An in silico and chemical approach towards small protein production and application in phosphoproteomicsElectronic supplementary information (ESI) available. See DOI: 10.1039/c4ra16934d

The human Pin1 WW domain (hPin1_WW) is a 38 residue protein which specifically recognizes ligands rich in proline and phosphorylated in Ser and Thr residues. This work presents a protocol for the improved chemical synthesis and modification of this protein through automated microwave assisted synthesis combined with the incorporation of pseudoproline units in the protein sequence. After purification, the protein was characterized by Mass Spectrometry and Circular Dichroism spectroscopy with results comparable to the same WW domain chemically synthesized by other strategies or biologically expressed. The protein was further immobilized on a matrix and tested for the selective binding and mild elution of phosphorylated sequences at Ser, Thr and Tyr residues. These results suggest that hPin1_WW is a useful protein scaffold for the purification of phosphorylated species in pTyr and pSer, which can be easily produced and modified by chemical methods. This work demonstrates the advantages of peptide chemical synthesis as a robust method for the production and modification of small proteins, such as the human Pin1 WW domain. The protein was immobilized on a solid support and used to enrich phosphorylated species.