FRINK Linked Data Fragments server

Matches in SemOpenAlex for { <https://semopenalex.org/work/W2118993579> ?p ?o ?g. }

• W2118993579 endingPage "1997" @default.
• W2118993579 startingPage "1983" @default.
• W2118993579 abstract "Toward multiplexed, comprehensive, and robust quantitation of the membrane proteome, we report a strategy combining gel-assisted digestion, iTRAQ (isobaric tags for relative and absolute quantitation) labeling, and LC-MS/MS. Quantitation of four independently purified membrane fractions from HeLa cells gave high accuracy (<8% error) and precision (<12% relative S.D.), demonstrating a high degree of consistency and reproducibility of this quantitation platform. Under stringent identification criteria (false discovery rate = 0%), the strategy efficiently quantified membrane proteins; as many as 520 proteins (91%) were membrane proteins, each quantified based on an average of 14.1 peptides per integral membrane protein. In addition to significant improvements in signal intensity for most quantified proteins, most remarkably, topological analysis revealed that the biggest improvement was achieved in detection of transmembrane peptides from integral membrane proteins with up to 19 transmembrane helices. To the best of our knowledge, this level of coverage exceeds that achieved previously using MS and provides superior quantitation accuracy compared with other methods. We applied this approach to the first proteomics delineation of phenotypic expression in a mouse model of autosomal dominant polycystic kidney disease (ADPKD). By characterizing kidney cell plasma membrane from wild-type versus PKD1 knock-out mice, 791 proteins were quantified, and 67 and 37 proteins showed ≥2-fold up-regulation and down-regulation, respectively. Some of these differentially expressed membrane proteins are involved in the mechanisms underlying major abnormalities in ADPKD, including epithelial cell proliferation and apoptosis, cell-cell and cell-matrix interactions, ion and fluid secretion, and membrane protein polarity. Among these proteins, targeting therapeutics to certain transporters/receptors, such as epidermal growth factor receptor, has proven effective in preclinical studies of ADPKD; others are known drug targets in various diseases. Our method demonstrates how comparative membrane proteomics can provide insight into the molecular mechanisms underlying ADPKD and the identification of potential drug targets, which may lead to new therapeutic opportunities to prevent or retard the disease. Toward multiplexed, comprehensive, and robust quantitation of the membrane proteome, we report a strategy combining gel-assisted digestion, iTRAQ (isobaric tags for relative and absolute quantitation) labeling, and LC-MS/MS. Quantitation of four independently purified membrane fractions from HeLa cells gave high accuracy (<8% error) and precision (<12% relative S.D.), demonstrating a high degree of consistency and reproducibility of this quantitation platform. Under stringent identification criteria (false discovery rate = 0%), the strategy efficiently quantified membrane proteins; as many as 520 proteins (91%) were membrane proteins, each quantified based on an average of 14.1 peptides per integral membrane protein. In addition to significant improvements in signal intensity for most quantified proteins, most remarkably, topological analysis revealed that the biggest improvement was achieved in detection of transmembrane peptides from integral membrane proteins with up to 19 transmembrane helices. To the best of our knowledge, this level of coverage exceeds that achieved previously using MS and provides superior quantitation accuracy compared with other methods. We applied this approach to the first proteomics delineation of phenotypic expression in a mouse model of autosomal dominant polycystic kidney disease (ADPKD). By characterizing kidney cell plasma membrane from wild-type versus PKD1 knock-out mice, 791 proteins were quantified, and 67 and 37 proteins showed ≥2-fold up-regulation and down-regulation, respectively. Some of these differentially expressed membrane proteins are involved in the mechanisms underlying major abnormalities in ADPKD, including epithelial cell proliferation and apoptosis, cell-cell and cell-matrix interactions, ion and fluid secretion, and membrane protein polarity. Among these proteins, targeting