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• W2160167428 abstract "Endogenous regeneration and repair mechanisms are responsible for replacing dead and damaged cells to maintain or enhance tissue and organ function, and one of the best examples of endogenous repair mechanisms involves skeletal muscle. Although the molecular mechanisms that regulate the differentiation of satellite cells and myoblasts toward myofibers are not fully understood, cell surface proteins that sense and respond to their environment play an important role. The cell surface capturing technology was used here to uncover the cell surface N-linked glycoprotein subproteome of myoblasts and to identify potential markers of myoblast differentiation. 128 bona fide cell surface-exposed N-linked glycoproteins, including 117 transmembrane, four glycosylphosphatidylinositol-anchored, five extracellular matrix, and two membrane-associated proteins were identified from mouse C2C12 myoblasts. The data set revealed 36 cluster of differentiation-annotated proteins and confirmed the occupancy for 235 N-linked glycosylation sites. The identification of the N-glycosylation sites on the extracellular domain of the proteins allowed for the determination of the orientation of the identified proteins within the plasma membrane. One glycoprotein transmembrane orientation was found to be inconsistent with Swiss-Prot annotations, whereas ambiguous annotations for 14 other proteins were resolved. Several of the identified N-linked glycoproteins, including aquaporin-1 and β-sarcoglycan, were found in validation experiments to change in overall abundance as the myoblasts differentiate toward myotubes. Therefore, the strategy and data presented shed new light on the complexity of the myoblast cell surface subproteome and reveal new targets for the clinically important characterization of cell intermediates during myoblast differentiation into myotubes. Endogenous regeneration and repair mechanisms are responsible for replacing dead and damaged cells to maintain or enhance tissue and organ function, and one of the best examples of endogenous repair mechanisms involves skeletal muscle. Although the molecular mechanisms that regulate the differentiation of satellite cells and myoblasts toward myofibers are not fully understood, cell surface proteins that sense and respond to their environment play an important role. The cell surface capturing technology was used here to uncover the cell surface N-linked glycoprotein subproteome of myoblasts and to identify potential markers of myoblast differentiation. 128 bona fide cell surface-exposed N-linked glycoproteins, including 117 transmembrane, four glycosylphosphatidylinositol-anchored, five extracellular matrix, and two membrane-associated proteins were identified from mouse C2C12 myoblasts. The data set revealed 36 cluster of differentiation-annotated proteins and confirmed the occupancy for 235 N-linked glycosylation sites. The identification of the N-glycosylation sites on the extracellular domain of the proteins allowed for the determination of the orientation of the identified proteins within the plasma membrane. One glycoprotein transmembrane orientation was found to be inconsistent with Swiss-Prot annotations, whereas ambiguous annotations for 14 other proteins were resolved. Several of the identified N-linked glycoproteins, including aquaporin-1 and β-sarcoglycan, were found in validation experiments to change in overall abundance as the myoblasts differentiate toward myotubes. Therefore, the strategy and data presented shed new light on the complexity of the myoblast cell surface subproteome and reveal new targets for the clinically important characterization of cell intermediates during myoblast differentiation into myotubes. Endogenous regeneration and repair mechanisms are responsible for replacing dead and damaged cells to maintain or enhance tissue and organ function. One of the best examples of endogenous repair mechanisms involves skeletal muscle, which has innate regenerative capacity (for reviews, see Refs. 1Chargé S.B. Rudnicki M.A. Cellular and molecular regulation of muscle regeneration.Physiol. Rev. 2004; 84: 209-238Crossref PubMed Scopus (1784) Google Scholar, 2Hawke T.J. Garry D.J. Myogenic satellite cells: physiology to molecular biology.J. Appl. Physiol. 