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• W2098202316 abstract "By interacting with the cytoplasmic tail of a Golgi-processed form of transforming growth factor-α (TGFα), Naked2 coats TGFα-containing exocytic vesicles and directs them to the basolateral corner of polarized epithelial cells where the vesicles dock and fuse in a Naked2 myristoylation-dependent manner. These TGFα-containing Naked2-associated vesicles are not directed to the subapical Sec6/8 exocyst complex as has been reported for other basolateral cargo, and thus they appear to represent a distinct set of basolaterally targeted vesicles. To identify constituents of these vesicles, we exploited our finding that myristoylation-deficient Naked2 G2A vesicles are unable to fuse at the plasma membrane. Isolation of a population of myristoylation-deficient, green fluorescent protein-tagged G2A Naked2-associated vesicles was achieved by biochemical enrichment followed by flow cytometric fluorescence-activated vesicle sorting. The protein content of these plasma membrane de-enriched, flow-sorted fluorescent G2A Naked2 vesicles was determined by LC/LC-MS/MS analysis. Three independent isolations were performed, and 389 proteins were found in all three sets of G2A Naked2 vesicles. Rab10 and myosin IIA were identified as core machinery, and Na+/K+-ATPase α1 was identified as an additional cargo within these vesicles. As an initial validation step, we confirmed their presence and that of three additional proteins tested (annexin A1, annexin A2, and IQGAP1) in wild-type Naked2 vesicles. To our knowledge, this is the first large scale protein characterization of a population of basolaterally targeted exocytic vesicles and supports the use of fluorescence-activated vesicle sorting as a useful tool for isolation of cellular organelles for comprehensive proteomics analysis. By interacting with the cytoplasmic tail of a Golgi-processed form of transforming growth factor-α (TGFα), Naked2 coats TGFα-containing exocytic vesicles and directs them to the basolateral corner of polarized epithelial cells where the vesicles dock and fuse in a Naked2 myristoylation-dependent manner. These TGFα-containing Naked2-associated vesicles are not directed to the subapical Sec6/8 exocyst complex as has been reported for other basolateral cargo, and thus they appear to represent a distinct set of basolaterally targeted vesicles. To identify constituents of these vesicles, we exploited our finding that myristoylation-deficient Naked2 G2A vesicles are unable to fuse at the plasma membrane. Isolation of a population of myristoylation-deficient, green fluorescent protein-tagged G2A Naked2-associated vesicles was achieved by biochemical enrichment followed by flow cytometric fluorescence-activated vesicle sorting. The protein content of these plasma membrane de-enriched, flow-sorted fluorescent G2A Naked2 vesicles was determined by LC/LC-MS/MS analysis. Three independent isolations were performed, and 389 proteins were found in all three sets of G2A Naked2 vesicles. Rab10 and myosin IIA were identified as core machinery, and Na+/K+-ATPase α1 was identified as an additional cargo within these vesicles. As an initial validation step, we confirmed their presence and that of three additional proteins tested (annexin A1, annexin A2, and IQGAP1) in wild-type Naked2 vesicles. To our knowledge, this is the first large scale protein characterization of a population of basolaterally targeted exocytic vesicles and supports the use of fluorescence-activated vesicle sorting as a useful tool for isolation of cellular organelles for comprehensive proteomics analysis. Transforming growth factor-α (TGFα), 1The abbreviations used are: TGFα, transforming growth factor-α; DiD, 1,1′-dioctadecyl-3,3,3′,3′-tetramethylindodicarbocyanine