MassPREP Mix %TFA 0.1% mg/ml < 70%DFVGYGVKDFVGVGVK 0.1%TFA 0.1% 1 4 mg/ml 1 ml/min UV 20 µl/min MS LC ACQUITY UPLC H-Class Bio

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1%TFA c QC ACQUITY UPLC H-Class Bio XSelect 5 µm XBridge 5 µm MassPREP TM RP Charged Surface Hybrid: CSH

1 C 18 Matthew A. Lauber, Stephan M. Koza, and Kenneth J. Fountain Waters Corporation, Milford, MA, USA 2 BEH130 C 18 & 0.1%TFA c QC ACQUITY UPLC H-Class Bio XSelect 5 µm XBridge 5 µm MassPREP TM RP Charged Surface Hybrid: CSH CSH130 C 18 peptide separation technology PST TFA TFA TFA TFA 4-5 TFA 7 BEH C 18 CSH TM C 18 BEH C 18 BEH C 18 ph Charged Surface Hybrid: CSH C 18 BEH C 18 BEH TFA BEH C 18 CSH C TFA 1

2 MassPREP Mix %TFA 0.1% mg/ml < 70%DFVGYGVKDFVGVGVK 0.1%TFA 0.1% 1 4 mg/ml 1 ml/min UV 20 µl/min MS LC ACQUITY UPLC H-Class Bio ACQUITY UPLC TUV 500 nl Xevo G2 Q-Tof TM 214 nm 250 nm MassPREP Mix A 0.1% (v/v) TFA B 0.1% (v/v) TFA in 90:10 ACN / A 0.1% (v/v) 2 Hz 1 s XBridge mm 5 µm 130Å XSelect mm 5 µm 130Å B 0.1% (v/v) in 90:10 ACN/ %A %B µl, C 18 2

3 DFYGYVKDFVGVGVK A 0.1% (v/v) TFA B 0.1% (v/v) TFA in 90:10 ACN/ %A %B A 0.1% (v/v) B 0.1% (v/v) in 90:10 ACN/ A 99:1 (v/v) / - 1% MS Xevo G2 QTof ESI Resolution 3.00 kv 25 V L/h 800 L/h NaI 1 µg/µl m/z Hz m/z MassLynx v4.1 B 90:9:1 (v/v) ACN/ / - 1% %A %B C 18 3

4 TFA 5 µm 4.6 mm 1 9 MassPREP Mix TFA 0.1% 0.1%TFA BEH % TFA TFA C 18 1 TFA 0.1%TFA 0.1% Mix 0.1%TFA RASG-1 RGDSPASSKP 2 Angiotensin 1-7 DRVYIHP 3 Bradykinin RPPGFSPFR 4 Angiotensin II DRVYIHPF 5 Angiotensin I DRVYIHPFHL 6 Renin Substrate DRVYIHPFHLLVYS 7 Enolase T35 WLTGPQLADLYHSLMK 8 Enolase T37 YPIVSIEDPFAEDDWEAWSHFFK 9 Melittin GIGAVLKVLTTGLPALISWIKRKRQQ 1 MassPREP Mix C 18 4

5 2.0 Vo BEH130 C18 w 4,Avg 0.75 min 0.1% w 4,Avg 1.53 min A A 214 Vo CSH130 C18 w 4,Avg 0.50 min 0.1% w 4,Avg 0.61 min 2.0 A A214 A 214 Vo Vo min 1 500µg MassPREP BEH130 5 µm mm RASG-1 Angiotensin [1-7] (4, min) Bradkykinin Angiotensin II Angiotensin I Renin Substrate Enolase T35 Enolase T37 Melittin (4, min) !"#!!"'# $"!! $"%#$"#!$"'#%"!!%"%#%"#!%"'# Analytical 0!"#!!"'# $"!! $"%#$"#!$"'#%"!!%"%#%"#!%"'# 2 MassPREP 30 µg 500 µg C 18 5

6 1000 DFVGYGVKDFVGVGVK, pi 6, 1.7 kda 250 nmbeh CSH 0.1% 3 BEH 50 µg % BEH 3 0.1% BEH CSH 0.1% CSH 8-9 BEH CSH % BEH130 C18 A250 w h 0.62 min w h 1.14 min 0.6 CSH130 C18 A250 w h 0.47 min w h 0.62 min DFVGYGVKDFVGVGVK (pi 6, 1.7 kda) 50 µg 4 C 18 6