therapeutics to certain transporters/receptors, such as epidermal growth factor receptor, has proven effective in preclinical studies of ADPKD; others are known drug targets in various diseases. Our method demonstrates how comparative membrane proteomics can provide insight into the molecular mechanisms underlying ADPKD and the identification of potential drug targets, which may lead to new therapeutic opportunities to prevent or retard the disease. Many membrane proteins are implicated in particular disease states and often are attractive therapeutic targets (1Stevens T.J. Arkin I.T. Do more complex organisms have a greater proportion of membrane proteins in their genomes?.Proteins Struct. Funct. Genet. 2000; 39: 417-420Crossref PubMed Scopus (214) Google Scholar). Comprehensive and quantitative profiles of membrane proteins facilitate our understanding of their roles in regulating biological processes and in cellular signaling. However, the analysis of membrane proteins is experimentally challenging because of their hydrophobic nature and low abundance, which seriously complicate their solubilization, sample handling, preparation, separation, and analysis (2Blonder J. Goshe M.B. Moore R.J. Pasa-Tolic L. Masselon C.D. Lipton M.S. Smith R.D. Enrichment of integral membrane proteins for proteomic analysis using liquid chromatography-tandem mass spectrometry.J. Proteome Res. 2002; 1: 351-360Crossref PubMed Scopus (209) Google Scholar, 3Han D.K. Eng J. Zhou H.L. Aebersold R. Quantitative profiling of differentiation-induced microsomal proteins using isotope-coded affinity tags and mass spectrometry.Nat. Biotechnol. 2001; 19: 946-951Crossref PubMed Scopus (829) Google Scholar, 4Washburn M.P. Wolters D. Yates J.R. Large-scale analysis of the yeast proteome by multidimensional protein identification technology.Nat. Biotechnol. 2001; 19: 242-247Crossref PubMed Scopus (4099) Google Scholar, 5Wu C.C. Yates J.R. The application of mass spectrometry to membrane proteomics.Nature Biotechnology. 2003; 21: 262-267Crossref PubMed Scopus (510) Google Scholar). Although ∼20–30% of open reading frames of most sequenced genomes are estimated to encode integral membrane proteins (6Krogh A. Larsson B. von Heijne G. Sonnhammer E.L.L. Predicting transmembrane protein topology with a hidden Markov model: application to complete genomes.J. Mol. Biol. 2001; 305: 567-580Crossref PubMed Scopus (9290) Google Scholar), the membrane proteome has not been mapped as comprehensively as the soluble proteome (2Blonder J. Goshe M.B. Moore R.J. Pasa-Tolic L. Masselon C.D. Lipton M.S. Smith R.D. Enrichment of integral membrane proteins for proteomic analysis using liquid chromatography-tandem mass spectrometry.J. Proteome Res. 2002; 1: 351-360Crossref PubMed Scopus (209) Google Scholar, 3Han D.K. Eng J. Zhou H.L. Aebersold R. Quantitative profiling of differentiation-induced microsomal proteins using isotope-coded affinity tags and mass spectrometry.Nat. Biotechnol. 2001; 19: 946-951Crossref PubMed Scopus (829) Google Scholar). For membrane proteomics studies, conventional strategies using two-dimensional gel electrophoresis (2DE) 1The abbreviations used are: 2DE, two-dimensional gel electrophoresis; iTRAQ, isobaric tags for relative and absolute quantitation; SCX, strong cation exchange; TMHMM, transmembrane hidden Markov model; TMH, transmembrane helix; ADPKD, autosomal dominant polycystic kidney disease; Na+/K+-ATPase α1 and β1, sodium/potassium-transporting ATPase α1 and β1 chain; SILAC, stable isotope labeling by amino acids in cell culture; BALF, bronchoalveolar lavage fluid; APS, ammonium persulfate; TEMED, N,N,N‘,N‘-tetramethylenediamine; TEABC, triethylammonium bicarbonate; ABC, ammonium bicarbonate; MMTS, methyl methanethiosulfonate; FA, formic acid; IPI, International Protein Index; R.S.D., relative standard deviation; Wt, wild type; EGFR, epidermal growth factor receptor; COX, cyclooxygenase; PKD, polycystic kidney disease; R, ratio. have had some success in profiling the bacterial outer membrane (7Phadke N.D. Molloy M.P. Steinhoff S.A. Ulintz P.J. Andrews P.C. Maddock J.R. Analysis of the outer membrane proteome of Caulobacter crescentus by two-dimensional electrophoresis and mass spectrometry.Proteomics. 