecyl sulfate (SDS) to lyse the membranes and solubilize hydrophobic membrane proteins, the high efficiency of the optimized acetone precipitation method in sample cleanup and protein recovery, and the advantages of sodium deoxycholate (SDC) in improving protein solubilization/digestion as well as being compatible with trypsin activity. Compared with two other representative sample preparation methods, the SDC-assisted membrane-lysing method and the tube gel method, the newly established CSP method exhibited superiority in the recovery and identification of hydrophobic peptides, larger peptides, and highly hydrophobic membrane proteins with multiple transmembrane domains. The CSP method has characteristics of easy operation, low cost, and suitability for treating protein samples in various volumes, particularly large volumes, thereby having potential in the analysis of membrane proteomes with mass spectrometry.

Advances in membrane proteomics and cancer biomarker discovery: Current status and future perspective

Membrane proteomic analysis has been proven to be a promising tool for identifying new and specific biomarkers that can be used for prognosis and monitoring of various cancers. Membrane proteins are of great interest particularly those with functional domains exposed to the extracellular environment. Integral membrane proteins represent about one-third of the proteins encoded by the human genome and assume a variety of key biological functions, such as cell-to-cell communication, receptor-mediated signal transduction, selective transport, and pharmacological actions. More than two-thirds of membrane proteins are drug targets, highlighting their immensely important pharmaceutical significance. Most plasma membrane proteins and proteins from other cellular membranes have several PTMs; for example, glycosylation, phosphorylation, and nitrosylation, and moreover, PTMs of proteins are known to play a key role in tumor biology. These modifications often cause change in stoichiometry and microheterogeneity in a protein molecule, which is apparent during electrophoretic separation. Furthermore, the analysis of glyco- and phosphoproteome of cell membrane presents a number of challenges mainly due to their low abundance, their large dynamic range, and the inherent hydrophobicity of membrane proteins. Under pathological conditions, PTMs, such as phosphorylation and glycosylation are frequently altered and have been recognized as a potential source for disease biomarkers. Thus, their accurate differential expression analysis, along with differential PTM analysis is of paramount importance. Here we summarize the current status of membrane-based biomarkers in various cancers, and future perspective of membrane biomarker research.

Identification of Flotillin-1 as a novel biomarker for lymph node metastasis and prognosis of lung adenocarcinoma by quantitative plasma membrane proteome analysis

To identify a novel lung adenocarcinoma (AdC) biomarker, iTRAQ-tagging combined with 2D LC-MS/MS analysis was used to identify differentially expressed plasma membrane (PM) proteins in primary lung AdCs and paraneoplastic normal lung tissues (PNLTs). As a result, 36 differentially expressed membrane proteins were identified. Two differential PM proteins flotillin-1 and caveolin-1 were selectively validated by Western blotting. As there has been no report on the association of flotillin-1 with lung AdC, immunohistochemistry was further performed to detect the expression of flotillin-1 in the archival tissue specimens including 42 cases of PNLTs, 62 cases of primary lung AdCs with lymph node metastasis (LNM AdCs), and 46 cases of primary lung AdCs without lymph node metastasis (non-LNM AdCs), and the correlation of flotillin-1 expression levels in lung AdCs with clinicopathological features and clinical outcomes were evaluated. The results showed that up-regulation of flotillin-1 expression in lung AdCs was significantly correlated with advanced clinical stage, lymph node metastasis, increased postoperative relapse and decreased overall survival. Cox regression analysis revealed that the expressional level of flotillin-1 was an independent prognostic factor. The data suggest that flotillin-1 is a potential novel biomarker for lymph node metastasis and prognosis of lung AdC, and flotillin-1 up-regulation might play an important role in the pathogenesis of lung AdC.

Sample preparation for the analysis of membrane proteomes by mass spectrometry

The low abundance and highly hydrophobic nature of most membrane proteins make their analysis more difficult than that for common soluble proteins. Successful membrane protein identification is largely dependent on the sample preparation including the enrichment and dissolution of the membrane proteins. A series of conventional and newly developed methods has been applied to the enrichment of low-abundance membrane proteins at membrane and/or protein levels and to the dissolution of hydrophobic membrane proteins. However, all the existing methods have inherent advantages and limitations. Up to now, there has been no unique method that can universally be employed to solve all the problems and more efforts are needed in improving sample preparation for the analysis of membrane proteomes.