perchlorate; FAVS, fluorescence-activated vesicle sorting; FDR, false discovery rate; MDCK, Madin-Darby canine kidney; TGN, trans Golgi network; GFP, green fluorescent protein; EGFP, enhanced green fluorescent protein; ER, endoplasmic reticulum; WT, wild-type; M-PER, Mammalian Protein Extraction Reagent; HEK, human embryonic kidney; SCX, strong cation exchange; G, sorting gate; EM, electron microscopy; Exp, experiment; ID, identity; VDAC, voltage-dependent anion-selective channel; PMT, photomultiplier tube; Bis-Tris, 2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)propane-1,3-diol; PF, paraformaldehyde. 1The abbreviations used are: TGFα, transforming growth factor-α; DiD, 1,1′-dioctadecyl-3,3,3′,3′-tetramethylindodicarbocyanine perchlorate; FAVS, fluorescence-activated vesicle sorting; FDR, false discovery rate; MDCK, Madin-Darby canine kidney; TGN, trans Golgi network; GFP, green fluorescent protein; EGFP, enhanced green fluorescent protein; ER, endoplasmic reticulum; WT, wild-type; M-PER, Mammalian Protein Extraction Reagent; HEK, human embryonic kidney; SCX, strong cation exchange; G, sorting gate; EM, electron microscopy; Exp, experiment; ID, identity; VDAC, voltage-dependent anion-selective channel; PMT, photomultiplier tube; Bis-Tris, 2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)propane-1,3-diol; PF, paraformaldehyde. one of seven mammalian epidermal growth factor receptor ligands, is directed preferentially to the basolateral surface of polarized epithelial cells (1Li C. Franklin J.L. Graves-Deal R. Jerome W.G. Cao Z. Coffey R.J. Myristoylated Naked2 escorts transforming growth factor α to the basolateral plasma membrane of polarized epithelial cells.Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 5571-5576Crossref PubMed Scopus (55) Google Scholar, 2Li C. Hao M. Cao Z. Ding W. Graves-Deal R. Hu J. Piston D.W. Coffey R.J. Naked2 acts as a cargo recognition and targeting protein to ensure proper delivery and fusion of TGF-α-containing exocytic vesicles at the lower lateral membrane of polarized MDCK cells.Mol. Biol. Cell. 2007; 18: 3081-3093Crossref PubMed Scopus (34) Google Scholar, 3Harris R.C. Chung E. Coffey R.J. EGF receptor ligands.Exp. Cell Res. 2003; 284: 2-13Crossref PubMed Scopus (597) Google Scholar, 4Dempsey P.J. Meise K.S. Coffey R.J. Basolateral sorting of transforming growth factor-α precursor in polarized epithelial cells: characterization of cytoplasmic domain determinants.Exp. Cell Res. 2003; 285: 159-174Crossref PubMed Scopus (37) Google Scholar). It contains a dileucine basolateral sorting motif in its 39-amino acid cytoplasmic tail, the region of TGFα that is most highly conserved across species (4Dempsey P.J. Meise K.S. Coffey R.J. Basolateral sorting of transforming growth factor-α precursor in polarized epithelial cells: characterization of cytoplasmic domain determinants.Exp. Cell Res. 2003; 285: 159-174Crossref PubMed Scopus (37) Google Scholar). We have found that Naked2, but not Naked1, recognizes basolateral sorting determinants in the cytoplasmic tail of Golgi-processed TGFα, coating these TGFα-containing vesicles and directing these vesicles to the basolateral surface of polarized epithelial cells (1Li C. Franklin J.L. Graves-Deal R. Jerome W.G. Cao Z. Coffey R.J. Myristoylated Naked2 escorts transforming growth factor α to the basolateral plasma membrane of polarized epithelial cells.Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 5571-5576Crossref PubMed Scopus (55) Google Scholar). Naked1 and -2 are mammalian members of the Naked family (Naked Cuticle in Drosophila) that are reported to act as inducible antagonists of canonical Wnt signaling in Drosophila, zebrafish, and mammals (5Zeng W. Wharton Jr., K.A. Mack J.A. Wang K. Gadbaw M. Suyama K. Klein P.S. Scott M.P. nakedcuticle encodes an inducible antagonist of Wnt signalling.Nature. 