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.

Keywords: comparative proteomics • proteolytic ¹⁸ O labeling • secreted proteome • adipose cell • electrospray-ionization mass spectrometry