Molecular architecture of mouse and human pancreatic zymogen granules: protein components and their copy numbers.

A molecular model of pancreatic zymogen granule (ZG) is critical for understanding its functions. We have extensively characterized the composition and membrane topology of rat ZG proteins. In this study, we report the development of targeted proteomics approaches to quantify representative mouse and human ZG proteins using LC-SRM and heavy isotope-labeled synthetic peptides. The absolute quantities of mouse Rab3D and VAMP8 were determined as 1242 ± 218 and 2039 ± 151 (mean ± SEM) copies per ZG. The size distribution and the averaged diameter of ZGs 750 ± 23 nm (mean ± SEM) were determined by atomic force microscopy. The absolute quantification of Rab3D was then validated using semi-quantitative Western blotting with purified GST-Rab3D proteins as an internal standard. To extend our proteomics analysis to human pancreas, ZGs were purified using human acini obtained from pancreatic islet transplantation center. One hundred and eighty human ZG proteins were identified for the first time including both the membrane and the content proteins. Furthermore, the copy number per ZG of human Rab3D and VAMP8 were determined to be 1182 ± 45 and 485 ± 15 (mean ± SEM). The comprehensive proteomic analyses of mouse and human pancreatic ZGs have the potential to identify species-specific ZG proteins. The determination of protein copy numbers on pancreatic ZGs represents a significant advance towards building a quantitative molecular model of a prototypical secretory vesicle using targeted proteomics approaches. The identification of human ZG proteins lays a foundation for subsequent studies of altered ZG compositions and secretion in pancreatic diseases.
Competing Interests: Compliance with Ethics StandardsJin-sook Lee, Joseph A. Caruso, Garrett Hubbs, Patricia Schnepp, James Woods, Jingye Fang, Chunying Li, Kezhong Zhang, Paul M. Stemmer, Bhanu Jena, and Xuequn Chen declare that they have no conflict of interest.The animal experiments conducted using mouse pancreas are approved by the Wayne State University Institutional Animal Care and Use Committee (IACUC), and are conducted following the Guide for the Care and Use of Laboratory Animals (National Research Council, revised 2010). All efforts are made to reduce animal suffering and minimize the total number of animals used.This article does not contain any studies with human participants performed by any of the authors.