An investigation of the role of the adiponectin variable domain on the stability of the collagen-like
domain
Paul W. R. Harris, L. Hampe, M. Radjainia, Margaret A. Brimble and Alok K. Mitra.
Supporting Information.
Figure S1. LC-MS of purified adiponectin fragment 39Cys-74Ala (2). The mass at 719.2 (M+5H)5+is the
desired peptide. The mass at 715.6 (M+5H)5+ is the inseparable aspartimide byproduct. Conditions:
linear gradient of 5 to 65%B over 21 mins where A = 0.1% formic acid in water and B = 0.1% formic
acid in acetonitrile.
Figure S2. LC-MS of purified adiponectin fragment 39Cys-74Ala (2) using Dmb backbone strategy at
63
Gly and 60Gly. Calc. (M+5H)5+ 719.3, Found 719.0. .Conditions: linear gradient of 5 to 65%B over
21 mins where A = 0.1% formic acid in water and B = 0.1% formic acid in acetonitrile.
Figure S3. LC-MS of purified adiponectin fragment 39Cys-74Ala analogue (3) whereby all three Asp are
substituted for Glu, Calc. (M+5H)5+ 727.8, Found 727.6. Conditions: linear gradient of 5 to 65%B
over 21 mins where A = 0.1% formic acid in water and B = 0.1% formic acid in acetonitrile.
Figure S4. Mass spectrum of crude (GPO)9 (O = hydroxyproline) Calc. (M+2H)2+ 1212.3, Found
1212.1, Calc. (M+3H)3+ 808.5, Found 808.6. Dimeric (GPO)9 Calc. (M+3H)3+ 1616.0, Found 1615.9.
Trimeric (GPO)9 Calc. (M+5H)5+ 1454.5, Found 1454.7. Conditions: linear gradient of 5 to 65%B over
21 mins where A = 0.1% formic acid in water and B = 0.1% formic acid in acetonitrile.
Figure S5. LC-MS of purified (GPO)9 (O = hydroxyproline) Calc. (M+2H)2+ 1212.3, Found 1211.9, Calc.
(M+3H)3+ 808.5, Found 808.3. Dimeric (GPO)9 Calc. (M+3H)3+ 1616.0, Found 1615.7.
Conditions: linear gradient of 5 to 65%B over 21 mins where A = 0.1% formic acid in water and B =
0.1% formic acid in acetonitrile at 75 °C.
Figure S6. LC-MS of purified CAGWMAGIPGHPGHN-(GPO)9 (O = hydroxyproline) 5 Calc. (M+2H)2+
1955.6, Found 1955.8, Calc. (M+3H)3+ 1304.1, Found 1304.2. Trimeric CAGWMAGIPGHPGHN-(GPO)9
Calc. (M+7H)7+ 1676.4, Found 1676.6, Calc. (M+8H)8+ 1467.0, Found 1467.1. Conditions: linear
gradient of 5 to 65%B over 21 mins where A = 0.1% formic acid in water and B = 0.1% formic acid in
acetonitrile.
Figure S7. LC-MS of purified CAGWMAGIPGHPGHN-(GPO)9 (O = hydroxyproline) 5 perfomed under
slow gradient conditions (1%B per min). 1 is attributed to monomeric 5 Calc. (M+3H)3+ 1304.1, Found
1304.2.. 2 is attributed to trimeric 5, Calc. (M+6H)6+ 1955.6, Found 1955., (M+7H)7+ 1676.4, Found
1676.6, Calc. (M+8H)8+ 1467.0, Found 1467.1, Calc. (M+9H)9+ 1304.1, Found 1304.3. Conditions:
linear gradient of 5 to 65%B over 60 mins where A = 0.1% formic acid in water and B = 0.1% formic
acid in acetonitrile.
Figure S8. LC-MS of purified CAGWMAGIPGHPGHN-(GPO)9 (O = hydroxyproline) 5. Conditions: linear
gradient of 5 to 65%B over 21 mins where A = 0.1% formic acid in water and B = 0.1% formic acid in
acetonitrile at 75 °C.
Figure S9. LC-MS of purified EDDVTTTEELAPALVPPPKGTCAGWMAGIPGHPGHN-(GPO)9 (O =
hydroxyproline) 6. Conditions: linear gradient of 5 to 65%B over 21 mins where A = 0.1% formic acid
in water and B = 0.1% formic acid in acetonitrile at 75 °C.