Table 1.
SYK phosphospecific analysis using mass spectrometry
Reactions to assess the sites of SYK autophosphorylation were conducted in the presence or absence of ppITAM and/or LYN and quenched at the indicated reaction time points. Each reaction was then subjected to proteolysis. Phosphoproteomic analysis of the resulting ionizable peptides was performed to assess the fractional phosphorylation at the indicated serine, threonine, or tyrosine residues.
| Phosphorylation sites | SYK | SYK + ATP | SYK + ATP + ppITAM | SYK + ATP + LYN | SYK + ATP + ppITAM + LYN | SYK + ATP + ppITAM + LYN | SYK + ATP + ppITAM + LYN |
|---|---|---|---|---|---|---|---|
| Reaction time | Upa | 5 min | 5 min | 5 min | 15 s | 30 s | 5 min |
| pTyr-364 | 1 | 1 | 1 | 2 | 1 | 2 | 5 |
| pTyr-203 | 2 | 2 | 2 | 2 | 2 | 2 | 2 |
| pTyr-630 (629) | 2 | 3 | 2 | 2 | 2 | 2 | 2 |
| pTyr-631 | <1 | <1 | <1 | <1 | <1 | <1 | <1 |
| pTyr-131 | NDb | ND | <1 | 17 | ND | <1 | 5 |
| pTyr-323 | <1 | <1 | 37 | 40 | 7 | 16 | 70 |
| pTyr-296 | ND | 30 | 7 | 43 | ND | 2 | 68 |
| pTyr-352 | ND | <1 | 2 | 10 | 18 | 25 | 22 |
| pTyr-348 + pTyr-352 | ND | <1 | 3 | 32 | 4 | 10 | 43 |
| pTyr-345 + pTyr-348 + pTyr-352 | ND | ND | <1 | <1 | <1 | <1 | <1 |
| pTyr-526 | ND | ND | ND | 25 | 2 | 5 | 21 |
| pTyr-525 + pTyr-526 | ND | ND | ND | 7 | ND | <1 | 3 |
a The notation “up” denotes unphosphorylated.
b ND indicates not determined.