2001; 1: 705-720Crossref PubMed Google Scholar), organelle membranes (8Bunai K. Nozaki M. Kakeshita H. Nemoto T. Yamane K. Quantitation of de novo localized N-15-labeled lipoproteins and membrane proteins having one and two transmembrane segments in a Bacillus subtilis secA temperature-sensitive mutant using 2D-PAGE and MALDI-TOF MS.J. Proteome Res. 2005; 4: 826-836Crossref PubMed Scopus (10) Google Scholar), and plasma membrane (9Helling S. Schmitt E. Joppich C. Schulenborg T. Mullner S. Felske-Muller S. Wiebringhaus T. Becker G. Linsenmann G. Sitek B. Lutter P. Meyer H.E. Marcus K. 2-D differential membrane proteome analysis of scarce protein samples.Proteomics. 2006; 6: 4506-4513Crossref PubMed Scopus (42) Google Scholar). However, the serious under-representation of membrane proteins reported in the above studies reveals the drawback of low sensitivity. To alleviate the drawbacks of 2DE, stable isotopic labeling in conjunction with off-line or on-line multidimensional LC-MS/MS analysis offers greater sensitivity. The two most common ways of incorporating stable isotopes into biological samples are through either ICAT or metabolic stable isotope labeling by amino acids in cell culture (SILAC) (10Ong S.E. Blagoev B. Kratchmarova I. Kristensen D.B. Steen H. Pandey A. Mann M. Stable isotope labeling by amino acids in cell culture, SILAC, as a simple and accurate approach to expression proteomics.Mol. Cell. Proteomics. 2002; 1: 376-386Abstract Full Text Full Text PDF PubMed Scopus (4625) Google Scholar). However, many proteins, especially membrane proteins, have few (if any) cysteine residues, resulting in smaller numbers of quantifiable proteins in ICAT. Furthermore the high concentration (>0.05%) of denaturing reagents (e.g. SDS) required for ICAT labeling, especially for membrane proteins, is incompatible with ICAT alkylation of cysteine residues because of reduced accessibility after formation of the SDS-protein complex. Although an optimized strategy using a smaller ICAT analog was recently reported by Ramus et al. (11Ramus C. de Peredo A.G. Dahout C. Gallagher M. Garin J. An optimized strategy for ICAT quantification of membrane proteins.Mol. Cell. Proteomics. 2006; 5: 68-78Abstract Full Text Full Text PDF PubMed Scopus (29) Google Scholar), the quantification efficiency of this labeling strategy on the proteome scale remains to be evaluated. Alternatively SILAC offers more options for selection of isotopically labeled amino acids such as arginine, leucine, lysine, serine, methionine, and tyrosine (12Beynon R.J. Pratt J.M. Metabolic labeling of proteins for proteomics.Mol. Cell. Proteomics. 2005; 4: 857-872Abstract Full Text Full Text PDF PubMed Scopus (174) Google Scholar). Although some successes have been reported in analysis of the membrane proteome (13Liang X.Q. Zhao J. Hajivandi M. Wu R. Tao J. Amshey J.W. Pope R.M. Quantification of membrane and membrane-bound proteins in normal and malignant breast cancer cells isolated from the same patient with primary breast carcinoma.J. Proteome Res. 2006; 5: 2632-2641Crossref PubMed Scopus (55) Google Scholar, 14Smart E.J. Ying Y.S. Mineo C. Anderson R.G.W. A detergent-free method for purifying caveolae membrane from tissue-culture cells.Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 10104-10108Crossref PubMed Scopus (676) Google Scholar), the major drawback of SILAC is that it is most applicable to cell culture rather than tissue or body fluids. Recently a new amine labeling strategy at the peptide level using isobaric tags (iTRAQ) was developed for multiplexed protein quantitation (15Ross P.L. Huang Y.L.N. Marchese J.N. Williamson B. Parker K. Hattan S. Khainovski N. Pillai S. Dey S. Daniels S. Purkayastha S. Juhasz P. Martin S. Bartlet-Jones M. He F. Jacobson A. Pappin D.J. Multiplexed protein quantitation in Saccharomyces cerevisiae using amine-reactive isobaric tagging reagents.Mol. Cell. Proteomics. 2004; 3: 1154-1169Abstract Full Text Full Text PDF PubMed Scopus (3721) Google Scholar). In contrast to other quantitative methods based on MS intensity of the isotopically paired peptides, the relative intensity of signature reporter ions of m/z 114, 115, 116 and 117 in an MS/MS spectrum is used to quantify protein levels. Since its development, it has become popular because of the following advantages. First, the multiplexing reagents target the N terminus as well as the lysine side chains of peptides, and thus each peptide fragment can be labeled with better efficiency compared with protein-level labeling. Second, the intensity of both the precursor ion and resultant MS/MS fragments is greatly enhanced by summing of four isobarically iTRAQ-labeled sample sets, thereby increasing the number of peptides identified and quantified and thus increasing the quantification accuracy. The iTRAQ strategy has been widely applied for comparative analysis characterization of the soluble proteome (16Aggarwal K. Choe L.H. Lee K.H. Quantitative analysis of protein expression using amine-specific isobaric tags in Escherichia coli cells expressing rhsA elements.Proteomics. 