Table 2.
Major peaks in secondary mass spectrum derived from collisional fragmentation of the DsRed chromopeptide ion (parent mass 2185.08) and the corresponding ion from the K83R mutant (parent mass 2187.11)
| Assignment | Calculated m/z | Observed m/z | SD | Calculated −observed m/z | Relative amplitude |
|---|---|---|---|---|---|
| Distinctive ions in secondary MS derived from DsRed chromopeptide ion | |||||
| x4c18(i) | 246.088 | 246.120 | .015 | −0.032 | 14 |
| y5b18-NH3(ii) | 310.083 | 310.076 | .013 | 0.007 | 9 |
| PQF + 17 y8c15(iii) | 390.214 | 390.208 | .010 | 0.006 | 13 |
| x4c18(iv) | 462.199 | 462.198 | .013 | 0.001 | 22 |
| (y8-2H)2+ | 466.717 | 466.724 | .006 | −0.007 | 8 |
| (y9-2H)2+ | 510.233 | 510.236 | .004 | −0.003 | 6 |
| y5-2H-NH3(v) | 543.220 | 543.224 | .011 | −0.004 | 100 |
| y5-2H (vi) | 560.247 | 560.256 | .014 | −0.009 | 11 |
| (y10-2H)2+ | 566.775 | 566.788 | .012 | −0.013 | 2 |
| (y11-2H)2+ | 623.317 | 623.320 | .004 | −0.003 | 1 |
| (y12-2H)2+ | 680.831 | 680.830 | .017 | 0.001 | 2 |
| y8-2H | 932.427 | 932.457 | .023 | −0.030 | 25 |
| y9-2H | 1019.459 | 1019.485 | .014 | −0.026 | 10 |
| y10-2H | 1132.543 | 1132.546 | .023 | −0.003 | 3 |
| Distinctive ions in secondary MS derived from K83R chromopeptide ion | |||||
| y82+ | 467.725 | 467.723 | .006 | 0.002 | 17 |
| y92+ | 511.241 | 511.240 | .004 | 0.001 | 11 |
| y102+ | 567.783 | 567.790 | .026 | −0.007 | 3 |
| y112+ | 624.325 | 624.349 | .035 | −0.024 | 1 |
| y122+ | 681.839 | 681.860 | .016 | −0.021 | 3 |
| y8 | 934.442 | 934.461 | .026 | −0.019 | 30 |
| y9 | 1021.474 | 1021.506 | .007 | −0.032 | 19 |
| y10 | 1134.558 | 1134.556 | .039 | 0.002 | 6 |
Separate wild-type and K83R mutant peptide digests were prepared and the resulting spectra were obtained on three different days. Fig. 3 gives the structures of the ions assigned as i–vi. C-terminal fragment ions (yn species, where n denotes the peptide bond cleaved counting from the C terminus of the peptide at Lys-70) already include the dehydration and dehydrogenation necessary to generate the K83R or GFP-like chromophore 1; yn − 2H indicates the corresponding ion with one additional dehydrogenation. Twenty-nine b-type ions and internal ions are omitted because they did not include the residues modified by the chromophore and therefore were observed in both of the secondary mass spectra. The ions that did include the chromophore generally differed from their calculated mass-to-charge by less than .01 m/z, within the measurement error.