2000; 403: 789-795Crossref PubMed Scopus (176) Google Scholar, 6Ishikawa A. Kitajima S. Takahashi Y. Kokubo H. Kanno J. Inoue T. Saga Y. Mouse Nkd1, a Wnt antagonist, exhibits oscillatory gene expression in the PSM under the control of Notch signaling.Mech. Dev. 2004; 121: 1443-1453Crossref PubMed Scopus (82) Google Scholar, 7Van Raay T.J. Coffey R.J. Solnica-Krezel L. Zebrafish Naked1 and Naked2 antagonize both canonical and non-canonical Wnt signaling.Dev. Biol. 2007; 309: 151-168Crossref PubMed Scopus (42) Google Scholar). Based on studies of basolateral trafficking of LDLR and VSVG protein (8Grindstaff K.K. Yeaman C. Anandasabapathy N. Hsu S.C. Rodriguez-Boulan E. Scheller R.H. Nelson W.J. Sec6/8 complex is recruited to cell-cell contacts and specifies transport vesicle delivery to the basal-lateral membrane in epithelial cells.Cell. 1998; 93: 731-740Abstract Full Text Full Text PDF PubMed Scopus (443) Google Scholar, 9Yeaman C. Grindstaff K.K. Nelson W.J. Mechanism of recruiting Sec6/8 (exocyst) complex to the apical junctional complex during polarization of epithelial cells.J. Cell Sci. 2004; 117: 559-570Crossref PubMed Scopus (142) Google Scholar), it is thought that basolaterally targeted vesicles and their attendant cargo are directed to a subapical Sec6/8 exocyst targeting patch where the vesicles dock and fuse. In addition, LDLR and VSVG utilize the AP-1 adaptor machinery, either at the TGN and/or recycling endosome, for their successful delivery to the basolateral surface (10Meyer C. Zizioli D. Lausmann S. Eskelinen E.L. Hamann J. Saftig P. von Figura K. Schu P. mu1A-adaptin-deficient mice: lethality, loss of AP-1 binding and rerouting of mannose 6-phosphate receptors.EMBO J. 2000; 19: 2193-2203Crossref PubMed Scopus (349) Google Scholar, 11Traub L.M. Kornfeld S. The trans-Golgi network: a late secretory sorting station.Curr. Opin. Cell Biol. 1997; 9: 527-533Crossref PubMed Scopus (192) Google Scholar). TGFα-containing Naked2-associated vesicles appear to represent a distinct set of basolaterally targeted vesicles based on the following four observations. First, Naked2 contains basolateral sorting information; residues 1–173 of Naked2 fused to Na+/H+ exchanger regulatory factor-1 are able to completely redirect Na+/H+ exchanger regulatory factor-1 from the apical cytoplasm to the basolateral plasma membrane (2Li C. Hao M. Cao Z. Ding W. Graves-Deal R. Hu J. Piston D.W. Coffey R.J. Naked2 acts as a cargo recognition and targeting protein to ensure proper delivery and fusion of TGF-α-containing exocytic vesicles at the lower lateral membrane of polarized MDCK cells.Mol. Biol. Cell. 2007; 18: 3081-3093Crossref PubMed Scopus (34) Google Scholar). Second, Naked2 vesicles are delivered directly to the lower lateral membrane of polarized MDCK cells and not to a subapical Sec6/8 targeting patch. Third, this delivery does not require AP-1B because Naked2-associated vesicles are found at the basolateral surface of polarized LLC-PK1 cells that lack μ1B, an essential component of the heterotetrameric AP-1B complex (12Sugimoto H. Sugahara M. Folsch H. Koide Y. Nakatsu F. Tanaka N. Nishimura T. Furukawa M. Mullins C. Nakamura N. Mellman I. Ohno H. Differential recognition of tyrosine-based basolateral signals by AP-1B subunit μ1B in polarized epithelial cells.Mol. Biol. Cell. 2002; 13: 2374-2382Crossref PubMed Scopus (62) Google Scholar). Finally, docking and fusion of these vesicles is dependent upon Naked2 myristoylation because Naked2-associated vesicles accumulate asymmetrically at the basolateral corner of MDCK cells stably expressing myristoylation-deficient G2A Naked2. These multiple functions of Naked2 have led us to designate it a cargo recognition and targeting protein (CaRT) for TGFα-containing, basolaterally targeted exocytic vesicles (2Li C. Hao M. Cao Z. Ding W. Graves-Deal R. Hu J. Piston D.W. Coffey R.J. Naked2 acts as a cargo recognition and targeting protein to ensure proper delivery and fusion of TGF-α-containing exocytic vesicles at the lower lateral membrane of polarized MDCK cells.Mol. Biol. Cell. 2007; 18: 3081-3093Crossref PubMed Scopus (34) Google Scholar). The distinctive properties of these Naked2 vesicles spurred us to determine the protein composition of these vesicles. Previous attempts to analyze basolaterally targeted vesicles have been unsuccessful likely because of their low abundance and transient nature with rapid fusion to acceptor membranes. Herein we exploited the fact that myristoylation-deficient G2A Naked2-enhanced green fluorescent protein (EGFP)-associated vesicles are “trapped” in time and space to biochemically isolate a population of these vesicles that are de-enriched for plasma membrane, ER, and Golgi constituents. We then used fluorescence-activated vesicle sorting (FAVS) as a novel means to purify a discrete pool of G2A Naked2-containing exocytic vesicles for large scale LC/LC-MS/MS analysis. By application of stringent acceptance criteria, we identified 389 proteins in these G2A Naked2-containing vesicles. We selected six of these proteins to study in wild-type (WT) Naked2-expressing MDCK cells, and we confirmed the presence of all six in WT Naked2-containing vesicles. These included proteins previously linked to basolateral exocytosis (Rab10, myosin IIA, Na+/K+-ATPase α1, IQGAP1, and annexin A2) as well as annexin A1 that has been associated with apical trafficking. We propose that FAVS may be a useful tool for isolation of cellular organelles for comprehensive LC/LC-MS/MS analysis. All cell culture reagents were from HyClone (Logan, UT). All chemicals, including TCA, anti-mouse myosin IIA antibody, and protease inhibitor mixture, were from Sigma unless otherwise stated. Chemicals for electrophoresis were purchased from Bio-Rad. A rabbit anti-human Naked2 antibody, R44, was made in collaboration with Covance (Princeton, NJ) as described by Li et al. (1Li C. Franklin J.L. Graves-Deal R. Jerome W.G. Cao Z. Coffey R.J. Myristoylated Naked2 escorts transforming growth factor α to the basolateral plasma membrane of polarized epithelial cells.Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 5571-5576Crossref PubMed Scopus (55) Google Scholar). Monoclonal antibodies to annexins A1 and A2 were purchased from Abcam (Cambridge, MA). Anti-Na+/K+-ATPase α1, -E-cadherin, and -IQGAP1 were obtained from Upstate (Temecula, CA), and anti-caveolin-1 was obtained from Santa Cruz Biotechnology (Santa Cruz, CA). Golgin97, anti-mouse CD147, anti-rabbit IgG, and DiD (in oil) were purchased from Invitrogen-Molecular Probes. Protein A beads and M-PER (Mammalian Protein Extraction Reagent) were purchased from Pierce. Cy3-conjugated donkey anti-sheep IgG and horseradish peroxidase-conjugated donkey anti-mouse and -rabbit IgG were obtained from Jackson ImmunoResearch Laboratories (West Grove, PA). The Rab11a antibody has been characterized previously (13Lapierre L.A. Avant K.M. Caldwell C.M. Ham A.J. Hill S. Williams J.A. Smolka A.J. Goldenring J.R. Characterization of immunoisolated human gastric parietal cells tubulovesicles: identification of regulators of apical recycling.Am. J. Physiol. 