Quantitative proteomic analysis of the secretory proteins from rat adipose cells using a 2D liquid chromatography-MS/MS approach

Xiaoli Chen, Samuel W. Cushman, Lewis K. Pannell, Sonja Hess

Research output: Contribution to journalArticlepeer-review

92 Scopus citations

Abstract

We have developed two-dimensional liquid chromatography-tandem mass spectrometry (2D-LC-MS/ MS) and 18O proteolytic labeling strategies to identify and compare levels of secretory proteins with low abundance in the conditioned medium of rat adipose cells without or with insulin stimulation. Culture medium was concentrated and secreted proteins were separated on a RP-HPLC followed by LC-MS/MS analysis. For 18O proteolytic labeling, 16O- to 18O-exchange in the digested peptides from eight individual fractions was carried out in parallel in H216O and H218O with immobilized trypsin, and the ratios of isotopically distinct peptides were measured by mass spectrometry. A total of 84 proteins was identified as secreted adipokines. This large number of secretory proteins comprise multiple functional categories. Comparative proteomics of 18O proteolytic labeling allows the detection of different levels of many secreted proteins as exemplified here by the difference between basal and insulin treatment of adipose cells. Taken together, our proteomic approach is able to identify and quantify the comprehensive secretory proteome of adipose cells. Thus, our data support the endocrine role of adipose cells in pathophysiological states through the secretion of signaling molecules.

Original languageEnglish (US)
Pages (from-to)570-577
Number of pages8
JournalJournal of Proteome Research
Volume4
Issue number2
DOIs
StatePublished - Mar 2005

Keywords

  • Adipose cell
  • Comparative proteomics
  • Electrospray-ionization mass spectrometry
  • Proteolytic O labeling
  • Secreted proteome

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