Direct visualisation of peptide hormones in cultured pancreatic islet alpha- and beta-cells by intact-cell mass spectrometry.
The application of intact-cell mass spectrometry (ICM) by matrix-assisted laser desorption/ionisation time-of-flight (MALDI-TOF) mass spectrometry to achieve direct protein-profiling of bacterial species is now well established. However, this methodology has not to our knowledge been applied to the analysis of mammalian cells in routine culture. Here, we describe a novel application of ICM by which we have identified proteins in intact cells from two lines representative of pancreatic islet alpha- and beta-cells. Adherent alphaTC1 clone 9 and betaTC6 F7 cells were harvested into phosphate-buffered saline (PBS) using enzyme-free dissociation buffer before 1 microL of cell suspension was spotted onto MALDI plates. Cells were overlaid with sinapinic acid then washed with pure water before application of a final coat of sinapinic acid. Data in the 2000-20,000 m/z range were acquired in linear mode on a Voyager DE-Pro mass spectrometer. The proteins which ionised were composed in large part of peptide hormones (e.g. insulin and glucagon) known to be packaged into the secretory granules of the beta- and alpha-cells respectively. However, in addition to visualising the peptides expected to be associated with these cells, a mass consistent with oxyntomodulin was identified in the cultured alpha-cells, a finding not previously reported to our knowledge. In summary, this paper describes, for the first time, a rapid and direct method useful for identifying secretory products in intact endocrine cells.
Buchanan
Christina M
CM
School of Biological Sciences and Maurice Wilkins Centre for Molecular Biodiscovery, Faculty of Science, University of Auckland, Private Bag 92-019, Auckland, New Zealand.
Malik
Arpita S
AS
Cooper
Garth J S
GJ
D000818:Animals; D002460:Cell Line; D003373:Coumaric Acids / Q000737:chemistry; D005934:Glucagon / Q000032:analysis / Q000378:metabolism*; D050416:Glucagon-Secreting Cells / Q000737:chemistry / Q000378:metabolism* / Q000648:ultrastructure; D007328:Insulin / Q000032:analysis / Q000378:metabolism*; D050417:Insulin-Secreting Cells / Q000737:chemistry / Q000378:metabolism* / Q000648:ultrastructure; D008855:Microscopy, Electron, Scanning; D053772:Oxyntomodulin / Q000737:chemistry / Q000378:metabolism; D010449:Peptide Mapping; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000295:instrumentation / Q000379:methods*
D003373:Coumaric Acids; D007328:Insulin; D053772:Oxyntomodulin; C073734:sinapinic acid; D005934:Glucagon