2001; 91: 534-551Crossref PubMed Google Scholar, 3Le Grand F. Rudnicki M.A. Skeletal muscle satellite cells and adult myogenesis.Curr. Opin. Cell Biol. 2007; 19: 628-633Crossref PubMed Scopus (325) Google Scholar, 4Seale P. Rudnicki M.A. A new look at the origin, function, and “stem-cell” status of muscle satellite cells.Dev. Biol. 2000; 218: 115-124Crossref PubMed Scopus (480) Google Scholar). Skeletal muscle repair begins with satellite cells, a heterogeneous population of mitotically quiescent cells located in the basal lamina that surrounds adult skeletal myofibers (5Mauro A. Satellite cell of skeletal muscle fibers.J. Biophys. Biochem. Cytol. 1961; 9: 493-495Crossref PubMed Google Scholar, 6Bischoff R. Interaction between satellite cells and skeletal muscle fibers.Development. 1990; 109: 943-952Crossref PubMed Google Scholar), that, when activated, rapidly proliferate (7Appell H.J. Forsberg S. Hollmann W. Satellite cell activation in human skeletal muscle after training: evidence for muscle fiber neoformation.Int. J. Sports Med. 1988; 9: 297-299Crossref PubMed Google Scholar). The progeny of activated satellite cells, known as myogenic precursor cells or myoblasts, undergo several rounds of division prior to withdrawal from the cell cycle. This is followed by fusion to form terminally differentiated multinucleated myotubes and skeletal myofibers (7Appell H.J. Forsberg S. Hollmann W. Satellite cell activation in human skeletal muscle after training: evidence for muscle fiber neoformation.Int. J. Sports Med. 1988; 9: 297-299Crossref PubMed Google Scholar, 8Horsley V. Pavlath G.K. Forming a multinucleated cell: molecules that regulate myoblast fusion.Cells Tissues Organs. 2004; 176: 67-78Crossref PubMed Scopus (182) Google Scholar). These cells effectively repair or replace damaged cells or contribute to an increase in skeletal muscle mass. The molecular mechanisms that regulate differentiation of satellite cells and myoblasts toward myofibers are not fully understood, although it is known that the cell surface proteome plays an important biological role in skeletal muscle differentiation. Examples include how cell surface proteins modulate myoblast elongation, orientation, and fusion (for a review, see Ref. 8Horsley V. Pavlath G.K. Forming a multinucleated cell: molecules that regulate myoblast fusion.Cells Tissues Organs. 2004; 176: 67-78Crossref PubMed Scopus (182) Google Scholar). The organization and fusion of myoblasts is mediated, in part, by cadherins (for reviews, see Refs. 9Geiger B. Ayalon O. Cadherins.Annu. Rev. Cell Biol. 1992; 8: 307-332Crossref PubMed Google Scholar and 10Kaufmann U. Martin B. Link D. Witt K. Zeitler R. Reinhard S. Starzinski-Powitz A. M-cadherin and its sisters in development of striated muscle.Cell Tissue Res. 1999; 296: 191-198Crossref PubMed Scopus (0) Google Scholar), which enhance skeletal muscle differentiation and are implicated in myoblast fusion (11Redfield A. Nieman M.T. Knudsen K.A. Cadherins promote skeletal muscle differentiation in three-dimensional cultures.J. Cell Biol. 1997; 138: 1323-1331Crossref PubMed Scopus (0) Google Scholar). Neogenin, another cell surface protein, is also a likely regulator of myotube formation via the netrin ligand signal transduction pathway (12Kang J.S. Yi M.J. Zhang W. Feinleib J.L. Cole F. Krauss R.S. Netrins and neogenin promote myotube formation.J. Cell Biol. 2004; 167: 493-504Crossref PubMed Scopus (121) Google Scholar, 13Krauss R.S. Cole F. Gaio U. Takaesu G. Zhang W. Kang J.S. Close encounters: regulation of vertebrate skeletal myogenesis by cell-cell contact.J. Cell Sci. 2005; 118: 2355-2362Crossref PubMed Scopus (122) Google Scholar), and the family of sphingosine 1-phosphate receptors (Edg receptors) are known key signal transduction molecules involved in regulating myogenic differentiation (14Rapizzi E. Donati C. Cencetti F. Nincheri P. Bruni P. Sphingosine 1-phosphate differentially regulates proliferation of C2C12 reserve cells and myoblasts.Mol. Cell. Biochem. 