7 10 1 CSH BEH 3 BEH 0.1% CSH %TFA 3 TFA TFA DFVGYGVKDFVGVGVK 4 MS CSH 0.1% BEH 0.1 A Target 3,4,5 co-elute A 250 1,2 3 Target 0.1% A A Target 3,4 co-elute Target 6 1% 20 min %TFA DFVGYGVKDFVGVGVK 1mg ESI-MS : (1, Da), (2, Da), (3, Da), (4, Da), (5, Da), and (6, Da). C 18 7

8 DFVGYGVKDFVGVGVK 4 mg 5 50 mm ID 0.5 g CSH BEH CSH 50 µg % 1.0 1% A250 A250 4 mg 4 mg mg C 18 8

9 5 µm 5 µm TFA ph 0.1%TFA C 18 Reference 1. Cornish J, Callon KE, Lin CQ, Xiao CL, Mulvey TB, Cooper GJ, Reid IR. Trifluoroacetate, a contaminant in purified proteins, inhibits proliferation of osteoblasts and chondrocytes. Am J Physiol. 1999; 277 (5 Pt 1): E Pini A, Lozzi L, Bernini A, Brunetti J, Falciani C, Scali S, Bindi S, Di Maggio T, Rossolini GM, Niccolai N, Bracci L. Efficacy and toxicity of the antimicrobial peptide M33 produced with different counter-ions. Amino Acids. 2012; 43(1): Reichert JP, Tartat A, Dunn MK. Development trends for peptide therapeutics: A comprehensive quantitative analysis of peptide therapeutics in clinical development. Peptide Therapeutics Foundation Fields GB, Carr SA, Marshak DR, Smith AJ, Stults JT, Williams LC, Williams KR, Young JD. In Techniques in Protein Chemistry IV. Hogue-Angeletti R, Ed. San Diego, 1993; Kent, SBH. Chemical Synthesis of Peptides and Proteins. Ann. Rev. Biochem. 1988; (57): Roux S, Zekri E, Rousseau B, Paternostre M, Cintrat JC, Fay N. Elimination and exchange of trifluoroacetate counter-ion from cationic peptides: a critical evaluation of different approaches. J Pept Sci. 2008; 14 (3): Alon H, Butilca GM, Eidelman C, Elster S, Ivchenko A, Shusman S, Tovi A, Zaoui GA. Counterion Exchange Process for Peptides. Patent Pending, 2006; Lauber MA, Koza SM, Fountain KJ. Peptide Mapping and Small Protein Separations with Charged Surface Hybrid (CSH) C 18 and TFA-Free Mobile Phases. Waters Application Note en January. 9. Lauber MA, Koza SM, Fountain KJ. Increasing Peak Capacity in Reversed-Phase Peptide Separations with Charged Surface Hybrid (CSH) C 18 Columns. Waters Application Note en January. 10. Gritti F, Guiochon G. Adsorption behavior of the three species of the biprotic peptide Phe-Ala onto an end-capped C 18 -bonded organic/inorganic hybrid stationary phase. Anal Chem. 2009; 81(24): Waters ACQUITY ACQUITY UPLC Xevo MassLynx XSelect XBridge The Science of What's Possible Waters Corporation CSH MassPREP Q-Tof Waters Corporation 2013 Waters Corporation. Printed in Japan JA PDF

Waters Sigma-Aldrich 1.0 mg/ml LC Waters Alliance HPLC ACQUITY UPLC H-Class Bio 30 cm Alliance HPLC UV ACQUITY UPLC TUV mm nm XBridge Protein B

Waters Sigma-Aldrich 1.0 mg/ml LC Waters Alliance HPLC ACQUITY UPLC H-Class Bio 30 cm Alliance HPLC UV ACQUITY UPLC TUV mm nm XBridge Protein B HPLC 3. µm BEH Stephan Koza, Susan Serpa, Hua Yang, Edouard Bouvier, and Kenneth J. Fountain Waters Corporation, Milford, MA, USA HPLC 200 0 HPLC 2 2010 UPLC 200 SEC 1 UPLC SE-UPLC 2 µm BEH BEH 100% SE-UPLC