Skip to main content
. 2015 Jun 24;3(4):e00154. doi: 10.1002/prp2.154

Figure 1.

Figure 1

Ethyl acetate with 5% acetic acid (but not 60% ethanol) efficiently concentrates the 6F peptide produced in transgenic tomatoes. Freeze-dried transgenic tomatoes expressing β-glucuronidase (EV), or expressing the 6F peptide (Tg6F) were extracted overnight at room temperature in either ethyl acetate/acetic acid, or in aqueous ethanol as described in Materials and Methods. The ethyl acetate/acetic acid extract was dried under argon; the aqueous ethanol extract was dried under vacuum as described in Materials and Methods. The remaining solids were suspended in water and analyzed on 18% SDS PAGE gels as described in Materials and Methods. (A) Image of the Sypro Ruby-stained gel of the tomato concentrates prepared with ethyl acetate/acetic acid (100 μg or 200 μg of protein per lane). (B) Image of the Sypro Ruby-stained gel of tomato concentrates prepared with aqueous ethanol (100 μg protein per lane). Authentic chemically synthesized 6F peptide at 5, 10, or 20 μg per lane was included as standards in (A) and (B). (C) Summary of the results of LC/MS analysis of the trypsin digest of the authentic 6F standard (6F Standard, top of panel C, 5 μg), the trypsin digest of the corresponding band from the Tg6F lane (Tg6F, middle of panel C, 100 μg), and the trypsin digest of the corresponding band from the EV lane (EV, bottom of C, 100 μg) performed as described in Materials and Methods. To simplify data presentation, the spectra collected between 13 and 25 min from all three chromatograms were averaged. All the tryptic peptides eluted within this window. The arrows point to signals identified on the basis of molecular weight concordance as being derived from 6F peptide: EFF (residues 16–18), found m/z 442.2 (detected in both 6F Standard and Tg6F, but not in EV), calculated for MH+ 442.197 Da (monoisotopic, mi); DWLK (residues 1–4), found m/z 561.3 (detected in both 6F Standard and Tg6F, but not in EV), calculated for MH+ 561.303 Da (mi); FFEK (residues 10–13), found m/z 570.3 (detected in both 6F Standard and Tg6F, but not in EV), calculated for MH+ 570.292 Da (mi); AFYDK (residues 5–9), found m/z 643.3 (detected in Tg6F, but not in 6F Standard or EV), calculated for MH+ 643.308 Da (mi); FKEFF (residues 14–18), found m/z 717.4 (detected in both 6F Standard and Tg6F, but not in EV), calculated for MH+ 717.361 Da (mi); DWLKAFYDK (residues 1–9), found m/z 1185.6 and 1185.7 (detected in 6F Standard and Tg6F, respectively, but not in EV), calculated for MH+ 1185.594 Da (mi); AFYDKFFEK (residues 5–13), found m/z 1194.6 and 1194.7 (detected in 6F Standard and Tg6F, respectively, but not in EV), calculated for MH+ 1194.583 Da (mi). These partially overlapping tryptic peptides provided 100% sequence coverage for both the 6F standard and the Tg6F sample. These results are representative of two experiments.