Figure 7.
ERp46 generates thiols in the EGF-2 domain of the β3 subunit of the αIIbβ3 integrin. (A) The MS/MS spectrum of a 5+ charged ion (m/z 618.72) corresponding to the peptide GECLCGQCVCHSSDFGK from integrin β3 with all 4 cysteines alkylated by iodoTMT reagent. The y- and b-ion series in (A) enabled the confident identification of the peptide sequence. During the MS/MS stage of acquisition to derive fragment ions and sequence information, a unique reporter ion mass is also generated. These reporter ions are in the low mass region of the MS/MS spectrum. The intensity of iodoTMT tag (126, 127, 130, and 131) in (B) indicate the relative quantitation of redox changes. IodoTMT-126 or iodoTMT-130 represent the initial abundance of thiols in the peptide, whereas iodoTMT-127 and iodoTMT-131 represent the thiols in the peptide labeled after reduction of disulfide bonds in each sample with DTT/TCEP. (C) Representative MS3 spectrum of the first Cys (Cys501) in the peptide showing the increase in iodoTMT-130 induced by ERp46 relative to iodoTMT-126 in the sample without ERp46. IodoTMT-127 and iodoTMT-131 represent the thiols labeled after reduction of disulfide bonds in the samples. (D) ERp46 generates thiols in Cys503, Cys501, Cys508, and Cys521 of the EGF-2 domain as measured by MS3; mean ± SEM, n = 3; *P < .05, **P < .01, t test. These cysteines form the Cys473-Cys503, Cys486-Cys501, and Cys508-Cys521 disulfide bonds. (E-G) Thiols are decreased in the β3 subunit of nonactivated (NA) and activated (Act) ERp46-null platelets but are unchanged in PDI-null platelets. Platelets from Pf4-Cre/ERp46fl/fl mice, Pf4-Cre/PDIfl/fl mice, or Cre− littermates were activated with collagen (10 μg/mL) for 10 minutes without stirring followed by MPB labeling. After platelet lysis, the MPB was pulled down with streptavidin-agarose beads. The samples were analyzed by blotting with rabbit anti-β3 and anti-ERp46 or anti-PDI antibodies and the band densities calculated by densitometry using Odyssey V3.0. (E and G) Representative blots (F and H) cumulative data for nonactivated (NA) and activated platelets; mean streptavidin SEM, n = 4 (E-F), n = 6 (G-H), ***P < .001, ****P < .0001, Student t test.

ERp46 generates thiols in the EGF-2 domain of the β3 subunit of the αIIbβ3 integrin. (A) The MS/MS spectrum of a 5+ charged ion (m/z 618.72) corresponding to the peptide GECLCGQCVCHSSDFGK from integrin β3 with all 4 cysteines alkylated by iodoTMT reagent. The y- and b-ion series in (A) enabled the confident identification of the peptide sequence. During the MS/MS stage of acquisition to derive fragment ions and sequence information, a unique reporter ion mass is also generated. These reporter ions are in the low mass region of the MS/MS spectrum. The intensity of iodoTMT tag (126, 127, 130, and 131) in (B) indicate the relative quantitation of redox changes. IodoTMT-126 or iodoTMT-130 represent the initial abundance of thiols in the peptide, whereas iodoTMT-127 and iodoTMT-131 represent the thiols in the peptide labeled after reduction of disulfide bonds in each sample with DTT/TCEP. (C) Representative MS3 spectrum of the first Cys (Cys501) in the peptide showing the increase in iodoTMT-130 induced by ERp46 relative to iodoTMT-126 in the sample without ERp46. IodoTMT-127 and iodoTMT-131 represent the thiols labeled after reduction of disulfide bonds in the samples. (D) ERp46 generates thiols in Cys503, Cys501, Cys508, and Cys521 of the EGF-2 domain as measured by MS3; mean ± SEM, n = 3; *P < .05, **P < .01, t test. These cysteines form the Cys473-Cys503, Cys486-Cys501, and Cys508-Cys521 disulfide bonds. (E-G) Thiols are decreased in the β3 subunit of nonactivated (NA) and activated (Act) ERp46-null platelets but are unchanged in PDI-null platelets. Platelets from Pf4-Cre/ERp46fl/fl mice, Pf4-Cre/PDIfl/fl mice, or Cre− littermates were activated with collagen (10 μg/mL) for 10 minutes without stirring followed by MPB labeling. After platelet lysis, the MPB was pulled down with streptavidin-agarose beads. The samples were analyzed by blotting with rabbit anti-β3 and anti-ERp46 or anti-PDI antibodies and the band densities calculated by densitometry using Odyssey V3.0. (E and G) Representative blots (F and H) cumulative data for nonactivated (NA) and activated platelets; mean streptavidin SEM, n = 4 (E-F), n = 6 (G-H), ***P < .001, ****P < .0001, Student t test.

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