2007; 292: G1249-G1262Crossref PubMed Scopus (57) Google Scholar). The rabbit polyclonal antibody against Rab10 (VU132/134) was raised against a specific peptide sequence at the carboxyl-terminal variable domain, CKTPVKEPNSENVDIS. Keyhole limpet hemocyanin was covalently attached to the amino-terminal cysteine for immunization of the rabbits (Covance). The antiserum displayed a single 23-kDa band upon Western blotting of MDCK and HeLa cell lysates (data not shown). The HaloTag vector, pHT2, was purchased from Promega (Promega, Madison, WI). MDCK Tet-Off cells T23 1628 (Clontech) were stably transfected with WT human Naked2 (codons 1–451) or G2A (in which the second residue glycine was mutated to alanine) cDNAs; EGFP was fused to the carboxyl terminus of both Naked2 cDNAs. Cells were grown in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal bovine serum, glutamine, non-essential amino acids, 100 units/ml penicillin, 100 μg/ml streptomycin, and 500 μg/ml Geneticin or 200 μg/ml hygromycin B (Roche Applied Science). Cells were maintained in doxycycline (1 μg/ml). Experiments were performed 48 h after removal of doxycycline at which time there was maximum expression of transfected Naked2 as described previously (1Li C. Franklin J.L. Graves-Deal R. Jerome W.G. Cao Z. Coffey R.J. Myristoylated Naked2 escorts transforming growth factor α to the basolateral plasma membrane of polarized epithelial cells.Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 5571-5576Crossref PubMed Scopus (55) Google Scholar). HEK 293 cells and HCA-7 cells were grown in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal bovine serum. For polarization experiments with MDCK cells, 1 × 105 cells were seeded on 12-mm Transwell filters (0.4 μm; Corning Costar, Corning, NY) and cultured for 4–5 days with replenishment of medium every other day until trans-epithelial electrical resistance exceeded 200 ohms/cm2 using the Millicell Electrical Resistance System (Millipore Corp., Bedford, MA). MDCK cells were transiently co-transfected with WT Naked2-HaloTag™ and G2A Naked2-EGFP cDNAs, and fluorescence was observed 24 h later. The Goldenring laboratory kindly provided the monomeric dsRed-Rab10 (C2 Clontech vector) plasmid. The schema for purifying Naked2-EGFP vesicles from MDCK cells is summarized in Fig. 1. We modified the OptiPrep™ protocol (S37; Axis Shield, Oslo, Norway) as described below. For each preparation, MDCK cells were plated on 24 × 150-mm plastic dishes and grown to 70% confluence at which time doxycycline was removed for 48 h. 5 × 108 cells were rinsed three times in 1× PBS and then washed once in Solution D (78 mm KCl, 4 mm MgCl2, 8 mm CaCl2, 10 mm EGTA, and 50 mm HEPES-KOH, pH 7.0). The cells were scraped off with a rubber policeman in Solution E (0.25 m sucrose, 78 mm KCl, 4 mm MgCl2, 8 mm CaCl2, 10 mm EGTA, and 50 mm HEPES-KOH, pH 7.0). The cell suspension was mixed with an equal volume of acid-washed glass beads (Sigma) and agitated at top speed eight times for 2 min on a BioSpec Mini-Beadbeater-8TM vortex mixer (BioSpec Products Inc., Bartlesville, OK). In preliminary experiments (data not shown), we determined that the efficiency of vesicle isolation was greater using glass beads (80%) than a Dounce homogenizer (30%; Wheaton Science Products, Millville, NJ). Lysates were then diluted 10-fold with Solution E and centrifuged at 4 °C sequentially at 1,000 × g (10 min; SS-34 rotor, Sorvall), 5,000 × g (40 min; SS-34 rotor, Sorvall), and 100,000 × g (2 h; SW 28 rotor, Beckman). This vesicle-enriched pellet was resuspended in 9 ml of digestion buffer (75 mm KCl, 50 mm Tris-HCl, 3 mm MgCl2, and 10 mm DTT, pH 8.3) and digested with RNase A (20 μg/ml) at 37 °C for 30 min at which time the digestion was stopped by the addition of 10 mm EDTA. Approximately 1.5 ml of the resuspended pellet then was placed on top of a discontinuous 10, 20, 25, 30, and 40% iodixanol gradient solution (2 ml each in a 12-ml-volume tube) and centrifuged at 90,000 × g at 4 °C for 16 h in a TH641 rotor (Beckman). Successive 500-μl aliquots were taken from the top of the ultracentrifuge tubes, and 23 fractions were collected. All operations were performed either on ice or at 4 °C. The