Isolation & Subfractionation of ZGs
Zymogen granules are best suited for subcellular (and suborganellar) proteomics, as they represent abundant organelles of the exocrine pancreas, which can be easily isolated and purified owing to their large size and density. In addition, their isolation yields large quantities of organelles and proteins. Furthermore, intact ZGs can be lysed and further separated into a ZG content (ZGC) and ZGM fraction.
Isolation procedures for ZGs from dog, bovine and guinea pig pancreas,[37,38] as well as for porcine and rat (mouse) pancreas,[40–43] have been described. Subcellular fractionation is the most widely used method, which after tissue homogenization and differential centrifugation results in a relatively pure ZG fraction (≥90%). To achieve a higher degree of purity, sucrose or percoll, gradient fractionation is often applied.[40–42] Table 1 lists the major ZG proteins with amylase being the most abundant. Coomassie staining after SDS-PAGE, immunoblotting and enzyme activity measurements for marker proteins (e.g., amylase) are generally performed to monitor the efficiency of the isolation procedure and the enrichment of ZG proteins as well as the presence of potential contaminations in the obtained fractions. In addition, electron microscopy of the ZG fractions is used to test for organelle integrity and the presence of contaminating subcellular structures (Figure 1B). Potential contaminants of ZG fractions are mitochondria and ER, which often adhere nonspecifically to the ZGM, as well as large lysosomes.
Zymogen granule content and ZGM subfractions can be achieved after gentle lysis of the intact ZGs at pH 8 (Figure 1C & D). This reflects the physiological conditions, as the ZGC proteins become soluble in the alkaline environment of the pancreatic duct, where bicarbonate is secreted to neutralize stomach acid in the small intestine. ZGM fractions have been further purified by potassium bromide and/or carbonate treatment at pH 11 and subsequent gradient centrifugation to enrich transmembrane and membrane-anchored proteins or peripheral ZGM proteins.[33,34,43,44] However, some ZGC proteins are still found in the ZGM fraction and vice versa. This may be due to the observation that some ZGC proteins exist in a soluble and membrane-associated pool.[33,45] Furthermore, some ZGM proteins are enzymatically cleaved and released in a soluble form (e.g., GP-2, a major glycosylphosphatidylinositol [GPI]-anchored ZGM glycoprotein).
Chen and co-workers developed a quantitative proteomics approach to monitor the enrichment of membrane proteins during purification and to discriminate between soluble, membrane-associated and intrinsic membrane proteins.[34,43] They applied isobaric tags for relative and absolute quantification (iTRAQ) to the purification process: the digested peptides from crude ZGM, a KBr- and a Na2CO3-washed ZGM fraction were labeled with 114 and 117 iTRAQ reagents, respectively, then separated by 2D liquid chromatography (2D-LC) and analyzed by matrix-assisted laser desorption ionization (MALDI)-tandem mass spectrometry (MS; MS/MS). The identified proteins were classified in two groups: group 1, with a 117:114 ratio lower than 1 (soluble or peripheral membrane proteins), and group 2, with a 117:114 ratio greater than 1 (postulated intrinsic ZGM proteins). In contrast to group 1, proteins from group 2 contained either known or predicted transmembrane domains or post-translational modifications for membrane insertion (e.g., prenylation and GPI anchor), thus supporting their approach. This strategy does not discriminate between genuine ZGM proteins and potential contaminants from other subcellular membranes.
Mass spectrometry-based proteomics can also be applied to aid in the differentiation of organelle-associated proteins from background proteins ('contaminants'; subtractive proteomics)[34,47] and to directly monitor protein distributions among subcellular fractions (protein correlation profile). The application of these techniques to ZGs has the potential of providing a better understanding of the suborganellar localization of the proteome and, in the near future, may allow the profiling of dynamic changes of the organelle proteome following different stimuli as well as in disease states.
Expert Rev Proteomics. 2010;7(5):735-747. © 2010
Expert Reviews Ltd.
Cite this: Proteomic Analysis of Zymogen Granules - Medscape - Oct 01, 2010.