Table 1.
DepY recognizes diethoxyphospho-tyrosine independent of the amino acid sequence around the modified tyrosine*
| Antigen | ELISA | Western blot | # OP-Tyr/protein | %OP-Tyr |
|---|---|---|---|---|
| Hu albumin-QYDVRK-OP | +++ | ++++ | 7 | |
| Hu albumin control | − | − | 0 | |
| Hu albumin-YGGFL-OP | +++ | ++++ | 5 | |
| Bovine albumin-LYGLPR-OP | +++ | ++++ | 16 | |
| Bovine albumin control | − | − | 0 | |
| Lysozyme-GGYR-OP | ++++ | NA | 4, 10 & 16 | |
| Lysozyme-RARYEM-OP | ++++ | NA | 6 & 9 | |
| Lysozyme-PYYMRR-OP | +++ | ++ | 1 | |
| Lysozyme control | − | − | 0 | |
| Ovalbumin-RSLYAS-OP | +++ | NA | 18 & 22 | |
| Ovalbumin-YPF-OP | ++++ | NA | 6 & 14 | |
| Ovalbumin-YGGFL-OP | ++++ | NA | 5, 9 & 12 | |
| Ovalbumin control | − | NA | 0 | |
| Casein-OP (OP on Tyr) | +++ | +++ | 10–95% | |
| Casein control | − | − | 0 | |
| Bovine tubulin-OP (OP on Tyr + Lys) | +++ | +++++ | 12–26% | |
| Bovine tubulin control | − | − | 0 | |
| Porcine tubulin-OP (OP on Tyr + Lys) | +++ | +++++ | 6–69% | |
| Porcine tubulin control | − | − | 0 | |
| Human albumin-OP (OP on Tyr + Lys) | + | − | 21–98% | |
| Lysozyme-OP (OP on Tyr + Lys) | +++ | − | 3–5% | |
| Aprotinin-OP (OP on Tyr + Lys) | ++ | + | 10% | |
| Aprotinin control | − | − | 0 | |
| Mouse albumin-OP (OP on Tyr + Lys) | +++ | NA | 83% | |
| Mouse albumin control | − | NA | 0 |
OP refers to the diethoxyphospho adduct on tyrosine. The OP adduct is exclusively on tyrosine in the peptides and in casein. The OP adduct is on both tyrosine and lysine in human albumin, bovine tubulin, porcine tubulin, lysozyme, and aprotinin. However, monoclonal antibody depY does not recognize OP-Lys adducts. The (+) symbol represents signal intensity, where (+++++) represents the highest and (+) the lowest positive signal. The symbol (−) represents a negative result. NA is the symbol for data not available. # OP-Tyr/protein is the number of diethoxyphospho-Tyr peptides crosslinked per protein molecule as determined by MALDI-TOF mass spectrometry. %OP-Tyr refers to the percent OP labeling of multiple tyrosines in the protein where the identity of each OP-modified tyrosine and the extent of labeling was determined by LC-MS/MS; the location of the OPlabeled tyrosines and the % modified in each protein is described in Supporting Information Appendix S1.