Targeted Serum Proteomics for Obesity

Serum samples were processed following a previously published protocol that ensured maximum yield of signal (49 (link)). We targeted a curated selection of 22 mostly organ-specific proteins with known genetic variants associated with obesity or metabolic syndrome (Table S1). Prepared samples, along with spiked-in heavy-isotope-labeled synthetic standard peptides, were quantified using a triple-quadrupole mass spectrometer (Agilent 6490; Agilent, Santa Clara, CA) with a nanospray ion source and Chip Cube nano-HPLC. Three to four transitions were monitored for each target peptide (see Table S1). Two micrograms of tryptically digested Mars-14 (Agilent, Santa Clara, CA) depleted serum was eluted from a high-capacity nano-HPLC chip (160 nl, 150 mm by 75 μm inside diameter [i.d.]; Agilent, Santa Clara, CA) with a 30-min gradient of 3 to 40% acetonitrile as described previously (49 (link), 50 (link)). Raw selective reaction monitoring (SRM) mass spectrometry data were analyzed with the Skyline targeted proteomics environment (51 (link)). Each detected peptide was quantified by the light/heavy (L/H) ratio of monitored transitions, after adjusting for the volume of the original serum sample.