2005; 5: 2297-2308Crossref PubMed Scopus (71) Google Scholar, 17Hu J. Qian J. Borisov O. Pan S.Q. Li Y. Liu T. Deng L.W. Wannemacher K. Kurnellas M. Patterson C. Elkabes S. Li H. Optimized proteomic analysis of a mouse model of cerebellar dysfunction using amine-specific isobaric tags.Proteomics. 2006; 6: 4321-4334Crossref PubMed Scopus (66) Google Scholar), particularly for enriched subproteomes (18Chen X.Q. Walker A.K. Strahler J.R. Simon E.S. Tomanicek-Volk S.L. Nelson B.B. Hurley M.C. Ernst S.A. Williams J.A. Andrews P.C. Organellar proteomics. Analysis of pancreatic zymogen granule membranes.Mol. Cell. Proteomics. 2006; 5: 306-312Abstract Full Text Full Text PDF PubMed Scopus (108) Google Scholar). Whether it can offer enhanced identification confidence and quantitation accuracy for the membrane proteome will require further evaluation. Although shotgun proteomics for membrane proteins produces more satisfactory results than 2DE, the incompatibility of high concentrations of detergent/solvent with subsequent enzymatic digestion and LC-MS/MS analysis still presents substantial limitations. Proteolysis of integral membrane proteins usually results in low sequence coverage because their low aqueous solubility and high hydrophobicity render them relatively inaccessible to proteases. Several groups have attempted to circumvent this limitation by using organic solvents (2Blonder J. Goshe M.B. Moore R.J. Pasa-Tolic L. Masselon C.D. Lipton M.S. Smith R.D. Enrichment of integral membrane proteins for proteomic analysis using liquid chromatography-tandem mass spectrometry.J. Proteome Res. 2002; 1: 351-360Crossref PubMed Scopus (209) Google Scholar), organic acids (19Han J. Schey K.L. Proteolysis and mass spectrometric analysis of an integral membrane: aquaporin 0.J. Proteome Res. 2004; 3: 807-812Crossref PubMed Scopus (39) Google Scholar), microwave-assisted acid hydrolysis (20Zhong H. Marcus S.L. Li L. Microwave-assisted acid hydrolysis of proteins combined with liquid chromatography MALDI MS/MS for protein identification.J. Am. Soc. Mass Spectrom. 2005; 16: 471-481Crossref PubMed Scopus (140) Google Scholar), or detergent-containing aqueous solution (3Han D.K. Eng J. Zhou H.L. Aebersold R. Quantitative profiling of differentiation-induced microsomal proteins using isotope-coded affinity tags and mass spectrometry.Nat. Biotechnol. 2001; 19: 946-951Crossref PubMed Scopus (829) Google Scholar). Recent studies have shown that SDS concentrations up to 1% are compatible with in-solution digestion for membrane proteins (21Li N. Shaw A.R.E. Zhang N. Mak A. Li L. Lipid raft proteomics: Analysis of in-solution digest of sodium dodecyl sulfate-solubilized lipid raft proteins by liquid chromatography-matrix-assisted laser desorption/ionization tandem mass spectrometry.Proteomics. 2004; 4: 3156-3166Crossref PubMed Scopus (90) Google Scholar, 22Zhang N. Chen R. Young N. Wishart D. Winter P. Weiner J.H. Li L. Comparison of SDS- and methanol-assisted protein solubilization and digestion methods for Escherichia coli membrane proteome analysis by 2-D LC-MS/MS.Proteomics. 2007; 7: 484-493Crossref PubMed Scopus (108) Google Scholar). However, the use of SDS reduces resolution during reverse-phase LC and suppresses peptide ionization efficiency. Alternative digestion using methanol-assisted protein solubilization has been effective for detecting integral membrane proteins (22Zhang N. Chen R. Young N. Wishart D. Winter P. Weiner J.H. Li L. Comparison of SDS- and methanol-assisted protein solubilization and digestion methods for Escherichia coli membrane proteome analysis by 2-D LC-MS/MS.Proteomics. 