Subcellular fractionation enhances proteome coverage of pancreatic duct cells.
OBJECTIVE
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.
Department of Pathology, Children's Hospital Boston, Boston, MA, USA. joao_paulo@post.harvard.edu
Joao A
JA
Paulo
Aleksandr
A
Gaun
Vivek
V
Kadiyala
Ali
A
Ghoulidi
Peter A
PA
Banks
Darwin L
DL
Conwell
Hanno
H
Steen
D002467:Cell Nucleus / Q000648:ultrastructure; D002498:Centrifugation; D002853:Chromatography, Liquid; D003600:Cytosol / Q000378:metabolism; D013058:Mass Spectrometry; D008565:Membrane Proteins / Q000302:isolation & purification / Q000378:metabolism; D009687:Nuclear Proteins / Q000302:isolation & purification / Q000378:metabolism; D015388:Organelles / Q000378:metabolism / Q000648:ultrastructure; D010183:Pancreatic Ducts / Q000166:cytology / Q000378:metabolism / Q000648:ultrastructure; D020543:Proteome / Q000032:analysis*; D013347:Subcellular Fractions / Q000378:metabolism / Q000648:ultrastructure
D008565:Membrane Proteins; D009687:Nuclear Proteins; D020543:Proteome