link.springer.com · Web viewEquation S2: Signal Yield for Mass Spectrometry (SYMS) Calculation Equation S3: Adjusted SYMS Calculation Table S1: Empirical Adjustment of CE for Non-Derivatized

Running Title: Fragmentation of Dimethylamino Peptides

Supplemental Information

Peptide Dimethylation: Fragmentation Control via Distancing the

Dimethylamino Group

Adam J. McShane, Yuanyuan Shen, Mary Joan Castillo, and Xudong Yao*

Department of Chemistry, University of Connecticut, Storrs, CT, USA 06269

*Address reprint requests to:

Xudong Yao

Department of Chemistry

55 N. Eagleville Rd., Unit 3060

Storrs, CT 06269-3060

Phone: 860-486-6644

Fax: 860-486-2981

E-mail: [email protected]

Supplemental Information (SI) Legend

SI 1: Dim-2 Conventional Synthesis

SI 2: Dim-3 Microwave Synthesis




Figure S1a: Non-derivatized NSILTETLHR [M+2H]2+ MS/MS Spectrum (y ions that are doubly

charged are denoted as y2+)

Figure S1b: Non-derivatized NSILTETLHR [M+3H]3+ MS/MS Spectrum (y ions that are doubly


Figure S1c: Non-derivatized LSLVPDSEQGEAILPR [M+2H]2+ MS/MS Spectrum (y ions that

are doubly charged are denoted as y2+)

Figure S1d: Non-derivatized LSEPAELTDAVK [M+2H]2+ MS/MS Spectrum (y ions that are

doubly charged are denoted as y2+)

Figure S1e: Non-derivatized SVILLGR [M+2H]2+ MS/MS Spectrum (y ions that are doubly


Figure S1f: Non-derivatized YGGFLR [M+2H]2+ MS/MS Spectrum

Figure S2a: Dim-2 Derivatized NSILTETLHR [M+2H]2+ MS/MS Spectrum

Figure S2b: Dim-2 Derivatized NSILTETLHR [M+3H]3+ MS/MS Spectrum

Figure S2c: Dim-2 Derivatized LSLVPDSEQGEAILPR [M+2H]2+ MS/MS Spectrum

Figure S2d: Dim-2 Derivatized LSLVPDSEQGEAILPR [M+3H]3+ MS/MS Spectrum

1

Figure S2e: Dim-2 Derivatized LSEPAELTDAVK [M+2H]2+ MS/MS Spectrum (y ions carrying

the derivatizing group, on the lysine’s side chain amine, are denoted as *y)

Figure S2f: Dim-2 Derivatized SVILLGR [M+2H]2+ MS/MS Spectrum






the derivatizing group on the lysine’s side chain amine are denoted as *y)



Figure S4b: Dim-4 Derivatized LSLVPDSEQGEAILPR [M+2H]2+ MS/MS Spectrum


Figure S4d: Dim-4 Derivatized LSEPAELTDAVK [M+2H]2+ MS/MS Spectrum (y ions carrying

an imido lactone on the lysine’s side chain amine are denoted cy)

Figure S4e: Dim-4 Derivatized SVILLGR [M+2H]2+ MS/MS Spectrum





2











Figure S7a: Charge State Shift of SVILLGR and Dim-2-SVILLGR

Figure S7b: Charge State Shift of YGGFLR and Dim-2-YGGFLR

Figure S7c: Charge State Shift of LSLVPDSEQGEAILPR and Dim-2-LSLVPDSEQGEAILPR

Figure S8: Sigmoidal Dose-Response Fitted Precursor Survival Curve of the YGGFLR Peptides

Normalized to One

Equation S1: Percent Chemical Conversion (PCC) Calculation

Equation S2: Signal Yield for Mass Spectrometry (SYMS) Calculation

Equation S3: Adjusted SYMS Calculation

3

Table S1: Empirical Adjustment of CE for Non-Derivatized Peptides

Table S2: Empirical Adjustment of CE for Dimethylamino Derivatized Peptides

Table S3: Attributes of Chosen Peptides

Table S4: Variables for Sigmoidal Dose-Response Equation of the YGGFLR Peptides

4

SI 1

2-aminoacetic acid (8 mmol) was dissolved in 5 mL of 88% formic acid (125 mmol) and 1 mL of

