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High-throughput identification of IMCD proteins using LC-MS/MS.

Publication ,  Journal Article
Pisitkun, T; Bieniek, J; Tchapyjnikov, D; Wang, G; Wu, WW; Shen, R-F; Knepper, MA
Published in: Physiol Genomics
April 13, 2006

The inner medullary collecting duct (IMCD) is an important site of vasopressin-regulated water and urea transport. Here we have used protein mass spectrometry to investigate the proteome of the IMCD cell and how it is altered in response to long-term vasopressin administration in rats. IMCDs were isolated from inner medullas of rats, and IMCD proteins were identified by liquid chromatography/tandem mass spectrometry (LC-MS/MS). We present a WWW-based "IMCD Proteome Database" containing all IMCD proteins identified in this study (n = 704) and prior MS-based identification studies (n = 301). We used the isotope-coded affinity tag (ICAT) technique to identify IMCD proteins that change in abundance in response to vasopressin. Vasopressin analog (dDAVP) or vehicle was infused subcutaneously in Brattleboro rats for 3 days, and IMCDs were isolated for proteomic analysis. dDAVP and control samples were labeled with different cleavable ICAT reagents (mass difference 9 amu) and mixed. This was followed by one-dimensional SDS-PAGE separation, in-gel trypsin digestion, biotin-avidin affinity purification, and LC-MS/MS identification and quantification. Responses to vasopressin for a total of 165 proteins were quantified. Quantification, based on semiquantitative immunoblotting of 16 proteins for which antibodies were available, showed a high degree of correlation with ICAT results. In addition to aquaporin-2 and gamma-epithelial Na channel (gamma-ENaC), five of the immunoblotted proteins were substantially altered in abundance in response to dDAVP, viz., syntaxin-7, Rap1, GAPDH, heat shock protein (HSP)70, and cathepsin D. A 28-protein vasopressin signaling network was constructed using literature-based network analysis software focusing on the newly identified proteins, providing several new hypotheses for future studies.

Duke Scholars

Published In

Physiol Genomics

DOI

EISSN

1531-2267

Publication Date

April 13, 2006

Volume

25

Issue

2

Start / End Page

263 / 276

Location

United States

Related Subject Headings

  • Time Factors
  • Spectrometry, Mass, Electrospray Ionization
  • Reproducibility of Results
  • Rats, Sprague-Dawley
  • Rats, Brattleboro
  • Rats
  • Qa-SNARE Proteins
  • Proteomics
  • Proteins
  • Male
 

Citation

APA
Chicago
ICMJE
MLA
NLM
Pisitkun, T., Bieniek, J., Tchapyjnikov, D., Wang, G., Wu, W. W., Shen, R.-F., & Knepper, M. A. (2006). High-throughput identification of IMCD proteins using LC-MS/MS. Physiol Genomics, 25(2), 263–276. https://doi.org/10.1152/physiolgenomics.00214.2005
Pisitkun, Trairak, Jared Bieniek, Dmitry Tchapyjnikov, Guanghui Wang, Wells W. Wu, Rong-Fong Shen, and Mark A. Knepper. “High-throughput identification of IMCD proteins using LC-MS/MS.Physiol Genomics 25, no. 2 (April 13, 2006): 263–76. https://doi.org/10.1152/physiolgenomics.00214.2005.
Pisitkun T, Bieniek J, Tchapyjnikov D, Wang G, Wu WW, Shen R-F, et al. High-throughput identification of IMCD proteins using LC-MS/MS. Physiol Genomics. 2006 Apr 13;25(2):263–76.
Pisitkun, Trairak, et al. “High-throughput identification of IMCD proteins using LC-MS/MS.Physiol Genomics, vol. 25, no. 2, Apr. 2006, pp. 263–76. Pubmed, doi:10.1152/physiolgenomics.00214.2005.
Pisitkun T, Bieniek J, Tchapyjnikov D, Wang G, Wu WW, Shen R-F, Knepper MA. High-throughput identification of IMCD proteins using LC-MS/MS. Physiol Genomics. 2006 Apr 13;25(2):263–276.

Published In

Physiol Genomics

DOI

EISSN

1531-2267

Publication Date

April 13, 2006

Volume

25

Issue

2

Start / End Page

263 / 276

Location

United States

Related Subject Headings

  • Time Factors
  • Spectrometry, Mass, Electrospray Ionization
  • Reproducibility of Results
  • Rats, Sprague-Dawley
  • Rats, Brattleboro
  • Rats
  • Qa-SNARE Proteins
  • Proteomics
  • Proteins
  • Male