2008; 314: 193-199Crossref PubMed Scopus (31) Google Scholar, 15Meacci E. Nuti F. Donati C. Cencetti F. Farnararo M. Bruni P. Sphingosine kinase activity is required for myogenic differentiation of C2C12 myoblasts.J. Cell. Physiol. 2008; 214: 210-220Crossref PubMed Scopus (53) Google Scholar, 16Squecco R. Sassoli C. Nuti F. Martinesi M. Chellini F. Nosi D. Zecchi-Orlandini S. Francini F. Formigli L. Meacci E. Sphingosine 1-phosphate induces myoblast differentiation through Cx43 protein expression: a role for a gap junction-dependent and -independent function.Mol. Biol. Cell. 2006; 17: 4896-4910Crossref PubMed Scopus (0) Google Scholar, 17Donati C. Meacci E. Nuti F. Becciolini L. Farnararo M. Bruni P. Sphingosine 1-phosphate regulates myogenic differentiation: a major role for S1P2 receptor.FASEB J. 2005; 19: 449-451Crossref PubMed Scopus (93) Google Scholar). Given the important role of these proteins, identifying and characterizing the cell surface proteins present on myoblasts in a more comprehensive approach could provide insights into the molecular mechanisms involved in skeletal muscle development and repair. The identification of naturally occurring cell surface proteins (i.e. markers) could also foster the enrichment and/or characterization of cell intermediates during differentiation that could be useful therapeutically. Although it is possible to use techniques such as flow cytometry, antibody arrays, and microscopy to probe for known proteins on the cell surface in discrete populations, these methods rely on a priori knowledge of the proteins present on the cell surface and the availability/specificity of an antibody. Proteomics approaches coupled with mass spectrometry offer an alternative approach that is antibody-independent and allows for the de novo discovery of proteins on the surface. One approach, which was used in the current study, exploits the fact that a majority of the cell surface proteins are glycosylated (18Apweiler R. Hermjakob H. Sharon N. On the frequency of protein glycosylation, as deduced from analysis of the SWISS-PROT database.Biochim. Biophys. Acta. 1999; 1473: 4-8Crossref PubMed Scopus (1351) Google Scholar). The method uses hydrazide chemistry (19Gemeiner P. Viskupic E. Stepwise immobilization of proteins via their glycosylation.J. Biochem. Biophys. Methods. 1981; 4: 309-319Crossref PubMed Scopus (14) Google Scholar) to immobilize and enrich for glycoproteins/glycopeptides, and previous studies using this chemistry have successfully identified soluble glycoproteins (20Zhang H. Yi E.C. Li X.J. Mallick P. Kelly-Spratt K.S. Masselon C.D. Camp 2nd, D.G. Smith R.D. Kemp C.J. Aebersold R. High throughput quantitative analysis of serum proteins using glycopeptide capture and liquid chromatography mass spectrometry.Mol. Cell. Proteomics. 2005; 4: 144-155Abstract Full Text Full Text PDF PubMed Scopus (186) Google Scholar, 21Zhang H. Liu A.Y. Loriaux P. Wollscheid B. Zhou Y. Watts J.D. Aebersold R. Mass spectrometric detection of tissue proteins in plasma.Mol. Cell. Proteomics. 2007; 6: 64-71Abstract Full Text Full Text PDF PubMed Scopus (154) Google Scholar, 22Liu T. Qian W.J. Gritsenko M.A. Camp 2nd, D.G. Monroe M.E. Moore R.J. Smith R.D. Human plasma N-glycoproteome analysis by immunoaffinity subtraction, hydrazide chemistry, and mass spectrometry.J. Proteome Res. 2005; 4: 2070-2080Crossref PubMed Scopus (345) Google Scholar, 23Liu T. Qian W.J. Gritsenko M.A. Xiao W. Moldawer L.L. Kaushal A. Monroe M.E. Varnum S.M. Moore R.J. Purvine S.O. Maier R.V. Davis R.W. Tompkins R.G. Camp 2nd, D.G. Smith R.D. High dynamic range characterization of the trauma patient plasma proteome.Mol. Cell. Proteomics. 2006; 5: 1899-1913Abstract Full Text Full Text PDF PubMed Scopus (128) Google Scholar, 24Sun B. Ranish J.A. Utleg A.G. White J.T. Yan X. Lin B. Hood L. Shotgun glycopeptide capture approach coupled with mass spectrometry for comprehensive glycoproteomics.Mol. Cell. Proteomics. 2007; 6: 141-149Abstract Full Text Full Text PDF PubMed Scopus (146) Google Scholar) as well as cell surface glycoproteins (25Zhang W. Zhou G. Zhao Y. White M.A. Zhao Y. Affinity enrichment of plasma membrane for proteomics analysis.Electrophoresis. 