link.springer.com · web viewequation s2: signal yield for mass spectrometry (syms) calculation...
Post on 26-Apr-2018
214 Views
Preview:
TRANSCRIPT
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: x.yao@uconn.edu
Supplemental Information (SI) Legend
SI 1: Dim-2 Conventional Synthesis
SI 2: Dim-3 Microwave Synthesis
SI 3: Dim-4 Microwave Synthesis
SI 4: Dim-5 Microwave Synthesis
SI 5: Dim-6 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
charged are denoted as y2+)
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
charged are denoted as y2+)
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
Figure S3a: Dim-3 Derivatized NSILTETLHR [M+2H]2+ MS/MS Spectrum
Figure S3b: Dim-3 Derivatized NSILTETLHR [M+3H]3+ MS/MS Spectrum
Figure S3c: Dim-3 Derivatized LSLVPDSEQGEAILPR [M+2H]2+ MS/MS Spectrum
Figure S3d: Dim-3 Derivatized LSLVPDSEQGEAILPR [M+3H]3+ MS/MS Spectrum
Figure S3e: Dim-3 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 S3f: Dim-3 Derivatized SVILLGR [M+2H]2+ MS/MS Spectrum
Figure S4a: Dim-4 Derivatized NSILTETLHR [M+3H]3+ MS/MS Spectrum
Figure S4b: Dim-4 Derivatized LSLVPDSEQGEAILPR [M+2H]2+ MS/MS Spectrum
Figure S4c: Dim-4 Derivatized LSLVPDSEQGEAILPR [M+3H]3+ 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
Figure S5a: Dim-5 Derivatized NSILTETLHR [M+2H]2+ MS/MS Spectrum
Figure S5b: Dim-5 Derivatized NSILTETLHR [M+3H]3+ MS/MS Spectrum
Figure S5c: Dim-5 Derivatized LSLVPDSEQGEAILPR [M+2H]2+ MS/MS Spectrum
Figure S5d: Dim-5 Derivatized LSLVPDSEQGEAILPR [M+3H]3+ MS/MS Spectrum
2
Figure S5e: Dim-5 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 S5f: Dim-5 Derivatized SVILLGR [M+2H]2+ MS/MS Spectrum
Figure S6a: Dim-6 Derivatized NSILTETLHR [M+2H]2+ MS/MS Spectrum
Figure S6b: Dim-6 Derivatized NSILTETLHR [M+3H]3+ MS/MS Spectrum
Figure S6c: Dim-6 Derivatized LSLVPDSEQGEAILPR [M+2H]2+ MS/MS Spectrum
Figure S6d: Dim-6 Derivatized LSLVPDSEQGEAILPR [M+3H]3+ MS/MS Spectrum
Figure S6e: Dim-6 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 S6f: Dim-6 Derivatized SVILLGR [M+2H]2+ MS/MS Spectrum
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
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
5
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 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
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 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
mL of 37% formaldehyde (36 mmol). The solution was heated via microwave irradiation at
110°C, 100 W for 2h. 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 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
Figure S2a: Dim-2 Derivatized NSILTETLHR [M+2H]2+ MS/MS Spectrum
14
Figure S2b: Dim-2 Derivatized NSILTETLHR [M+3H]3+ MS/MS Spectrum
15
Figure S2c: Dim-2 Derivatized LSLVPDSEQGEAILPR [M+2H]2+ MS/MS Spectrum
16
Figure S2d: Dim-2 Derivatized LSLVPDSEQGEAILPR [M+3H]3+ MS/MS Spectrum
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
Figure S2f: Dim-2 Derivatized SVILLGR [M+2H]2+ MS/MS Spectrum
19
Figure S3a: Dim-3 Derivatized NSILTETLHR [M+2H]2+ MS/MS Spectrum
20
Figure S3b: Dim-3 Derivatized NSILTETLHR [M+3H]3+ MS/MS Spectrum
21
Figure S3c: Dim-3 Derivatized LSLVPDSEQGEAILPR [M+2H]2+ MS/MS Spectrum
22
Figure S3d: Dim-3 Derivatized LSLVPDSEQGEAILPR [M+3H]3+ MS/MS Spectrum
23
Figure S3e: Dim-3 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)
24
Figure S3f: Dim-3 Derivatized SVILLGR [M+2H]2+ MS/MS Spectrum
25
Figure S4a: Dim-4 Derivatized NSILTETLHR [M+3H]3+ MS/MS Spectrum
26
Figure S4b: Dim-4 Derivatized LSLVPDSEQGEAILPR [M+2H]2+ MS/MS Spectrum
27
Figure S4c: Dim-4 Derivatized LSLVPDSEQGEAILPR [M+3H]3+ MS/MS Spectrum
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
Figure S5a: Dim-5 Derivatized NSILTETLHR [M+2H]2+ MS/MS Spectrum
31
Figure S5b: Dim-5 Derivatized NSILTETLHR [M+3H]3+ MS/MS Spectrum
32
Figure S5c: Dim-5 Derivatized LSLVPDSEQGEAILPR [M+2H]2+ MS/MS Spectrum
33
Figure S5d: Dim-5 Derivatized LSLVPDSEQGEAILPR [M+3H]3+ MS/MS Spectrum
34
Figure S5e: Dim-5 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)
35
Figure S5f: Dim-5 Derivatized SVILLGR [M+2H]2+ MS/MS Spectrum
36
Figure S6a: Dim-6 Derivatized NSILTETLHR [M+2H]2+ MS/MS Spectrum
37
Figure S6b: Dim-6 Derivatized NSILTETLHR [M+3H]3+ MS/MS Spectrum
38
Figure S6c: Dim-6 Derivatized LSLVPDSEQGEAILPR [M+2H]2+ MS/MS Spectrum
39
Figure S6d: Dim-6 Derivatized LSLVPDSEQGEAILPR [M+3H]3+ MS/MS Spectrum
40
Figure S6e: Dim-6 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)
41
Figure S6f: Dim-6 Derivatized SVILLGR [M+2H]2+ MS/MS Spectrum
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
NSILTETLHR [M+3H]3+ 1.1
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
NSILTETLHR [M+2H]2+ 1.25
NSILTETLHR [M+3H]3+ 1.25
LSLVPDSEQGEAILPR [M+2H]2+ 1.1
LSLVPDSEQGEAILPR [M+3H]3+ 1.1
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
top related