Mass spectrometry analysis of fragments of recombinant FcγRIIa cytoplasmic tail generated by calpain cleavage
| Calpain, incubation time, h . | Observed, m/z . | Theoretical Mw, Da . | Amino acid sequence of fragment . |
|---|---|---|---|
| 0 | 8201.9 | 8201 | 212RISANSTDPVKAAQFEPPGRQMIAIRKRQLEETNNDYETADGGYMTLNPRAPTDDDKNIYLTLPPNDHVNSNN284 |
| 4 | 1187.4 | 1187 | 212RISANSTDPVK222 |
| 4 | 7034.5 | 7032 | 223AAQFEPPGRQMIAIRKRQLEETNNDYETADGGYMTLNPRAPTDDDKNIYLTLPPNDHVNSNN284 |
| 4 | 1984.7 | 1985 | 212RISANSTDPVKAAQFEPPG230 |
| 4 | 6235.7 | 6234 | 231RQMIAIRKRQLEETNNDYETADGGYMTLNPRAPTDDDKNIYLTLPPNDHVNSNN284 |
| 24 | 5635.3 | 5635 | 236IRKRQLEETNNDYETADGGYMTLNPRAPTDDDKNIYLTLPPNDHVNSNN284 |
| Calpain, incubation time, h . | Observed, m/z . | Theoretical Mw, Da . | Amino acid sequence of fragment . |
|---|---|---|---|
| 0 | 8201.9 | 8201 | 212RISANSTDPVKAAQFEPPGRQMIAIRKRQLEETNNDYETADGGYMTLNPRAPTDDDKNIYLTLPPNDHVNSNN284 |
| 4 | 1187.4 | 1187 | 212RISANSTDPVK222 |
| 4 | 7034.5 | 7032 | 223AAQFEPPGRQMIAIRKRQLEETNNDYETADGGYMTLNPRAPTDDDKNIYLTLPPNDHVNSNN284 |
| 4 | 1984.7 | 1985 | 212RISANSTDPVKAAQFEPPG230 |
| 4 | 6235.7 | 6234 | 231RQMIAIRKRQLEETNNDYETADGGYMTLNPRAPTDDDKNIYLTLPPNDHVNSNN284 |
| 24 | 5635.3 | 5635 | 236IRKRQLEETNNDYETADGGYMTLNPRAPTDDDKNIYLTLPPNDHVNSNN284 |
Recombinant FcγRIIa (212Arg-284Asn) was expressed and purified as described in “FcγRIIa and calmodulin fusion proteins,” and aliquots (10 μg) were treated for the indicated time with 0.1 U of calpain. Resultant peaks were analyzed by mass spectrometry in the m/z range of 1000 to 9000. The amino acid sequence corresponding to the observed m/z is shown. The FcγRIIa ITAM domain is underlined in bold, in the uncleaved sequence.
Mw indicates molecular weight.