Figure 2.

Experimental workflow. Spheroids were prepared with one of the three different pulsed isotopic cell culture media, and the red box specifies one spheroid population that is followed through the workflow. Each set of spheroids was subjected to serial trypsinization to harvest the three cellular populations. In this case, LC–MS/MS analysis of just the LHM spheroid is shown. Six rounds of serial trypsinization were performed and combined into three fractions, representing the three cellular populations. The cells were lysed in sodium dodecyl sulfate, and the proteins were quantified by BCA. Proteins were reduced, alkylated, and digested with the S-trap protocol, and the resulting peptides were analyzed by LC–MS/MS. The three distinct isotopic media are distinguishable in the MS mode.¹⁹