Shotgun analysis of membrane proteomes using a novel combinative strategy of solution-based sample preparation coupled with liquid chromatography–tandem mass spectrometry

Although much effort has been made in the field of membrane proteomics, the analysis of membrane proteins particularly integral membrane proteins with poor water solubility still presents a great challenge. In this paper, 2% SDS was used to extract membrane proteins and experimental conditions for the application of acetone precipitation method to the cleanup of SDS-solubilized membrane protein sample were optimized. For improving the re-dissolution and trypsinolysis of acetone-precipitated proteins, several commonly used additives, urea, methanol and sodium deoxycholate (SDC), were employed and compared. The results showed that, when the pre-cooled acetone-to-sample ratio was 6:1 (v/v) with one additional washing step, residual SDS in the protein sample could be lowered to below 0.01% and more than 90% of the proteins were precipitated and therefore recovered. 1% SDC-containing buffer could improve the re-dissolution and digestion of the acetone precipitated proteins more efficiently than the others. Using the combinative sample preparation strategy developed, 398 proteins were identified from the rat liver membrane-enriched fraction, including 188 membrane proteins. Compared with other three representative solution-based sample preparation methods commonly used in membrane proteomics, the newly developed combinative strategy increased the number of identified total proteins and membrane proteins on average by 29.2% and 28.5%, respectively. This combinative strategy was demonstrated to be easily operated at low cost and suitable for the analysis of membrane proteins varying in type and sample volume, etc.

Membrane proteomic analysis comparing squamous cell lung cancer tissue and tumour-adjacent normal tissue

Lung cancer is the leading cause of cancer-related deaths worldwide. Squamous cell carcinoma is one of the predominant histological subtypes of lung cancer. Detecting lung cancer at an early stage is essential for successful therapy and increasing survival. There are still no satisfactory biomarkers for the early detection of lung cancer. In this study, tumour tissue paired with tumour-adjacent normal bronchial epithelial tissue was obtained from patients with squamous cell lung carcinoma without metastasis. The proteins extracted from the cell membrane were separated by two-dimensional polyacrylamide gel electrophoresis (2-DE) and were analysed with the Image Master two-dimensional platinum software. Twenty-five significantly different protein spots were selected and identified with matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry (MALDI-TOF MS). A total of 19 proteins were successfully identified. Twelve proteins were up-regulated, and seven proteins were down-regulated in the cancerous tissue compared with the tumour-adjacent normal tissue. One up-regulated protein and one down-regulated protein in squamous cell lung carcinoma were verified by Western blot analysis and RT-PCR; the results were consistent with the 2-DE analysis. In conclusion, membrane proteomics identified a number of candidate biomarker proteins that were differentially expressed between squamous cell lung cancer tissue and adjacent normal tissue. These biomarker candidates have the potential to elucidate the underlying pathogenesis of squamous cell lung cancer.

Cover Picture: Electrophoresis 2'2012

AbstractIssue no. 2 is a regular issue consisting of 18 contributions distributed over 5 distinct parts. The issue has an “Emphasis on Proteins and Proteomics” with 7 contributions featured in Part I. This is followed by Part II that has 4 research articles on nanopore and nanoparticles based analyses. Part III has 2 contributions on agarose gel electrophoresis for the analysis of the structure fluidity of a phage T3 DNA packaging intermediate and analyzing RNA quality. Part IV has two research articles on microchip capillary electrophoresis with poly(ethylene glycol)‐coated microfluidic devices and poly(dimethylsiloxane) microchips using a mixture of ionic and zwitterionic surfactants. The last part (Part V) consists of 3 contributions on CE of some common controlled stimulants, CE test of a blood‐brain barrier model and the simultaneous determinations of nitrite and nitrate in human plasma by CE. This issue is populated by a collection of very important research articles. It is a must read.Featured articles include:The challenge to quantify proteins with charge trains due to isoforms or conformers ((10.1002/elps.201100321))Electrophoretically‐driven SDS removal and protein fractionation in the shotgun analysis of membrane proteomes ((10.1002/elps.201100364))Modeling and simulation of nanoparticle separation through a solid state nanopore ((10.1002/elps.201100201))A novel open‐tubular capillary electrochromatography with magnetic nanoparticle coating as stationary phase ((10.1002/elps.201100337))Direct visualization of electrophoretic mobility shift assays using nanoparticle‐aptamer conjugates ((10.1002/elps.201100308))

Electrophoretically driven SDS removal and protein fractionation in the shotgun analysis of membrane proteomes

SDS is mostly used to enhance the solubilization and extraction of membrane proteins due to its strong detergency and low cost. Nevertheless, SDS interferes with the subsequent procedures and needs to be removed from the samples. In this work, a special gradient gel electrophoresis (GGE) system was developed to remove SDS from the SDS-solubilized protein samples. As a proof-of-principle experiment, the GGE system was designed to be composed of an agarose loading layer, six polyacrylamide fractionation layers with different concentrations and a high-concentration polyacrylamide sealing layer. The advantages of the GGE system are that it not only can electrophoretically remove SDS efficiently so that the protein loss resulted from the repeated gel washing after electrophoresis was avoided, but also can reduce the complexity of the sample, prevent the precipitation of proteins after loading and avoid the loss of proteins with low molecular weight during the electrophoresis. Using GGE system, about 85% of SDS in the sample and gel was electrophoretically removed and the proteins were fractionated. Compared with the two representative gel-based sample cleanup methods reported in literature, GGE-based strategy significantly improved the identification efficiency of proteins in terms of the number and coverage of the identified proteins.

