S1 Negative-Ion Electron Capture Dissociation: Radical-Driven Fragmentation of Charge-Increased Gaseous Peptide Anions Hyun Ju Yoo, †,§,⊥ Ning Wang, †,§ Shuyi Zhuang, †,‡ Hangtian Song, † and Kristina Håkansson* ,† † Department of Chemistry, University of Michigan, Ann Arbor, Michigan 48109-1055, United States ‡ Department of Chemistry, Tsinghua University, Beijing, 100084, China SUPPORTING INFORMATION Materials and Methods Sample Preparation. The following peptides were used: neuromedin B (H-GNLWATGHF-NH 2 ), neuromedin C (H-GNHWAVGHLM-NH 2 ), neurokinin B (H-DMHDFFVGLM-OH), cholecystokinin (CCK, H-DYMGWMDF-NH 2 ), H-AKPSYP*P*TYK-OH (P* = hydroxyproline), eHWSYGLRPG-NH 2 (e = pyroglutamic acid), exorphin C (H-YPISL-OH), H-RRREEEpSEEEAA-OH, H-KRSpYEEHIP-OH, angiotensin I (H-DRVYIHPFHL-OH), H-RRApSVA-OH, H-TSTEPQpYQPGENL-NH 2 , bradykinin 2-9 (H-PPGFSPFR-OH), substance P-OH (H-RPKPQQFFGLM-OH), and sulfonated cholecystokinin (CCKS, H-DsYMGWMDF-NH 2 ). Most peptides were purchased from Sigma-Aldrich (St. Louis, MO), except H-TSTEPQpYQPGENL-NH 2 (which was from Millipore, Billerica, MA) and CCKS (from Advanced Chemtech, Louisville, NY). Bovine milk α- and β-casein were from Sigma-Aldrich. Coumarin tag (7-methoxycoumarin-3-carboxylic acid succinimidyl ester) was from Sigma-Aldrich. The above chemicals were used without further purification. Coumarin tagging reaction was performed according to a published procedure, 1 except for using longer reaction time (1 h). Trypsin (Promega, Madison, WI) digestion of α-/β-casein was performed for 12 h at 37 °C at an enzyme/substrate ratio of 1:50. The tryptic peptides H-FQpSEEQQQTEDELQDK-OH (β-casein 48-63), H-YLGYLEQLLR-OH (α-casein 106-115), H-FALPQYLK-OH (α-casein 189-196), H-TVDMEpSTEVFTK-OH (α-casein 153- 164), H-DIGpSEpSTEDQAMEDIK-OH (α-casein 58-73), and H-VPQLEIVPNpSAEER-OH (α-casein