optical densities were read at 600 and 280 nm. An aliquot from each fraction was used for Western blotting of Naked2 and various cellular markers. DiD is a lipophilic fluorescent dye that nonspecifically partitions into membrane bilayers. Pool 2 (fractions 18–22) from G2A Naked2-EGFP-expressing MDCK cells was subjected to DiD staining (final concentration, 12.5 μg/ml) at 37 °C for 10 min in Solution D. The EGFP and DiD dual-labeled Pool 2 vesicles were pelleted at 100,000 × g for 1 h. The pellet was resuspended in 7 ml of Solution E and successively sheared through 22-, 27- and 30-gauge syringes six times each before FAVS. Sorting was performed on a BD Biosciences FACSAria customized with a forward scatter PMT and standardized for linearity and sensitivity using eight peak beads (Spherotech, Lake Forest, IL). Resolution of particle size was refined using green fluorescent beads ranging in size from 40 to 700 nm (Duke Scientific, Fremont, CA). A custom high salt sheath (Solution D), which was compatible with the vesicles, was filtered through a 100-nm filter prior to installation into the Aria sheath reservoir. Two additional in-line filters (200 nm) were used to assure the sheath had the lowest background possible. Unstained and single stained (DiD or GFP only) vesicles were used to compensate for spectral overlap. Doubly positive DiD-counterstained G2A Naked2-EGFP-tagged vesicles were gated and subjected to pulse processing analysis for doublet discrimination. Extensive preliminary studies were performed to determine and validate that the sorting gates contained the double-labeled vesicles of interest. Briefly vesicles were sorted using a variety of sort gating strategies, and the isolated vesicles were concentrated separately and imaged using confocal microscopy. These preliminary studies allowed us to define a target mask with a linear range of fluorescence intensities that enabled significant enrichment of the double-labeled vesicles. Vesicles falling outside the target mask included significant numbers of doublet vesicles that, even upon repeated sorting, could not yield a single peak and thus were excluded from subsequent analysis. Finally double positive vesicles were gated to remove those vesicles greater than 0.55 SD from their mean fluorescence intensities. Pool 2 from G2A Naked2-sorted vesicles was concentrated using 20% TCA at 4 °C overnight. A transparent pellet was obtained after centrifugation at 15,000 rpm for 20 min at 4 °C. The pellet was washed three times with 10 ml of ice-chilled acetone, dried in a fume hood for 10 min, and then resuspended in 100 μl of 1× lauryl dodecyl sulfate NuPAGE sample buffer (Invitrogen, 4×) with reducing agent DTT (final concentration, 50 μm). The sample was heated for 10 min at 85 °C and then run ∼1.5 cm into a 10% SDS-polyacrylamide gel. After staining with Colloidal Coomassie Blue (Invitrogen), the entire stained protein region was excised, chopped into 1-mm cubes, and placed in 0.5-ml Eppendorf tubes containing 150 μl of 100 mm ammonium bicarbonate. Samples were reduced with 10 μl of 45 mm DTT for 20 min at 55 °C and alkylated with 10 μl of 100 mm iodoacetamide for 20 min at room temperature in the dark. Samples were destained with 100 μl of 50% acetonitrile and 50 mm ammonium bicarbonate. The gel pieces were then dehydrated with 100% acetonitrile and digested with trypsin (10 μl of 0.01 ng/μl Trypsin Gold (Promega) in 25 mm ammonium bicarbonate) overnight at 37 °C. The peptides were extracted with two rounds of 60% acetonitrile and 0.1% trifluoroacetic acid. They were dried down and reconstituted in 0.1% formic acid. The resulting peptides were analyzed by multidimensional chromatography-tandem mass spectrometry using a combination of off-line cation exchange separation and on-line reverse