and GFPhs-r5M-Aha (C) incorporated with Met, Hpg and Aha, respectively. Inset table of each spectra shows calculated and found masses. The peaks corresponding to found masses with Met, Hpg and Aha incorporated proteins might be due to cleavage of 8 residues. We generally could observe these peaks with almost all of the samples of GFPhs-r5M variants. The GFPhs-r5M containing Hpg and Aha showed the mass shift of 222 and 25 Da respectively, compared to GFPhs-r5M with Met. (TIF) Table S1 Oligonucleotides used for saturation genesis of internal Met-free GFP construction. (TIF) muta- References 1. Merkel L, Beckmann HSG, Wittmann V, Budisa N (2008) Efficient N-terminal glycoconjugation of proteins by the N-end rule. ChemBioChem 9: 1220–1224. 2. Saerens D, Huang L, Bonroy K, Muyldermans S (2008) Antibody fragments as probe in biosensor development. Sensors 8: 4669–4686. 3. Tomizaki KY, Usui K, Mihara H (2005) Protein-detecting microarrays: Current accomplishments and requirements. 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