2007; 7: 484-493Crossref PubMed Scopus (108) Google Scholar, 23Goshe M.B. Blonder J. Smith R.D. Affinity labeling of highly hydrophobic integral membrane proteins for proteome-wide analysis.J. Proteome Res. 2003; 2: 153-161Crossref PubMed Scopus (76) Google Scholar). Although methanol (up to 60%) is easily removed after proteolysis, both SDS and methanol, even at low levels, substantially reduce protease activity. For example, the activity of trypsin (the most commonly used protease) falls to ∼20, 1, and 30% in the presence of 0.1% SDS, 0.5% SDS, and 50% methanol, respectively. 2Data from Waters Corp., Library Number 720000468EN. Recently Lu and Zhu (24Lu X.N. Zhu H.N. Tube-gel digestion. A novel proteomic approach for high throughput analysis of membrane proteins.Mol. Cell. Proteomics. 2005; 4: 1948-1958Abstract Full Text Full Text PDF PubMed Scopus (164) Google Scholar) described a Tube-Gel digestion protocol that incorporates protein into a polyacrylamide gel matrix in a glass tube without electrophoresis. The digestion efficiency is surprisingly good: a single 2.5-h LC-MS/MS analysis using Tube-Gel digestion yields results comparable to 20 LC-MS/MS analyses from SDS-PAGE fractionation (24Lu X.N. Zhu H.N. Tube-gel digestion. A novel proteomic approach for high throughput analysis of membrane proteins.Mol. Cell. Proteomics. 2005; 4: 1948-1958Abstract Full Text Full Text PDF PubMed Scopus (164) Google Scholar). Inspired by Tube-Gel digestion, we reported a gel-assisted digestion that is more compatible with high concentrations of a variety of detergents and allows more efficient removal of detergent (25Lu Y.T. Han C.L. Wu C.L. Yu T.M. Chien C.W. Liu C.L. Chen Y.J. Proteomic profiles of bronchoalveolar lavage fluid from patients with ventilator-associated pneumonia by gel-assisted digestion and 2D-LC/MS/MS.Proteomics Clin. Appl. 2008; 2 (in press)Crossref Scopus (12) Google Scholar). The method was applied to the first global description of the bronchoalveolar lavage fluid (BALF) proteome in patients with ventilator-associated pneumonia (25Lu Y.T. Han C.L. Wu C.L. Yu T.M. Chien C.W. Liu C.L. Chen Y.J. Proteomic profiles of bronchoalveolar lavage fluid from patients with ventilator-associated pneumonia by gel-assisted digestion and 2D-LC/MS/MS.Proteomics Clin. Appl. 2008; 2 (in press)Crossref Scopus (12) Google Scholar). It effectively overcame interference by the intrinsic high salt concentration in BALF, and the 206 identified proteins represent the most comprehensive proteome map of BALF. Thus, with good digestion efficiency, gel-assisted digestion may be ideal for peptide-level labeling, such as iTRAQ, with certain protocol modifications to ensure compatibility in the labeling reaction. In this study, we report a strategy integrating enhanced gel-assisted digestion and iTRAQ labeling for multiplex quantitative profiling of the membrane proteome. Specifically we aimed to achieve 1) higher identification/quantification numbers, 2) enhanced quantification for integral membrane proteins, and 3) broad compatibility with a variety of solubilization reagents with robust applicability to diverse biological systems. First we evaluated the reproducibility of gel-assisted digestion and its compatibility with iTRAQ labeling for multiplex quantitation in comparison with the most common in-solution or Tube-Gel digestion techniques. Performance assessment, including coverage of quantified peptides and proteins, quantitation accuracy, and precision, was evaluated via large scale quantitation of replicate membrane fractions from HeLa cells. Finally the new approach was applied to analyze the membrane proteome for the mouse model of autosomal dominant polycystic kidney disease (ADPKD). ADPKD is the most prevalent and potentially lethal inherited human renal disease (26Iglesias C.G. Torres V.E. Offord K.P. Holley K.E. Beard C.M. Kurland L.T. Epidemiology of adult polycystic kidney disease, Olmsted County, Minnesota: 1935–1980.Am. J. Kidney Dis. 1983; 2: 630-639Abstract Full Text PDF PubMed Scopus (302) Google Scholar). However, current treatments are mostly limited to renal replacement therapy by dialysis, which extends survival by an average of 7 years, or by transplantation coupled with aggressive management of hypertension (27Sweeney W.E. Chen Y. Nakanishi K. Frost P. Avner E.D. Treatment of polycystic kidney disease with a novel tyrosine kinase inhibitor.Kidney Int. 2000; 57: 33-40Abstract Full Text Full Text PDF PubMed Scopus (190) Google Scholar). Although genetic manifestations of the disease have revealed that a mutation in the gene PKD1 contributes the major etiology of the disease (28Watnick T. He N. Wang K.R. Liang Y. Parfrey P. Hefferton D. St George-Hyslop P. Germino G. Pei Y. Mutations of PKD1 in ADPKD2 cysts suggest a pathogenic effect of trans-heterozygous mutations.Nat. Genet. 