The protein interactome of the human C3orf33 protein was explored in U2OS cells through affinity purification-mass spectrometry (AP-MS). Specifically, U2OS cells stably expressing C3orf33 with either an N-terminus or a C-terminus 3XFlag tag were initially lysed using RIPA buffer. Subsequently, the proteins associated with C3orf33 were purified with anti-flag magnetic beads. Proteins were then eluted from the beads using competition with 3×Flag peptides and subsequently analyzed with mass spectrometry.