2003; 24: 2855-2863Crossref PubMed Scopus (81) Google Scholar, 26Lewandrowski U. Moebius J. Walter U. Sickmann A. Elucidation of N-glycosylation sites on human platelet proteins: a glycoproteomic approach.Mol. Cell. Proteomics. 2006; 5: 226-233Abstract Full Text Full Text PDF PubMed Scopus (131) Google Scholar, 27Zhang H. Li X.J. Martin D.B. Aebersold R. Identification and quantification of N-linked glycoproteins using hydrazide chemistry, stable isotope labeling and mass spectrometry.Nat. Biotechnol. 2003; 21: 660-666Crossref PubMed Scopus (1195) Google Scholar, 28Schiess R. Mueller L.N. Schmidt A. Mueller M. Wollscheid B. Aebersold R. Analysis of cell surface proteome changes via label-free, quantitative mass spectrometry.Mol. Cell Proteomics. 2009; 8: 624-638Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar). A recently optimized hydrazide chemistry strategy by Wollscheid et al. (29Wollscheid B. Bausch-Fluck D. Henderson C. O'Brien R. Bibel M. Schiess R. Aebersold R. Watts J.D. Mass-spectrometric identification and relative quantification of N-linked cell surface glycoproteins.Nat. Biotechnol. 2009; 27: 378-386Crossref PubMed Scopus (374) Google Scholar) termed cell surface capturing (CSC) 1The abbreviations used are:CSCcell surface capturingTMtransmembraneFBSfetal bovine serumLTQlinear trap quadrupoleFAformic acidBis-Tris2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)propane-1,3-diolCDcluster of differentiationGPIglycosylphosphatidylinositolECMextracellular matrixNCAMneural cell adhesion molecule.1The abbreviations used are:CSCcell surface capturingTMtransmembraneFBSfetal bovine serumLTQlinear trap quadrupoleFAformic acidBis-Tris2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)propane-1,3-diolCDcluster of differentiationGPIglycosylphosphatidylinositolECMextracellular matrixNCAMneural cell adhesion molecule. technology, reports the ability to identify cell surface (plasma membrane) proteins specifically with little (<15%) contamination from non-cell surface proteins. The specificity stems from the fact that the oligosaccharide structure is labeled using membrane-impermeable reagents while the cells are intact rather than after cell lysis. Consequently, only extracellular oligosaccharides are labeled and subsequently captured. Utilizing information regarding the glycosylation site then allows for a rapid elimination of nonspecifically captured proteins (i.e. non-cell surface proteins) during the data analysis process, a feature that makes this approach unique to methods where no label or tag is used. Additionally, the CSC technology provides information about glycosylation site occupancy (i.e. whether a potential N-linked glycosylation site is actually glycosylated), which is important for determining the protein orientation within the membrane and, therefore, antigen selection and antibody design. cell surface capturing transmembrane fetal bovine serum linear trap quadrupole formic acid 2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)propane-1,3-diol cluster of differentiation glycosylphosphatidylinositol extracellular matrix neural cell adhesion molecule. cell surface capturing transmembrane fetal bovine serum linear trap quadrupole formic acid 2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)propane-1,3-diol cluster of differentiation glycosylphosphatidylinositol extracellular matrix neural cell adhesion molecule. To uncover information about the cell surface of myoblasts and to identify potential markers of myoblast differentiation, we used the CSC technology on the mouse myoblast C2C12 cell line model system (30Yaffe D. Saxel O. Serial passaging and differentiation of myogenic cells isolated from dystrophic mouse muscle.Nature. 1977; 270: 725-727Crossref PubMed Google Scholar, 31Blau H.M. Pavlath G.K. Hardeman E.C. Chiu C.P. Silberstein L. Webster S.G. Miller S.C. Webster C. Plasticity of the differentiated state.Science. 1985; 230: 758-766Crossref PubMed Scopus (582) Google Scholar). This adherent cell line derived from satellite cells has routinely been used as a model for skeletal muscle development (e.g. Refs. 1Chargé S.B. Rudnicki M.A. Cellular and molecular regulation of muscle regeneration.Physiol. Rev. 2004; 84: 209-238Crossref PubMed Scopus (1784) Google Scholar, 32Burattini S. Ferri P. Battistelli M. Curci R. Luchetti F. Falcieri E. C2C12 murine myoblasts as a model of skeletal muscle development: morpho-functional characterization.Eur. J. Histochem. 