2000; 25: 143-144Crossref PubMed Scopus (109) Google Scholar), most of the efforts to date have focused on PKD1 genetic heterogeneity and the potential contribution of modifier genes (29Torres V.E. Harris P.C. Pirson Y. Autosomal dominant polycystic kidney disease.Lancet. 2007; 369: 1287-1301Abstract Full Text Full Text PDF PubMed Scopus (1042) Google Scholar). Systematic mapping of phenotypic expression of the disease and uncovering perturbed cellular networks remain to be further investigated, and to our knowledge no large scale proteomics or genomics studies have been reported. Here we present the first comparative proteomics study of the plasma membrane from the kidney of control and PKD1 knock-out mice. We identified many differentially expressed membrane proteins in the knock-out mouse that may provide insight into the molecular mechanism underlying the development and progression of ADPKD as well as an opportunity for the development of a more effective pathophysiology-based therapy. Monomeric acrylamide/bisacrylamide solution (40%, 29:1) was purchased from Bio-Rad. Trypsin (modified, sequencing grade) was obtained from Promega (Madison, WI). The BCA and Bradford protein assay reagent kits were obtained from Pierce. SDS was purchased from GE Healthcare. Ammonium persulfate (APS), iodoacetamide, and TEMED were purchased from Amersham Biosciences. EDTA and methanol were purchased from Merck. Tris(2-carboxyethyl)phosphine hydrochloride, triethylammonium bicarbonate (TEABC), ammonium bicarbonate (ABC), PBS, sodium carbonate (Na2CO3), sucrose, potassium chloride (KCl), magnesium chloride hexahydrate (MgCl2), dl-DTT, HEPES, methyl methanethiosulfonate (MMTS), TFA, and HPLC grade ACN were purchased from Sigma-Aldrich. Formic acid (FA) and potassium dihydrogen phosphate (KH2PO4) were purchased from Riedel de Haen (Seelze, Germany). Water was obtained from a Milli-Q® Ultrapure Water Purification System (Millipore, Billerica, MA). The PKD1L3/L3 mice were generated according to Jiang et al. (30Jiang S.T. Chiou Y.Y. Wang E. Lin H.K. Lin Y.T. Chi Y.C. Wang C.K.L. Tang M.J. Li H. Defining a link with autosomal-dominant polycystic kidney disease in mice with congenitally low expression of Pkd1.Am. J. Pathol. 2006; 168: 205-220Abstract Full Text Full Text PDF PubMed Scopus (95) Google Scholar). Briefly a loxP site and a loxP-flanked mc1-neo cassette were introduced into introns 30 and 34, respectively, of the PKD1 locus to generate a conditional targeted mutation. Homozygotes were generated by a cross between heterozygotes and heterozygotes, and homozygotes were identified by PCR of tail genomic DNA. Mice were sacrificed by CO2 inhalation to obtain kidney samples. Renal tissue (∼0.5 × 0.5 cm in size) was suspended in 5 ml of 10 mm homogenization buffer at pH 7.4 (0.25 m sucrose and 0.1 m EDTA) and then homogenized at 4 °C in a Tissue-Tearor homogenizer (BioSpec Products, Bartlesville, OK). Renal extract was centrifuged at 1,000 × g for 30 min at 4 °C, and then the supernatant was centrifuged at 100,000 × g for an additional 60 min at 4 °C in a Beckman L5-65 ultracentrifuge. The resulting crude membrane fraction pellet was resuspended in 50 μl of 6 m urea, 5 mm EDTA, and 2% (w/v) SDS in 0.1 m TEABC for gel-assisted digestion. HeLa cells were first washed three times with 10 ml of PBS and then scraped into hypotonic buffer (10 mm HEPES, pH 7.5, 1.5 mm MgCl2, 10 mm KCl, and 1× protease inhibitor mixture (Calbiochem)) and homogenized with 50 strokes of a Dounce homogenizer. The membrane fraction was purified by two-step centrifugation. First nuclei were pelleted by centrifugation at 3,000 × g for 10 min at 4 °C. The postnuclear supernatant was mixed with 1.8 m sucrose to a final concentration of 0.25 m sucrose and was centrifuged for 1 h at 13,000 × g at 4 °C to pellet remaining membranes. The pellet was washed twice with 1 ml of ice-cold 0.1 m Na2CO3 (pH 11.5), dissolved in 50 μl of 90% (v/v) FA prior to the Bradford assay to determine the membrane protein concentration, and then vacuum-dried to obtain a membrane pellet for subsequent proteolysis reactions. Four digestion methods were compared in this study. For Tube-Gel digestion (24Lu X.N. Zhu H.N. Tube-gel digestion. A novel proteomic approach for high throughput analysis of membrane proteins.Mol. Cell. Proteomics. 