I (green, response to applied stimulus; red, no response; grey, not tested; applied peptide concentration: ORN #1?12, 1 mM; ORN #13?21, 5 mM; ORN #22?24, 10 mM; ORN #25?31, 200 mM). [AA mix: amino acidOlfactory Responses to Amino Acids and PeptidesFigure 3. Peptide stimulation evokes calcium transients with lower maximum amplitude than stimulation with amino acids. (A) The maximum amplitude of [Ca2+]i increases upon peptide application (green, group I, 1 mM; orange, group II, 200 mM) is much lower than upon application of amino acids (200 mM; number of responses averaged: L-arginyl-L-methionine (Arg-Met), 2; L-arginyl-L-methionyl-L-arginine (Arg-MetArg), 4; L-methionyl-L-arginyl-L-methionine (Met-Arg-Met), 9; L-methionyl-L-arginine (Met-Arg), 9; L-arginyl-L-lysine (Arg-Lys), 4; L-arginyl-L-lysyl-Larginine (Arg-Lys-Arg), 7; L-lysyl-L-arginyl-L-lysine (Tubastatin A site Lys-Arg-Lys), 7; L-lysyl-L-arginine (Lys-Arg), 2; out of 12 ORNs, four OE slices; Docosahexaenoyl ethanolamide L-arginyl-glycine (ArgGly), 10; glycyl-L-arginine (Gly-Arg), 4; L-methionyl-glycine (Met-Gly), 4; glycyl-glycine (Gly-Gly), 4; glycyl-glycyl-glycine (Gly-Gly-Gly), 2; out of six ORNs, four OE slices). (B) Of the five group II peptides only the dipeptide L-arginyl-glycine (Arg-Gly) featured a stimulus-induced maximum amplitude of [Ca2+]i increases comparable to stimulation with L-arginine (only ORNs exclusively sensitive to the amino acid L-arginine, i.e. #27?30 taken into account). In contrast, the dipeptide glycyl-L-arginine (Gly-Arg) showed a weak response (averaging of multiple applications of glycyl-L-arginine (GlyArg); *, p,0.05; **, p,0.001, paired t-test, 
error bars represent standard deviation). [AA: amino acids, Arg: L-arginine]. doi:10.1371/journal.pone.0053097.gL-arginine, L-lysine and L-methionine and group I peptides. Of the 42 amino acid-responsive ORNs, 62 responded to Larginine, 79 to L-methionine and 43 to L-lysine. As some ORNs responded to more than one amino acid, the frequencies sum up to values higher than 100 . A clearly smaller fraction of ORNs (29 ) also responded to at least one of the eight group I peptides (Figure 2A). In a second set of experiments we applied Larginine, L-methionine and glycine and group II peptides. In order to reduce the size and possibly the steric hindrance of the peptides at the receptor binding site, we chose to include 23727046 glycine, the smallest amino acid found in proteins. In this second set, of 28 amino acid-responsive ORNs, 57 responded to L-arginine, 79 to L-methionine and 32 to glycine. As in the first set of experiments only a small subset of ORNs (21 ) also responded to at least one of the five group II peptides (Figure 2A). The matrix in Figure 2B depicts the exact response profile of all peptide-sensitive ORNs. Figure 3A depicts the mean maximum amplitudes of the peptide-induced increases of Ca2+-dependent fluorescence relative to the amplitude reached upon application of amino 
acid controls. Out of the group I peptides (green bars), even the peptide that elicited the highest mean amplitudes (L-methionyl-L-arginyl-Lmethionine) reached only about 32 of the amino acid-induced amplitudes. In comparison, the smaller group II peptides (orange bars), tendentially featured a slightly higher mean maximum amplitude. Thereby, the peptide L-arginyl-glycine showed an exceptionally high mean maximum amplitude. L-arginyl-glycine elicited responses in five of the six ORNs sensitive to group II peptides. Three of them were sensitive only