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

Jin-Sook Lee,Jingye Fang,Joseph A Caruso,Garrett Hubbs,Bhanu P Jena,Chunying Li,Xuequn Chen,James Woods,Paul M Stemmer,Kezhong Zhang,Patricia Schnepp

doi:10.1007/s41048-018-0055-1

Abstract

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.

Highlights

The acinar cells of the exocrine pancreas are the functional units of digestive enzyme synthesis, storage, and secretion
zymogen granule (ZG) protein bands were stained with Coomassie Blue reagent, and the entire lane were excised in thirty gel slices for in-gel tryptic digestion (Fig. 1B)
This approach minimized the protein loss during sample processing, which is critical to the precise determination of protein abundance on ZGs

Summary

Introduction

The acinar cells of the exocrine pancreas are the functional units of digestive enzyme synthesis, storage, and secretion. The zymogen granule (ZG) is the secretory organelle in the acinar cells responsible for transport, storage, and secretion of digestive enzymes and has long 94 | April 2018 | Volume 4 | Issue 2. The overall goal of our study is to build a quantitative, architectural model of the pancreatic ZG which will direct new hypotheses for subsequent functional analysis of this prototypic secretory granule. This model should comprise the complete protein components of the ZG, their membrane topologies, and their absolute quantities and protein complexes they are associated with

Objectives

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Results

Conclusion