TABLE 2.

Hinge peptide derivatives determined by Lys-C peptide map

The estimation of abundance for each peptide was based on the individual integrated percent peak area relative to the total integrated peak area of all H12 peptide derivatives. A few H12 peptide derivatives were detected in lower quantities, coeluting with the H12e peptides. However, due to poor b-ion signals in the corresponding MS² spectra, the identification of the N terminus of some peptides was not possible. Therefore, only two peptides could be identified and are listed in the table. The total integrated peak area of the H12e peptide was ∼8%. However, the quantities of the two identified peptides eluting in peak e could not be determined (ND). The abundance of the intact H12 peptide was estimated at ∼56%. Since the amino acid sequence (²²²PKSCDKTHTCPPC-AP) in the hinge region contain two Lys-C cleavage sites, some truncated hinge peptides with adducts at the N terminus may not be retained on the reversed-phase column because of their small size and were therefore not detectable.

Peptide	Lys-C peak no.	Adduct location	%
228THTCPPCAPELLGGPSVFLFPPKPK	a	SO3H at Cys231	∼12
230TCPPCAPELLGGPSVFLFPPKPK	b		∼5
231CPPCAPELLGGPSVFLFPPKPK	c	SO3H at Cys231	∼11
230TCPPCAPELLGGPSVFLFPPKPK	d	71 Da at Thr230	∼8
228THTCPPCAPELLGGPSVFLFPPKPK	e	45 Da at Thr228	ND
228THTCPPCAPELLGGPSVFLFPPKPK	e	Asp229	ND
232PPCAPELLGGPSVFLFPPKPK	f	16 Da at Pro232	Trace
233PCAPELLGGPSVFLFPPKPK	f	16 Da at Pro233	Trace