phase separation. The peptides were first fractionated using strong cation exchange chromatography (Luna SCX, Phenomenex, Torrance, CA) with a 100-μm × 10-cm column and using a 0–500 mm ammonium formate, pH 3.0–8.0, gradient in 25% acetonitrile as originally described by Adkins et al. (14) with minor modifications as described previously (13Lapierre L.A. Avant K.M. Caldwell C.M. Ham A.J. Hill S. Williams J.A. Smolka A.J. Goldenring J.R. Characterization of immunoisolated human gastric parietal cells tubulovesicles: identification of regulators of apical recycling.Am. J. Physiol. 2007; 292: G1249-G1262Crossref PubMed Scopus (57) Google Scholar). Ten equal fractions were collected from the SCX fractionation, and fractions 7–9 were combined. The flow-through and seven fractions were subjected to LC-MS analysis using a ThermoFinnigan LTQ ion trap mass spectrometer equipped with a Thermo MicroAS autosampler and Thermo Surveyor HPLC pump, Nanospray source, and Xcalibur 1.4 instrument control. The peptides were separated on a packed capillary tip, 100 μm × 11 cm, with C18 resin (Jupiter C18, 5 μm, 300 Å, Phenomenex, Torrance, CA) using an in-line solid phase extraction column (100 μm × 3 cm) packed with the same C18 resin using a 0.1% formic acid, acetonitrile gradient as described previously (14Adkins J.N. Varnum S.M. Auberry K.J. Moore R.J. Angell N.H. Smith R.D. Springer D.L. Pounds J.G. Toward a human blood serum proteome: analysis by multidimensional separation coupled with mass spectrometry.Mol. Cell. Proteomics. 2002; 1: 947-955Abstract Full Text Full Text PDF PubMed Scopus (711) Google Scholar). Centroided MS/MS scans were acquired using an isolation width of 2 m/z, an activation time of 30 ms, an activation Q of 0.250, and 30% normalized collision energy using one microscan and maximum injection time of 100 ms for each MS/MS scan. The mass spectrometer was tuned prior to analysis using the synthetic peptide TpepK (AVAGKAGAR) so that some parameters may have varied slightly from experiment to experiment, but typically the tune parameters were as follows: spray voltage of 1.8 KV, a capillary temperature of 160 °C, a capillary voltage of 50 V, and tube lens voltage of 120 V. The MS/MS spectra of the peptides were collected using data-dependent scanning in which one full MS spectrum was followed by three MS/MS spectra. Raw data files for these analyses are available from the ProteomeCommons Tranche network (http://www.proteomecommons.org/data-downloader.jsp?fileName=t7lfD%2BVtLYdFQT8Bhlq7Zok3c52%2BJWOeJKmWUcCBTA/d65bAVtmTiIZkg8QMoCpWGXy/77QFnszqvSxjkDiuCCwNPBgAAAAAAAAT3w==) using hash codes. The “ScanSifter” algorithm read tandem mass spectra stored as centroided peak lists from Thermo RAW files and transcoded them to mzData v1.05 files. Only MS/MS scans were written to the mzData files; MS scans were excluded. If 90% of the intensity of a tandem mass spectrum appeared at a lower m/z than that of the precursor ion, a single precursor charge was assumed; otherwise the spectrum was processed under both double and triple precursor charge assumptions. Proteins were identified using the MyriMatch v1.0.385 algorithm (15Tabb D.L. Fernando C.G. Chambers M.C. MyriMatch: highly accurate tandem mass spectral peptide identification by multivariate hypergeometric analysis.J. Proteome Res. 2007; 6: 654-661Crossref PubMed Scopus (434) Google Scholar) on a cluster of 12 dual core × 86 processors. The database used (available as part of the raw data download above) was based on the Ensembl Canis familiaris sequence database, release 44 (25,568 sequences), with protein descriptions added through links between Ensembl and other protein databases. The database was augmented with 74 common contaminant proteins including five proteases, 42 Ig constant regions, 15 human