37% formaldehyde (36 mmol). The solution was refluxed at 100°C for 1h. After heating, 1 mL

of concentrated HCl was added. The solvent was dried in vacuo, after which a white precipitate

was obtained. The precipitate was washed with 5 mL of glacial acetic acid 3 times. The product

was recrystallized in methanol and diethyl ether. The yield was 29.6%. 1H NMR (400 MHz,

D2O, 25C): δ = 4.06 (s, 2H), 3.01 (s, 6H) ppm; HRMS (DART) calculated [M+H]+ 104.0712,

found 104.0671.

SI 2

3-aminopropanoic acid (8 mmol) was dissolved in 5 mL of 88% formic acid (125 mmol) and 1

mL of 37% formaldehyde (36 mmol). The solution was heated via microwave irradiation at

110°C, 100 W for 1h. After microwave heating 1 mL of concentrated HCl was added. The

solvent was dried in vacuo, after which a white precipitate was obtained. The precipitate was

washed with 5 mL of glacial acetic acid 3 times. The product was recrystallized in methanol and

diethyl ether. The yield was 86.7%. 1H NMR (400 MHz, D2O, 25C): δ = 3.47 (t, J = 6.7 Hz,

2H), 2.96 (s, 6H), 2.93 (t, J = 6.7 Hz, 2H) ppm; HRMS (DART) calculated [M+H]+ 118.0868,

found 118.0871.

SI 3

4-aminobutanoic acid (8 mmol) was dissolved in 5 mL of 88% formic acid (125 mmol) and 1



5



diethyl ether. The yield was 84.1%. 1H NMR (400 MHz, D2O, 25C): δ = 3.22 (t, J = 8.2 Hz,

2H), 2.94 (s, 6H), 2.54 (t, J = 7.2 Hz, 2H), 2.12-2.01 (m, 2H) ppm; HRMS (DART) calculated

[M+H]+ 132.1025, found 132.1047.

SI 4

5-aminopentanoic acid (8 mmol) was dissolved in 5 mL of 88% formic acid (125 mmol) and 1





diethyl ether. The yield was 29.2%. 1H NMR (400 MHz, D2O, 2 C): δ = 3.19 (t, J = 8.1 Hz,

2H), 2.91 (s, 6H), 2.49 (t, J = 7.1 Hz, 2H), 1.84-1.68 (m, 4H) ppm; HRMS (DART) calculated

[M+H]+ 146.1181, found 146.1166.

SI 5

6-aminohexanoic acid (8 mmol) was dissolved in 5 mL of 88% formic acid (125 mmol) and 1




washed with 5 mL glacial of acetic acid 3 times. The product was recrystallized in methanol and

diethyl ether. The yield was 20.8%. 1H NMR (400 MHz, D2O, 2 C): δ = 3.17 (t, J = 8.1 Hz,

6

2H), 2.90 (s, 6H), 2.44 (t, J = 7.3 Hz, 2H), 1.82-1.63 (m, 4H), 1.48-1.38 (m, 2H) ppm; HRMS

(DART) calculated [M+H]+ 160.1338, found 160.1386.

7

Figure S1a: Non-derivatized NSILTETLHR [M+2H]2+ MS/MS Spectrum (y ions that are

doubly charged are denoted as y2+)

Figure S1b: Non-derivatized NSILTETLHR [M+3H]3+ MS/MS Spectrum (y ions that are doubly charged are denoted as y2+)

8

9

Figure S1c: Non-derivatized LSLVPDSEQGEAILPR [M+2H]2+ MS/MS Spectrum (y ions that are doubly charged are denoted as y2+)

10

Figure S1d: Non-derivatized LSEPAELTDAVK [M+2H]2+ MS/MS Spectrum (y ions that are doubly charged are denoted as y2+)

11

Figure S1e: Non-derivatized SVILLGR [M+2H]2+ MS/MS Spectrum (y ions that are doubly charged are denoted as y2+)

12

Figure S1f: Non-derivatized YGGFLR [M+2H]2+ MS/MS Spectrum

13


14


15


16


17

Figure S2e: Dim-2 Derivatized LSEPAELTDAVK [M+2H]2+ MS/MS Spectrum (y ions carrying the derivatizing group, on the lysine’s side chain amine, are denoted as *y)

