Organellar Proteomics

doi:10.1074/mcp.M500172-MCP200

Organellar Proteomics

Analysis of Pancreatic Zymogen Granule Membranes *S

From the ^‡Departments of Biological Chemistry, ^§Molecular and Integrative Physiology, and ^‖Cell and Developmental Biology, The University of Michigan, Ann Arbor, Michigan 48109

¶ To whom correspondence should be addressed: National Resource for Proteomics and Pathways, 1195SE North Ingalls Bldg., The University of Michigan, Ann Arbor, MI 48109. Tel.: 734-647-0951; Fax: 734-647-0951; E-mail: xuequnc{at}umich.edu

Abstract

The zymogen granule (ZG) is the specialized organelle in pancreatic acinar cells for digestive enzyme storage and regulated secretion and has been a model for studying secretory granule functions. In an initial effort to comprehensively understand the functions of this organelle, we conducted a proteomic study to identify proteins from highly purified ZG membranes. By combining two-dimensional gel electrophoresis and two-dimensional LC with tandem mass spectrometry, 101 proteins were identified from purified ZG membranes including 28 known ZG proteins and 73 previously unknown proteins, including SNAP29, Rab27B, Rab11A, Rab6, Rap1, and myosin Vc. Moreover several hypothetical proteins were identified that represent potential novel proteins. The ZG localization of nine of these proteins was further confirmed by immunocytochemistry. To distinguish intrinsic membrane proteins from soluble and peripheral membrane proteins, a quantitative proteomic strategy was used to measure the enrichment of intrinsic membrane proteins through the purification process. The iTRAQ™ ratios correlated well with known or Transmembrane Hidden Markov Model-predicted soluble or membrane proteins. By combining subcellular fractionation with high resolution separation and comprehensive identification of proteins, we have begun to elucidate zymogen granule functions through proteomic and subsequent functional analysis of its membrane components.

Footnotes

Published, MCP Papers in Press, November 8, 2005, DOI 10.1074/mcp.M500172-MCP200
↵1 The abbreviations used are: ZG, zymogen granule; iTRAQ, isobaric tag for relative and absolute quantification; SNARE, soluble N-ethylmaleimide-sensitive factor attachment protein receptor; TMHMM, Transmembrane Hidden Markov Model; VAMP, vesicle-associated membrane protein; 2D, two-dimensional; GPI, glycosylphosphatidylinositol; GE, gel electrophoresis; GTPαS, guanosine 5′-O-(1-thiotriphosphate).
↵* This work was supported by National Institutes of Health (NIH) Grant DK41122 (to J. A. W.), the National Resource for Proteomics and Pathways (under NIH Grant P41 RR018627 to P. C. A.), the Michigan Gastrointestinal Peptide Center (under NIH Grant P30 DK34933), and the Michigan Diabetes Research and Training Center (under NIH Grant P60 DK20572). The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
↵S The on-line version of this article (available at http://www.mcponline.org) contains supplemental material.
- Received June 3, 2005.
- Revision received October 31, 2005.