keratins, and 12 proteins from wool, cotton, and saliva. Green fluorescent protein (GFP_AEQVI) and a sequence for human Naked2 (Q969F2_HUMAN) with the G2A mutation were added; accession numbers are from UniProtKB. Each protein was included in both normal and reversed orientation for a total of 51,288 database entries. The database search encompassed tryptic peptides with any number of missed cleavage sites; in practice, this setting yielded identifications in which one-third of the lysine and arginine residues were found in positions other than the peptide carboxyl terminus. All cysteines were expected to undergo carboxamidomethylation and were assigned a mass of 160 kDa. All methionines were allowed to be oxidized. Precursor ions were required to fall within 1.25 m/z of the position expected from their average masses, and fragment ions were required to fall within 0.5 m/z of their monoisotopic positions. The database searches produced raw identifications in SQT format (16McDonald W.H. Tabb D.L. Sadygov R.G. MacCoss M.J. Venable J. Graumann J. Johnson J.R. Cociorva D. Yates III, J.R. MS1, MS2, and SQT—three unified, compact, and easily parsed file formats for the storage of shotgun proteomic spectra and identifications.Rapid Commun. Mass Spectrom. 2004; 18: 2162-2168Crossref PubMed Scopus (288) Google Scholar). Peptide identification filtering and protein assembly were conducted by the IDPicker algorithm (17Zhang B. Chambers M.C. Tabb D.L. Proteomic parsimony through bipartite graph analysis improves accuracy and transparency.J. Proteome Res. 2007; 6: 3549-3557Crossref PubMed Scopus (255) Google Scholar). Initial filtering took place in multiple stages. First IDPicker filtered raw peptide identifications to a target false discovery rate (FDR) of 5%. The peptide filtering used reversed matching information to determine thresholds that yielded an estimated 5% FDR for the identifications of each charge state by the formula FDR = (2R)/(R + F) where R is the number of passing reversed peptide identifications and F is the number of passing forward (normal orientation) peptide identifications. The second round of filtering removed proteins supported by less than three distinct peptide identifications in the aggregate of the three experiments. As a final criterion, protein identifications were required to be supported by at least one spectrum in each of the three SCX/LC-MS/MS experiments (methionine oxidations were considered sequence differences). Indistinguishable proteins were recognized and grouped. Parsimony rules were applied to generate a minimal list of proteins that explained all of the peptides that passed our entry criteria. No reversed proteins passed the second level criteria so that zero proteins were estimated to be falsely identified in this list, i.e. a 0% FDR. Cells were lysed in Pierce lysis buffer (M-PER). Protein A beads were blocked for 10 min in 2% BSA (TBS buffer, pH 8.0). For immunoprecipitations, lysates corresponding to 1 mg of total cellular protein were incubated overnight with primary antibody and then bound to protein A-agarose beads for 2 h at 4 °C. The beads were then washed five times with 1× lysis buffer-protease inhibitor mixture (0.2 mm 4-(2-aminoethyl)benzenesulfonyl fluoride, 0.1 mm EDTA, 13 μm bestatin, 1.4 μm E-64, 0.1 μm leupeptin, and 0.03 μm aprotinin). Immunoprecipitates were resolved by 7–12.5% SDS-PAGE and transferred onto ni" @default.
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• W2098202316 date "2008-09-01" @default.
• W2098202316 modified "2023-10-03" @default.
• W2098202316 title "Use of Fluorescence-activated Vesicle Sorting for Isolation of Naked2-associated, Basolaterally Targeted Exocytic Vesicles for Proteomics Analysis" @default.
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