18


19


20


21


22


23


24


25


26

Figure S4b: Dim-4 Derivatized LSLVPDSEQGEAILPR [M+2H]2+ MS/MS Spectrum

27


28

Figure S4d: Dim-4 Derivatized LSEPAELTDAVK [M+2H]2+ MS/MS Spectrum

29

Figure S4e: Dim-4 Derivatized SVILLGR [M+2H]2+ MS/MS Spectrum (y ions carrying an imido lactone on the lysine’s side chain amine are denoted cy)

30


31


32


33


34


35


36


37


38


39


40


41


42

Figure S7a: Charge State Shift of SVILLGR (top) and Dim-2-SVILLGR (bottom)

43

Figure S7b: Charge State Shift of YGGFLR (top) and Dim-2-YGGFLR (bottom)

44

Figure S7c: Charge State Shift of LSLVPDSEQGEAILPR (top) and Dim-2-

LSLVPDSEQGEAILPR (bottom)

45

Figure S8: Sigmoidal Dose-Response Fitted Precursor Survival Curves of YGGFLR

Peptides (Normalized)

46

Equation S1

After the reaction between the 5-peptide mix and the derivatizing dimethylated amino acid was

quenched, an equal amount of isotopic SVIL[L-13C615N]GR was added. After desalting, LC-MS

was performed. The following equation was then applied based on intensity to calculate the

PCC.

PCC= Intensity of Isotopic SVILLGR−Intensity of SVILLGRIntensity of Isotopic SVILLGR

×100 %

Equation S2

After the reaction between the 5-peptide mix and the derivatizing dimethylated amino acid was

quenched, an equal amount of isotopic SVIL[L-13C615N]GR was added. After desalting, LC-MS

was performed. The following equation was then applied based on intensity to calculate the

SYMS.

SYMS= Intensity of Derivatized SVILLGRIntensity of Isotopic SVILLGR

×100 %

SI Equation S3

To account for an inefficient reaction, the following equation was developed to predict SYMS if

the reaction went to 100% completion.

Adjusted SYMS=SYMSPCC

×100 %

47

Table S1

Non-Derivatized Peptide Multiplication Factor for CE

NSILTETLHR [M+2H]2+ 1.15


LSLVPDSEQGEAILPR [M+2H]2+ 1.1

LSEPAELTDAVK [M+2H]2+ 1.1

SVILLGR [M+2H]2+ 1.1

YGGFLR [M+2H]2+ 1.25

48

Table S2

Dimethylamino Derivatized Peptide Multiplication Factor for CE





LSEPAELTDAVK [M+2H]2+ 1.15

SVILLGR [M+2H]2+ 1.4

YGGFLR [M+2H]2+ 1.5

49

Table S3

Peptide Mass (Da) Number of Amino Acids

Isoelectric Point

NSILTETLHR 1183.32 10 7.55LSLVPDSEQGEAILPR 1723.93 16 3.93YGGFLR 711.81 6 9.34SVILLGR 756.94 7 10.55LSEPAELTDAVK 1272.41 12 3.93

50

Table S4

y=A 1+ A 2−A 11+10log ( x0− x ) × p

Peptide A1 A2 Logx0 p Reduced

χ2

Adjusted

R2

Non-

Derivatized

0.002328 0.999005 15.699651 -0.244595 0.00148

4

0.9916

Dim-2 0.021004 1.00113 20.392597 -0.190917 0.00608

4

0.9654

Dim-3 -0.014948 1.00080 21.466005 -0.176504 0.00521

8

0.9704

Dim-4 0.002769 0.995858 22.800792 -0.166370 0.00802

5

0.9574

Dim-5 -0.016008 0.996510 23.644762 -0.130546 0.01062 0.9408

Dim-6 -0.002919 0.999499 27.274779 -0.189113 0.00127

2

0.9916

51

Documents

link.springer.com · Web viewEquation S2: Signal Yield for Mass Spectrometry (SYMS) Calculation Equation S3: Adjusted SYMS Calculation Table S1: Empirical Adjustment of CE for Non-Derivatized