to the amino acid Largin.I (green, response to applied stimulus; red, no response; grey, not tested; applied peptide concentration: ORN #1?12, 1 mM; ORN #13?21, 5 mM; ORN #22?24, 10 mM; ORN #25?31, 200 mM). [AA mix: amino acidOlfactory Responses to Amino Acids and PeptidesFigure 3. Peptide stimulation evokes calcium transients with lower maximum amplitude than stimulation with amino acids. (A) The maximum amplitude of [Ca2+]i increases upon peptide application (green, group I, 1 mM; orange, group II, 200 mM) is much lower than upon application of amino acids (200 mM; number of responses averaged: L-arginyl-L-methionine (Arg-Met), 2; L-arginyl-L-methionyl-L-arginine (Arg-MetArg), 4; L-methionyl-L-arginyl-L-methionine (Met-Arg-Met), 9; L-methionyl-L-arginine (Met-Arg), 9; L-arginyl-L-lysine (Arg-Lys), 4; L-arginyl-L-lysyl-Larginine (Arg-Lys-Arg), 7; L-lysyl-L-arginyl-L-lysine (Lys-Arg-Lys), 7; L-lysyl-L-arginine (Lys-Arg), 2; out of 12 ORNs, four OE slices; L-arginyl-glycine (ArgGly), 10; glycyl-L-arginine (Gly-Arg), 4; L-methionyl-glycine (Met-Gly), 4; glycyl-glycine (Gly-Gly), 4; glycyl-glycyl-glycine (Gly-Gly-Gly), 2; out of six ORNs, four OE slices). (B) Of the five group II peptides only the dipeptide L-arginyl-glycine (Arg-Gly) featured a stimulus-induced maximum amplitude of [Ca2+]i increases comparable to stimulation with L-arginine (only ORNs exclusively sensitive to the amino acid L-arginine, i.e. #27?30 taken into account). In contrast, the dipeptide glycyl-L-arginine (Gly-Arg) showed a weak response (averaging of multiple applications of glycyl-L-arginine (GlyArg); *, p,0.05; **, p,0.001, paired t-test, error bars represent standard deviation). [AA: amino acids, Arg: L-arginine]. doi:10.1371/journal.pone.0053097.gL-arginine, L-lysine and L-methionine and group I peptides. Of the 42 amino acid-responsive ORNs, 62 responded to Larginine, 79 to L-methionine and 43 to L-lysine. As some ORNs responded to more than one amino acid, the frequencies sum up to values higher than 100 . A clearly smaller fraction of ORNs (29 ) also responded to at least one of the eight group I peptides (Figure 2A). In a second set of experiments we applied Larginine, L-methionine and glycine and group II peptides. In order to reduce the size and possibly the steric hindrance of the peptides at the receptor binding site, we chose to include 23727046 glycine, the smallest amino acid found in proteins. In this second set, of 28 amino acid-responsive ORNs, 57 responded to L-arginine, 79 to L-methionine and 32 to glycine. As in the first set of experiments only a small subset of ORNs (21 ) also responded to at least one of the five group II peptides (Figure 2A). The matrix in Figure 2B depicts the exact response profile of all peptide-sensitive ORNs. Figure 3A depicts the mean maximum amplitudes of the peptide-induced increases of Ca2+-dependent fluorescence relative to the amplitude reached upon application of amino acid controls. Out of the group I peptides (green bars), even the peptide that elicited the highest mean amplitudes (L-methionyl-L-arginyl-Lmethionine) reached only about 32 of the amino acid-induced amplitudes. In comparison, the smaller group II peptides (orange bars), tendentially featured a slightly higher mean maximum amplitude. Thereby, the peptide L-arginyl-glycine showed an exceptionally high mean maximum amplitude. L-arginyl-glycine elicited responses in five of the six ORNs sensitive to group II peptides. Three of them were sensitive only to the amino acid Largin.