2004; 48: 223-233PubMed Google Scholar, and 33Molkentin J.D. Olson E.N. Defining the regulatory networks for muscle development.Curr. Opin. Genet. Dev. 1996; 6: 445-453Crossref PubMed Scopus (381) Google Scholar), skeletal muscle differentiation (e.g. Refs. 34Capanni C. Del Coco R. Squarzoni S. Columbaro M. Mattioli E. Camozzi D. Rocchi A. Scotlandi K. Maraldi N. Foisner R. Lattanzi G. Prelamin A is involved in early steps of muscle differentiation.Exp. Cell Res. 2008; 314: 3628-3637Crossref PubMed Scopus (29) Google Scholar, 35Murray T.V. McMahon J.M. Howley B.A. Stanley A. Ritter T. Mohr A. Zwacka R. Fearnhead H.O. A non-apoptotic role for caspase-9 in muscle differentiation.J. Cell Sci. 2008; 121: 3786-3793Crossref PubMed Scopus (104) Google Scholar, 36Buas M.F. Kabak S. Kadesch T. Inhibition of myogenesis by Notch: evidence for multiple pathways.J. 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Additionally, these cells have been used in cell-based therapies (e.g. Refs. 40Bonacchi M. Nistri S. Nanni C. Gelsomino S. Pini A. Cinci L. Maiani M. Zecchi-Orlandini S. Lorusso R. Fanti S. Silvertown J. Bani D. Functional and histopathological improvement of the post-infarcted rat heart upon myoblast cell grafting and relaxin therapy.J. Cell Mol. Med. 2009; (in press)Crossref PubMed Scopus (29) Google Scholar, 41Formigli L. Perna A.M. Meacci E. Cinci L. Margheri M. Nistri S. Tani A. Silvertown J. Orlandini G. Porciani C. Zecchi-Orlandini S. Medin J. Bani D. Paracrine effects of transplanted myoblasts and relaxin on post-infarction heart remodelling.J. Cell. Mol. Med. 2007; 11: 1087-1100Crossref PubMed Scopus (74) Google Scholar, 42Reinecke H. Murry C.E. Transmural replacement of myocardium after skeletal myoblast grafting into the heart. Too much of a good thing?.Cardiovasc. Pathol. 2000; 9: 337-344Crossref PubMed Scopus (0) Google Scholar). Using the CSC technology, 128 cell surface N-linked glycoproteins were identified, including several that were found to change in overall abundance as the myoblasts differentiate toward myotubes. The current data also confirmed the occupancy of 235 N-linked glycosites of which 226 were previously unconfirmed. The new information provided by the current study is expected to facilitate the development of useful tools for studying the differentiation of myoblasts toward myotubes. Mouse myoblasts (C2C12 cell line) were cultured as described previously (43Minty A. Blau H. Kedes L. Two-level regulation of cardiac actin gene transcription: muscle-specific modulating factors can accumulate before gene activation.Mol. Cell. Biol. 1986; 6: 2137-2148Crossref PubMed Scopus (0) Google Scholar, 44Koban M.U. Brugh S.A. Riordon D.R. Dellow K.A. Yang H.T. Tweedie D. Boheler K.R. A distant upstream region of the rat multipartite Na(+)-Ca(2+) exchanger NCX1 gene promoter is sufficient to confer cardiac-specific expression.Mech. Dev. 2001; 109: 267-279Crossref PubMed Scopus (0) Google Scholar). C2C12 cells were cultivated in growth medium (Dulbecco's modified Eagle's medium, l-Glu, penicillin/streptomycin, 20% fetal bovine serum (FBS), 4.5g/liter glucose) in 5% CO2 and passaged at 70–80% confluency to maintain the undifferentiated myoblast population. Three biological replicates of undifferentiated C2C12 cells at ∼70% confluency were used. For differentiation, cells were switched under confluent conditions (>70–80%) to low serum conditions (5% FBS). Approximately 1 × 108 cells per biological replicate were taken through the CSC technology work flow as reported previously (28Schiess R. Mueller L.N. Schmidt A. Mueller M. Wollscheid B. Aebersold R. Analysis of cell surface proteome changes via label-free, quantitative mass spectrometry.Mol. Cell Proteomics. 2009; 8: 624-638Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar, 29Wollscheid B. Bausch-Fluck D. Henderson C. O'Brien R. Bibel M. Schiess R. Aebersold R. Watts J.D. Mass-spectrometric identification and relative quantification of N-linked cell surface glycoproteins.Nat. Biotechnol. 