2005; 4: 1948-1958Abstract Full Text Full Text PDF PubMed Scopus (164) Google Scholar), the membrane protein pellet was first dissolved in 50 μl of 25 mm ABC and 2% (w/v) SDS. The protein solution was mixed with 17.5 μl of acrylamide/bisacrylamide (40%, v/v, 29:1) solution, 2.5 μl of 1% (w/v) APS, and 1.07 μl of 100% TEMED in the tube without electrophoresis. The gel slice was cut into small pieces and washed several times with 25 mm ABC containing 50% (v/v) ACN. After drying in a SpeedVac, proteins were chemically reduced by the addition of 200 μl of 10 mm DTT at 60 °C for 30 min and alkylated by 200 μl of 55 mm iodoacetamide at room temperature in the dark for 30 min. The gel samples were further dehydrated with 100% ACN and then dried by SpeedVac. Proteolytic digestion was then performed with trypsin (protein:trypsin = 10:1, g/g) in 25 mm ABC with incubation overnight at 37 °C. Peptides were extracted from the gel using sequential extraction with 50 μl of 25 mm ABC, 100 μl of 0.1% (v/v) TFA in water, 150 μl of 0.1% (v/v) TFA in ACN, and 50 μl of 100% ACN, and the solutions were combined and concentrated by SpeedVac. For gel-assisted digestion, the membrane protein pellet was resuspended in 50 μl of 6 m urea, 5 mm EDTA, and 2% (w/v) SDS in 0.1 m TEABC and incubated at 37 °C for 30 min for complete dissolvation. Proteins were chemically reduced by adding 1.28 μl of 200 mm tris(2-carboxyethyl)phosphine and alkylated by adding 0.52 μl of 200 mm MMTS at room temperature for 30 min. To incorporate proteins into a gel directly in the Eppendorf vial, 18.5 μl of acrylamide/bisacrylamide solution (40%, v/v, 29:1), 2.5 μl of 10% (w/v) APS, and 1 μl of 100% TEMED was then applied to the membrane protein solution. The gel was cut into small pieces and washed several times with 1 ml of TEABC containing 50% (v/v) ACN. The gel samples were further dehydrated with 100% ACN and then completely dried by SpeedVac. Proteolytic digestion was then performed with trypsin (protein:trypsin = 10:1, g/g) in 25 mm TEABC with incubation overnight at 37 °C. Peptides were extracted from the gel using sequential extraction with 200 μl of 25 mm TEABC, 200 μl of 0.1% (v/v) TFA in water, 200 μl of 0.1% (v/v) TFA in ACN, and 200 μl of 100% ACN. The solutions were combined and concentrated in a SpeedVac. For methanol-assisted in-solution digestion (2Blonder J. Goshe M.B. Moore R.J. Pasa-Tolic L. Masselon C.D. Lipton M.S. Smith R.D. Enrichment of integral membrane proteins for proteomic analysis using liquid chromatography-tandem mass spectrometry.J. Proteome Res. 2002; 1: 351-360Crossref PubMed Scopus (209) Google Scholar), the membrane protein pellet was resuspended in 125 mm ABC. The suspension was first thermally denatured at 90 °C for 20 min, cooled on ice, and diluted by adding 100% methanol to produce a sample solution containing 60% (v/v) methanol. The final concentration of protein solution was 0.5 μg/μl. Tryptic digestion was performed at 37 °C for 5 h (protein:trypsin = 30:1, g/g). The suspension was saved, and the remaining pellet was subjected to digestion with trypsin (protein:trypsin = 50:1, g/g) for another 5 h. The supernatants of these two digestions were combined and concentrated by SpeedVac. For SDS-assisted in-solution digestion (31Zhang N. Li N. Li L. Liquid chromatography MALDI MS/MS for membrane proteome analysis.J. Proteome Res. 2004; 3: 719-727Crossref PubMed Scopus (58) Google Scholar), the membrane protein pellet was resuspended in 50 μl of 6 m urea, 5 mm EDTA, and 0.1% (w/v) SDS in 0.1 m TEABC and incubated at 37 °C for 30 min for complete dissolvation." @default.
• W2118993579 created "2016-06-24" @default.
• W2118993579 creator A5005102249 @default.
• W2118993579 creator A5022841259 @default.
• W2118993579 creator A5032536480 @default.
• W2118993579 creator A5037894454 @default.
• W2118993579 creator A5040673661 @default.
• W2118993579 creator A5062515455 @default.
• W2118993579 creator A5088296624 @default.
• W2118993579 date "2008-10-01" @default.
• W2118993579 modified "2023-10-14" @default.
• W2118993579 title "A Multiplexed Quantitative Strategy for Membrane Proteomics" @default.
