Figure 3. Reversed-phase HPLC purification and sequence analysis of [¹²⁵I]TID-labeled fragments containing the M4 segments.

Reversed-phase HPLC purification of β2V8-8 (A) and the tryptic digest of β2V8-13 (B) and α4V8-14 (C). The elution of peptides was monitored by absorbance at 210 nm (solid line) and elution of ¹²⁵I (closed circles) by γ-counting. D–F, ¹²⁵I (●) and PTH-amino acids released (□, ◇) during sequencing of HPLC-purified β2V8-8 (D) and fractions containing the peak of ¹²⁵I from the HPLC purification of the tryptic digest of β2V8-13 (E) and α4V8-14 (F). For β2V8-8, a single amino acid sequence begining at β2-Asp⁴²⁵ (□, I_o, 9.9 pmol; R, 93%) was detected (44,250 cpm loaded in the filter and 22,000 cpm left after 35 cycles), with ¹²⁵I release in cycle 21 corresponding to labeling of β2-Cys445 (590 cpm). E, For the ¹²⁵I peak fraction from the tryptic digest of β2V8-13, there were amino acid sequences beginning at β2-Tyr⁴²⁸ (□, I_o, 9 pmol; R, 92%) and at β2-Ser⁴¹⁵ (◇, I_o, 4.5 pmol; R, 92%), (46,200 cpm loaded in the filter and 26,000 cpm left after 35 cycles). Peaks of ¹²⁵I release in cycles 18 (507 cpm) and 31 (59 cpm) correspond to labeling of β2-Cys⁴⁴⁵ in the fragments beginning at β2-Tyr⁴²⁸ and β2-Ser⁴¹⁵, respectively. F, For the ¹²⁵I peak fraction from the tryptic digest of α4V8-14, the primary amino acid sequence begin at α4-Tyr⁵⁶⁵ (□, I_o, 4.3 pmol; R, 90%; 23,000 cpm loaded in the filter and 6188 cpm left after 30 cycles). The ¹²⁵I release in cycle 18 (72 cpm) corresponds to labeling of α4-Cys⁵⁸². The amino acid sequences of the detected fragment(s) are shown above each panel, with a line indicating the M4 region.