2009; 27: 378-386Crossref PubMed Scopus (374) Google Scholar) (Fig. 1). Cells were washed twice with labeling buffer (1× PBS (Quality Biological, Gaithersburg, MD), pH 6.5, 0.1% (v/v) FBS (Invitrogen)) followed by treatment for 15 min in 1.5 mm sodium meta-periodate (Pierce) in labeling buffer at 4 °C. Cells were washed with labeling buffer, collected, and centrifuged at 225 × g for 5 min at 25 °C. The pelleted cells were resuspended in 2.5 mg/ml biocytin hydrazide (Biotium, Hayward, CA) in labeling buffer for 1 h at 4 °C with gentle agitation, then washed with 1× PBS, and pelleted as above. Cells were resuspended in lysis buffer (10 mm Tris, pH 7.5, 0.5 mm MgCl2) and homogenized using a Dounce homogenizer. Cell lysate was centrifuged at 2500 × g for 10 min at 4 °C to remove the nucleus. The supernatant, containing the membranes, was centrifuged at 210,000 × g for 16 h at 4 °C. The membrane pellet was washed with 25 mm Na2CO3, resuspended in lysis buffer, and centrifuged at 210,000 × g for 30 min at 4 °C. The pellet was resuspended by sonication in 100 mm NH4HCO3, 5 mm tris(2-carboxyethyl)phosphine (Sigma), 0.1% (v/v) Rapigest (Waters). Proteins were then alkylated with 10 mm iodoacetamide for 30 min in the dark at 25 °C. The sample was then incubated with 1 µg of glycerol-free endoproteinase Lys-C (Calbiochem) at 37 °C for 4 h with end-over-end rotation and then with 20 µg of proteomics grade trypsin (Promega, Madison, WI) at 37 °C for 16 h with end-over-end rotation. The enzymes were inactivated by heating at 100 °C for 10 min followed by the addition of 10 µl of 1× Complete protease inhibitor mixture (Roche Applied Science). The peptide mixture was incubated with a 500-µl bead slurry of UltraLink Immobilized Streptavidin PLUS (Pierce) for 1 h at 25 °C. The beads were sequentially washed with the following: 5 m NaCl, 100 mm NH4HCO3, 5 m NaCl, 100 mm Na2CO3, 80% isopropanol, and 100 mm NH4HCO3. The beads were resuspended in 100 mm NH4HCO3 and 500 units of glycerol-free endoproteinase peptide-N-glycosidase F (New England Biolabs, Ipswich, MA) and incubated at 37 °C for 16 h with end-over-end rotation to release the peptides from the beads. The collected peptides were desalted and concentrated using a C18 UltraMicroSpin™ column (Nest Group, Southborough, MA) according to the manufacturer's instructions. In general, 1 × 108 cells provided sufficient peptide quantity for two to three individual LC-MS/MS analyses. For each biological replicate (n = 3), two technical replicates were analyzed by LC-MS/MS using either an LTQ-Orbitrap (Thermo, Waltham, MA) or an LTQ-FT (Thermo). For the LTQ-Orbitrap, desalted peptides were resuspended in 12 µl of 0.1% (v/v) aqueous formic acid (FA). Two times 5 µl were injected and analyzed on an Agilent 1200 nano-LC system (Agilent, Santa Clara, CA) connected to an LTQ-Orbitrap mass spectrometer (Thermo) equipped with a nanoelectrospray ion source (Thermo). Peptides were separated on a BioBasic (New Objective, Woburn, MA) C18 reversed phase HPLC column (75 µm × 10 cm) using a linear gradient from 5% B to 65% B in 60 min at a flow rate of 300 nl/min where mobile phase A was composed of 0.1% (v/v) aqueous FA and mobile phase B was 90% acetonitrile, 0.1% FA in water. Each MS1 scan was followed by CID (acquired in the LTQ part) of the five most abundant precursor ions with dynamic exclusion for 30 s. Only MS1 signals exceeding 10,000 counts triggered the MS2 scans. For MS1, 2 × 105 ions were accumulated in the Orbitrap over a maximum time of 500 ms and scanned at a resolution of 60,000 full-width half-maximum (at 400 m/z). MS2 spectra (via CID) were acquired in normal scan mode in the LTQ using a target setting of 104 ions and an accumulation time of 30 ms. The normalized collision energy was set to 35%, and one microscan was acquired for each spectrum. For the LTQ-FT, desalted peptides were resuspended in 12 µl of 0.1% (v/v) aqueous FA. Two times 4 µl were injected and analyzed on a Tempo™ Nano 1D+ HPLC system (Applied Biosystems/MDS Sciex, Foster City, CA) connected to a 7-tesla Finnigan LTQ-FT-ICR instrument (Thermo) equipped with a nanoelectrospray ion source (Thermo) using a C18 reverse phase