• W2118993579 cites W1484977079 @default.
• W2118993579 cites W1597678601 @default.
• W2118993579 cites W1797925867 @default.
• W2118993579 cites W1966120295 @default.
• W2118993579 cites W1966760595 @default.
• W2118993579 cites W1971676830 @default.
• W2118993579 cites W1976001215 @default.
• W2118993579 cites W197861672 @default.
• W2118993579 cites W1995089537 @default.
• W2118993579 cites W1999560996 @default.
• W2118993579 cites W2003584540 @default.
• W2118993579 cites W2005312965 @default.
• W2118993579 cites W2005515607 @default.
• W2118993579 cites W2008220701 @default.
• W2118993579 cites W2018070639 @default.
• W2118993579 cites W2019235980 @default.
• W2118993579 cites W2019277255 @default.
• W2118993579 cites W2030370943 @default.
• W2118993579 cites W2031148468 @default.
• W2118993579 cites W2032190558 @default.
• W2118993579 cites W2042042511 @default.
• W2118993579 cites W2045702517 @default.
• W2118993579 cites W2046602608 @default.
• W2118993579 cites W2048168245 @default.
• W2118993579 cites W2051042325 @default.
• W2118993579 cites W2051209896 @default.
• W2118993579 cites W2051795211 @default.
• W2118993579 cites W2054171565 @default.
• W2118993579 cites W2062017840 @default.
• W2118993579 cites W2069093132 @default.
• W2118993579 cites W2079565710 @default.
• W2118993579 cites W2080581150 @default.
• W2118993579 cites W2084377042 @default.
• W2118993579 cites W2086110602 @default.
• W2118993579 cites W2118635467 @default.
• W2118993579 cites W2119383430 @default.
• W2118993579 cites W2120016245 @default.
• W2118993579 cites W2123815804 @default.
• W2118993579 cites W2125851915 @default.
• W2118993579 cites W2134852489 @default.
• W2118993579 cites W2140770667 @default.
• W2118993579 cites W2140946052 @default.
• W2118993579 cites W2143108257 @default.
• W2118993579 cites W2151754704 @default.
• W2118993579 cites W2152770371 @default.
• W2118993579 cites W2153821477 @default.
• W2118993579 cites W2154113357 @default.
• W2118993579 cites W2160007089 @default.
• W2118993579 cites W2163616625 @default.
• W2118993579 cites W2204738624 @default.
• W2118993579 doi "https://doi.org/10.1074/mcp.m800068-mcp200" @default.
• W2118993579 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/18490355" @default.
• W2118993579 hasPublicationYear "2008" @default.
• W2118993579 type Work @default.
• W2118993579 sameAs 2118993579 @default.
• W2118993579 citedByCount "128" @default.
• W2118993579 countsByYear W21189935792012 @default.
• W2118993579 countsByYear W21189935792013 @default.
• W2118993579 countsByYear W21189935792014 @default.
• W2118993579 countsByYear W21189935792015 @default.
• W2118993579 countsByYear W21189935792016 @default.
• W2118993579 countsByYear W21189935792017 @default.
• W2118993579 countsByYear W21189935792018 @default.
• W2118993579 countsByYear W21189935792019 @default.
• W2118993579 countsByYear W21189935792020 @default.
• W2118993579 countsByYear W21189935792022 @default.
• W2118993579 crossrefType "journal-article" @default.
• W2118993579 hasAuthorship W2118993579A5005102249 @default.
• W2118993579 hasAuthorship W2118993579A5022841259 @default.
• W2118993579 hasAuthorship W2118993579A5032536480 @default.
• W2118993579 hasAuthorship W2118993579A5037894454 @default.
• W2118993579 hasAuthorship W2118993579A5040673661 @default.
• W2118993579 hasAuthorship W2118993579A5062515455 @default.
• W2118993579 hasAuthorship W2118993579A5088296624 @default.
• W2118993579 hasBestOaLocation W21189935791 @default.
• W2118993579 hasConcept C104317684 @default.
• W2118993579 hasConcept C185592680 @default.
• W2118993579 hasConcept C41008148 @default.
• W2118993579 hasConcept C46111723 @default.
• W2118993579 hasConcept C55493867 @default.
• W2118993579 hasConcept C70721500 @default.
• W2118993579 hasConcept C80311884 @default.
• W2118993579 hasConcept C86803240 @default.
• W2118993579 hasConceptScore W2118993579C104317684 @default.
• W2118993579 hasConceptScore W2118993579C185592680 @default.
• W2118993579 hasConceptScore W2118993579C41008148 @default.

• next