Utility of peptide-protein affinity complexes in proteomics: identification of interaction partners of a tumor suppressor peptide.
We used a N-biotinylated peptide analog of the C-terminal domain of the tumor suppressor protein, p21cip1/waf1 to elucidate peptide/protein interacting partners. The C-terminal domain of p21cip1/waf1 protein spanning 141-160 amino acid residues is known to bind PCNA and this interaction is important in many biological processes including cell-cycle control. This C-terminal 20-mer efficiently extracts PCNA in the presence of a variety of N- or C-terminally attached affinity tags. Using difference silver stained 2D gels combined with in-gel tryptic digests, we identified the difference spots using MALDI-TOF mass spectrometry-based peptide mass fingerprinting followed by a database search using PROFOUND against NCBIs human nonredundant protein sequence data bank. Identified spots include the p48 subunit of chromatin assembly factor-1, the heat shock 70 protein analog BiP, calmodulin, nucleolin and a spot similar in size to dimeric PCNA. In contrast, microcapillary ion-trap LC-MS/MS analysis of a tryptic digest of entire affinity extracts derived from both control and experimental runs followed by database searches using SEQUEST confirmed the presence of most of the above proteins. This strategy also identified hnRNPA1, HPSP90alpha, HSP40 and T-complex protein 1, a protein similar to prothymosin, and a possible allelic variant of the p21cip1/waf1 protein. The use of N-biotinylated peptide derived from the C-terminal domain of p21cip1/waf1 protein in proteomic analysis exemplified here suggests that peptides obtained from intracellular functional screens could also potentially serve as efficient baits to discover new drug targets.
Gururaja
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TL
Rigel Pharmaceuticals Inc, San Francisco, CA 94080, USA. tgururaja@rigel.com
Li
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W
Payan
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DG
Anderson
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DC
D000595:Amino Acid Sequence; D015153:Blotting, Western; D050759:Cyclin-Dependent Kinase Inhibitor p21; D016213:Cyclins / Q000737:chemistry / Q000378:metabolism*; D015180:Electrophoresis, Gel, Two-Dimensional; D046911:Macromolecular Substances; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism*; D019151:Peptide Library; D018809:Proliferating Cell Nuclear Antigen / Q000378:metabolism; D011485:Protein Binding; D040901:Proteomics; D017422:Sequence Analysis, DNA; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
D050759:Cyclin-Dependent Kinase Inhibitor p21; D016213:Cyclins; D046911:Macromolecular Substances; D010446:Peptide Fragments; D019151:Peptide Library; D018809:Proliferating Cell Nuclear Antigen