HPLC column (75 µm × 15 cm) packed in house (Magic C18 AQ 3 µm; Michrom Bioresources, Auburn, CA) using a linear gradient from 4% B to 35% B in 60 min at a flow rate of 300 nl/min where mobile phase A was composed of 0.15% aqueous FA and mobile phase B was 98% (v/v) acetonitrile, 0.15% (v/v) FA in water. Each MS1 scan (acquired in the ICR cell) was followed by CID (acquired in the LTQ) of the five most abundant precursor ions with dynamic exclusion for 30 s. Only MS1 signals exceeding 150 counts were allowed to trigger MS2 scans with wideband activation disabled. For MS1, 3 × 106 ions were accumulated in the ICR cell over a maximum time of 500 ms and scanned at a resolution of 100,000 full-width half-maximum (at 400 m/z). MS2 spectra were acquired in normal scan mode with a target setting of 104 ions and an accumulation time of 100 ms. Singly charged ions and ions with unassigned charge state were excluded from triggering MS2 events. The normalized collision energy was set to 32%, and one microscan was acquired for each spectrum. Raw MS data were searched against the International Protein Index mouse v3.47 database (45Kersey P.J. Duarte J. Williams A. Karavidopoulou Y. Birney E. Apweiler R. The International Protein Index: an integrated database for proteomics experiments.Proteomics. 2004; 4: 1985-1988Crossref PubMed Scopus (625) Google Scholar) (55,298 entries; August 26, 2008) using Sorcerer 2™-SEQUEST® (Sage-N Research, Milpitas, CA) with postsearch analysis performed using the Trans-Proteome Pipeline, implementing PeptideProphet (46Keller A. Nesvizhskii A.I. Kolker E. Aebersold R. Empirical statistical model to estimate the accuracy of peptide identifications made by MS/MS and database search.Anal. Chem. 2002; 74: 5383-5392Crossref PubMed Scopus (3610) Google Scholar) and ProteinProphet (47Nesvizhskii A.I. Keller A. Kolker E. Aebersold R. A statistical model for identifying proteins by tandem mass spectrometry.Anal. Chem. 2003; 75: 4646-4658Crossref PubMed Scopus (3298) Google Scholar) algorithms. All raw data peak extraction was performed using Sorcerer 2-SEQUEST default settings. Database search parameters were as follows: semienzyme digest using trypsin (after Lys or Arg) with up to two missed cleavages; monoisotopic precursor mass range of 400–4500 amu; and oxidation (Met), carbamidomethylation (Cys), and deamidation (Asn) allowed as differential modifications. Peptide mass tolerance was set to 50 ppm, fragment mass tolerance was set to 1 amu, fragment mass type was set to monoisotopic, and the maximum number of modifications was set to four per peptide. Advanced search options that were enabled included the following: XCorr score cutoff of 1.5, isotope check using a mass shift of 1.003355 amu, keep the top 2000 preliminary results for final scoring, display up to 200 peptide results in the result file, display up to five full protein descriptions in the result file, and display up to one duplicate protein reference in the result file. Error rates (false discovery rates) and protein probabilities (p) were calculated by ProteinProphet. Raw data from all three biological replicates were combined into a single database search. The ProteinProphet interact-prot.xml result files were input into ProteinCenter (Proxeon Bioinformatics, Odense, Denmark) and filtered to contain only proteins with protein probability scores of p > 0.48. To prevent redundancy in protein identifications, proteins were grouped according to “indistinguishable proteins,” resulting in 128 protein groups. For the final database, isoform notation is provided only when a peptide that is unique to a specific protein isoform was identified. The protein list in supplemental Table S1 displays only those proteins identified by peptides containing an observed deamidation at the asparagine(s) within the conserved sequence motif for N-linked" @default.
• W2160167428 created "2016-06-24" @default.
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• W2160167428 date "2009-11-01" @default.
• W2160167428 modified "2023-10-06" @default.
• W2160167428 title "The Mouse C2C12 Myoblast Cell Surface N-Linked Glycoproteome" @default.
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