Subcellular fractionation enhances proteome coverage of pancreatic duct cells

Joao A. Paulo, Aleksandr Gaun, Vivek Kadiyala, Ali Ghoulidi, Peter A. Banks, Darwin L. Conwell, Hanno Steen

Research output: Contribution to journalArticlepeer-review

19 Scopus citations

Abstract

Objectives Subcellular fractionation of whole cell lysates offers a means of simplifying protein mixtures, potentially permitting greater depth of proteomic analysis. Here we compare proteins identified from pancreatic duct cells (PaDC) following organelle enrichment to those identified from PaDC whole cell lysates to determine if the additional procedures of subcellular fractionation increase proteome coverage. Methods We used differential centrifugation to enrich for nuclear, mitochondrial, membrane, and cytosolic proteins. We then compared - via mass spectrometry-based analysis - the number of proteins identified from these four fractions with four biological replicates of PaDC whole cell lysates. Results We identified similar numbers of proteins among all samples investigated. In total, 1658 non-redundant proteins were identified in the replicate samples, while 2196 were identified in the subcellular fractionation samples, corresponding to a 30% increase. Additionally, we noted that each organelle fraction was in fact enriched with proteins specific to the targeted organelle. Conclusions Subcellular fractionation of PaDC resulted in greater proteome coverage compared to PaDC whole cell lysate analysis. Although more labor intensive and time consuming, subcellular fractionation provides greater proteome coverage, and enriches for compartmentalized sub-populations of proteins. Application of this subcellular fractionation strategy allows for a greater depth of proteomic analysis and thus a better understanding of the cellular mechanisms of pancreatic disease.

Original languageEnglish
Pages (from-to)791-797
Number of pages7
JournalBiochimica et Biophysica Acta - Proteins and Proteomics
Volume1834
Issue number4
DOIs
StatePublished - Apr 2013

Bibliographical note

Funding Information:
NIH financial support: NIH/NIDDK NRSA Fellowship NIH NIDDK 1 F32 DK085835-01A1 (JP), 1 R21 DK081703-01A2 (DC), and 5 P30 DK034854-24 (Harvard Digestive Diseases Center; DC).

Funding Information:
Funds were provided by the following NIH grants: 1 F32 DK085835-01A1 (JP), 1 R21 DK081703-01A2 (DC) and 5 P30 DK034854-24 (Harvard Digestive Diseases Center; DC), as well as a grant from the American College of Gastroenterology: ACG-042103580 (JP). In addition, we would like to thank the Burrill family for their generous support through the Burrill Research Grant. We would also like to thank members of the Steen Lab at Children's Hospital Boston, in particular Scott Brizard, and John FK Sauld, as well as Shadeah Suleiman (Research Manager) from the Center for Pancreatic Disease at Brigham and Women's Hospital for their technical assistance and critical reading of the manuscript.

Keywords

  • Chronic pancreatitis
  • Organelle enrichment
  • Pancreatic cancer

ASJC Scopus subject areas

  • Analytical Chemistry
  • Biophysics
  • Biochemistry
  • Molecular Biology

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