Fig. 1.

A, human colon carcinoma HCT116 cells were grown in DMEM containing either the normal light isotopes of carbon and nitrogen (i.e. ¹²C¹⁴N) (light), l-[¹³C₆,¹⁴N₄]arginine and l-[²H₄]lysine (medium), or l-[¹³C₆,¹⁵N₄]arginine and l-[¹³C₆,¹⁵N₂]lysine (heavy). Separate cytoplasmic, nuclear, and nucleolar fractions were isolated from each labeled cell population. B, equal amounts of total protein from each fraction were then recombined to recreate a whole cell extract but with Cyto, Nuc, and No arising from cells with different isotope labels. The recombined whole cell extract mixture was solubilized with loading buffer; proteins were separated using SDS-PAGE; and the resulting gel was cut into eight equal pieces, trypsin-digested, and analyzed by LC-MS/MS using an LTQ Orbitrap. Triplicate fractions and LC-MS/MS resulted in a proteome of 2,536 proteins.