Fibrinogen fragment D100, but not fragment ‘D98,’ is recognized by an antibody to the fibrinogen C-terminal γ-chain dodecapeptide (γ-12); the C-termini of D100 and ‘D98’ are primarily γ-411 and γ-405, respectively. (A) Fragment D100 and fragment ‘D98’ were electrophoresed in a 10% acrylamide gel. Separated proteins were then transferred to a nitrocellulose membrane and stained with Ponceau (left). After washing away the stain, the membrane was immunoblotted with mAb 7E9, which reacts with the fibrinogen γ-12 peptide. (B) MS-based quantitation of C-terminal peptides. D100 and ‘D98’ gel bands were treated with propionic anhydride, which modifies primary amines, and then digested with trypsin. Propionic anhydride-modified residues are marked with [+56]. C-terminal peptides of D100 and ‘D98’ were measured by parallel reaction monitoring⁷¹  and signals extracted and analyzed by Skyline.⁷²  Signals normalized to the amount of fibrinogen C-terminal γ-chain in the 2 samples are shown for each peptide. For the D100 sample, the most abundant peptide measured was the C-terminal γ-chain peptide γ-392-411 (LTIGEGQQHHLGGAK[+56]QAGDV) containing the intact γ-12 peptide. No signal for this peptide was identified in the ‘D98’ sample; rather, the most abundant signal was assigned to LTIGEGQQHHLGGA, which ends at γ-405. See supplemental Table 2 for additional validation of C termini, assignment of N termini, and peptides used for normalization.