Dataset Brief

Mass spectrometric characterization of recombinant rat 5-hydroxytryptamine receptor 1A (5-HT1AR) expressed in tsA201 human embryonic kidney cells

  1. Seok Heo1,
  2. Jae-Won Yang2,
  3. Marie L. Huber3,
  4. Melanie Planyavsky3,
  5. Keiryn L. Bennett3,
  6. Gert Lubec1,*

Article first published online: 19 OCT 2012

DOI: 10.1002/pmic.201200183

Issue

PROTEOMICS

PROTEOMICS

Volume 12, Issue 22, pages 3338–3342, November 2012

Additional Information

How to Cite

Heo, S., Yang, J.-W., Huber, M. L., Planyavsky, M., Bennett, K. L. and Lubec, G. (2012), Mass spectrometric characterization of recombinant rat 5-hydroxytryptamine receptor 1A (5-HT1AR) expressed in tsA201 human embryonic kidney cells. Proteomics, 12: 3338–3342. doi: 10.1002/pmic.201200183

Author Information

  1. 1

    Department of Pediatrics, Medical University of Vienna, Vienna, Austria

  2. 2

    Institute of Pharmacology, Medical University of Vienna, Vienna, Austria

  3. 3

    CeMM Research Center for Molecular Medicine of the Austrian Academy of Sciences, Vienna, Austria

* Correspondence: Professor Dr. Gert Lubec, Department of Pediatrics, Medical University of Vienna, Waehringer Guertel 18, A-1090 Vienna, Austria
E-mail: gert.lubec@meduniwien.ac.at
Fax: +43-1-40400-6065

Publication History

  1. Issue published online: 26 NOV 2012
  2. Article first published online: 19 OCT 2012
  3. Accepted manuscript online: 19 SEP 2012 07:08AM EST
  4. Manuscript Accepted: 3 SEP 2012
  5. Manuscript Revised: 31 JUL 2012
  6. Manuscript Received: 4 MAY 2012

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FilenameFormatSizeDescription
pmic7234-sup-0001-FigureS1.pdf286KFigure S1. A) Immunoblotting(right side) from the BN-PAGE (left side) revealed a single band at the approx. apparent MW of 480 kDa. B) Immunoblottingpatterns of the 2DE (BN/SDS-PAGE) from cellular membrane fraction and C) total cell lysateshow a single spot at the approx. MW of 75 kDacompatible with the 5-HT1AR-YPF. A rabbitpolyclonalantibody against mouse/rat 5-HT1AR was used.
pmic7234-sup-0002-FigureS2.pdf242KFigure S2. A) The BN Western blot pattern indicating the position for excision of bands in the right two lanes. B) The 3DE (BN/SDS/SDS-PAGE)immunoblottingof 5-HT1AR from the cellular membrane fraction showing a single spot C) from total celllysate.
pmic7234-sup-0003-FigureS3.pdf780KFigure S3. A) The gel and the correspondingimmunoblottingfrom proteinsimmunoprecipitatedby the GFP/YFP-antibody and the bracket indicates the presence of the tagged 5-HT1AR. Thecorrespondingnumbers indicate the bands excised for mass spectrometric analysis. B) Silver stained gel of different elution protocols. The best result was by SDS elution. C) The silver-stained 2DE gel of theimmunoprecipitatedproteins with an arrow indicating the 5-HT1AR. The corresponding numbers indicate the spots excised for massspectrometric analysis.
pmic7234-sup-0004-FigureS4.pdf52KFigure S4. Immunoblottingof 2DE IEF/SDS-PAGE of recombinant rat 5-HT1AR from transfectedtsA201 cells.
pmic7234-sup-0005-FigureS5.pdf710KFigure S5. Representative gel image of 1DE SDS-PAGE and 2DE dSDS-PAGE for mass spectrometricalanalysis.
pmic7234-sup-0006-FigureS6.pdf113KFigure S6. Sequence coverage, post-translationalmodifications and amino acid substitutions of recombinant rat 5-HT1AR obtained from bioinformatictools of Mascot v2.3 and Modiro™v1.1.
pmic7234-sup-0007-FigureS7.pdf1165KFigure S7. Sequence coverage and identified peptidesof YFP.
pmic7234-sup-0008-FigureS8.pdf114KFigure S8. MS/MS spectra of phosphorylatedand dephosphorylatedS301 by alkaline phosphatasetreatment from recombinant rat 5-HT1AR.
pmic7234-sup-0009-FigureS9.pdf44KFigure S9. Immunoblottinganalysis of recombinant rat 5-HT1AR from tsA201 cells after deglycosylatingenzyme treatment.
pmic7234-sup-0010-FigureS10.pdf1946KFigure S10. Representative mass spectra of identified post-translationalmodifications of recombinant rat 5-HT1AR expressed from tsA201 cells.
pmic7234-sup-0011-TableS1.doc37KTable S1. Sequence coverage of recombinant rat 5-HT1AR. Two different search engines (Mascot v2.3 and Modiro™ v1.1) were used. The conditions of all enzymes used in this study are listed. The sequence coverage was evaluated by combining sequence coverages of all spots of rat recombinant rat 5-HT1AR.
pmic7234-sup-0012-TableS2.xls235KTable S2. The list of identified peptides of recombinant rat 5-hydroxytryptamine receptor 1A (5-HT1AR) with Mascot v2.3
pmic7234-sup-0013-TableS3.xls1066KTable S3. The list of identified peptides of recombinant rat 5-hydroxytryptamine receptor 1A (5-HT1AR) with Modiro™ v1.11
pmic7234-sup-0014-S1.doc55KSupporting Information

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