Utility of peptide–protein affinity complexes in proteomics: identification of interaction partners of a tumor suppressor peptide*

摘要: We used a N-biotinylated peptide analog of the C-terminal domain tumor suppressor protein, p21cip1/waf1 to elucidate peptide/protein interacting partners. The protein spanning 141-160 amino acid residues is known bind PCNA and this interaction important in many biological processes including cell-cycle control. This 20-mer efficiently extracts presence variety N- or C-terminally attached affinity tags. Using difference silver stained 2D gels combined with in-gel tryptic digests, we identified spots using MALDI-TOF mass spectrometry-based fingerprinting followed by database search PROFOUND against NCBIs human nonredundant sequence data bank. Identified include p48 subunit chromatin assembly factor-1, heat shock 70 BiP, calmodulin, nucleolin spot similar size dimeric PCNA. In contrast, microcapillary ion-trap LC-MS/MS analysis digest entire derived from both control experimental runs searches SEQUEST confirmed most above proteins. strategy also hnRNPA1, HPSP90alpha, HSP40 T-complex 1, prothymosin, possible allelic variant protein. use proteomic exemplified here suggests that peptides obtained intracellular functional screens could potentially serve as efficient baits discover new drug targets.