Impairment on sperm quality and fertility of adult rats after antiandrogen exposure during prepuberty

This study evaluated the effects of antiandrogen exposure during the prepubertal period on reproductive development and reproductive competence in adults. Male rats were divided into two groups: flutamide, receiving 25mg/kg/day of flutamide by oral gavage and control, receiving vehicle daily. Dosing continued from PND 21 to 44, and animals were killed on PND 50 or PND 75–80. The epididymis, prostate, vas deferens and seminal vesicle weights were lower in Flutamide group on PND 50, while on PND 80 only seminal vesicle weight was reduced. Fertility assessed by IUI revealed a decrease in the fertility potential in the flutamide-treated adults. Flutamide accelerated sperm transit time through the epididymis, impairing sperm motility and storage. A quantitative analysis of the cauda sperm membrane proteome revealed a few significant changes in protein expression. Thus, exposure to flutamide during the prepubertal period compromises the function of the epididymis along with epididymal sperm quality at adulthood.

Insights into physiological traits of Bifidobacterium animalis subsp. lactis BB-12 through membrane proteome analysis

Bifidobacterium animalis subsp. lactis BB-12 is a widely used probiotic strain associated with a variety of health-promoting traits. There is, however, only limited knowledge available regarding the membrane proteome and the proteins involved in oligosaccharide transport in BB-12. We applied two enrichment strategies to improve the identification of membrane proteins from BB-12 cultures grown on glucose and on xylo-oligosaccharides, the latter being an emerging prebiotic substrate recently reported to be fermented by BB-12. Our approach encompassed consecutive steps of detergent- and carbonate-treatment in order to generate inside-out membrane vesicles and to interfere with binding of membrane-associated proteins to the membrane, respectively. Proteins in the enriched membrane fraction and membrane-associated fraction were digested by lysyl endopeptidase and trypsin followed by peptide sequencing by LC-ESI-Q-TOF MS/MS. Ninety of a total of 248 identified unique proteins were predicted to possess transmembrane segments (TMSs), and 56 of these have more than one TMS. Seventy-nine of the identified proteins are annotated to be involved in transport of amino acids, oligosaccharides, inorganic ions, nucleotides, phosphate or exopolysaccharides, or to belong to the F1F0-ATP-synthetase complex and the protein translocation machinery, respectively.

Characterization of the Asia Oceania Human Proteome Organisation Membrane Proteomics Initiative Standard using SDS‐PAGE shotgun proteomics

Although there are now multiple methods for the analysis of membrane proteomes, there is relatively little systematic characterization of proteomic workflows for membrane proteins. The Asia Oceania Human Proteome Organisation (AOHUPO) has therefore embarked on a Membrane Proteomics Initiative (MPI) using a large range of workflows. Here, we describe the characterization of the MPI mouse liver microsomal membrane Standard using SDS-PAGE prior to in-gel tryptic digestion and LC-ESI-MS/MS. The Na(2) CO(3) wash followed by SDS-PAGE prior to in-gel tryptic digestion and LC-MS/MS strategy was effective for the detection of membrane proteins with 47.1% of the identified proteins being transmembrane proteins. Gene Ontology term enrichment analysis showed that biological processes involving transport, lipid metabolism, cell communication, cell adhesion, and cellular component organization were significantly enriched. Comparison of the present data with the previously published reports on mouse liver proteomes confirmed that the MPI Standard provides an excellent resource for the analysis of membrane proteins in the AOHUPO MPI.

Plasma membrane proteomic analysis of human osteosarcoma and osteoblastic cells: revealing NDRG1 as a marker for osteosarcoma

Osteosarcoma (OS) is the most common primary malignant bone tumor in children and adolescents. To identify new biomarkers for early diagnosis of OS and novel therapeutic candidates, we carried out a plasma membrane proteomic study based on two-dimensional electrophoresis (2DE). The OS cell line MG-63 and the human osteoblastic cell line hFOB1.19 were adopted as the comparison model. We extracted plasma membrane by aqueous two-phase partition extraction. The proteins were separated through 2DE. We analyzed the differentially expressed proteins by Imagemaster software and then identified them by liquid chromatography-tandem mass spectrometry, and the location and function of differential proteins were searched through the Gene Ontology database. In total, 220 protein spots were separated by 2DE. Seven proteins with more than 2.0-folds of difference were successfully identified from 13 gel spots, with 6 up-regulated and 1 down-regulated. Gene Ontology analysis of the differentially expressed proteins indicated that these proteins were involved in seven kinds of functions including binding, structural, cell motility, receptor activity, electron carrier activity, NADH dehydrogenase (ubiquinone) activity, and transcription repressor activity. The up-regulation of NDRG1 was verified in osteosarcoma through Western blotting and by immunohistochemistry in paraffin-embedded tissues. The plasma membrane proteins identified in this study may provide new insights into osteosarcoma cancer biology and potential diagnostic and therapeutic biomarkers.