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CASSS
23 September, 2015
Ultrahigh Resolution Mass Spectrometry:
Extending the Size and Detail of
Biomolecule Structure Analysis
Alan G. Marshall
National High Magnetic Field Laboratory
Department of Chemistry & Biochemistry
Florida State University
The Tool:
Fourier Transform
Ion Cyclotron Resonance
Mass Spectrometry
NMR or EMR
B
ICR
B

= B
qB
=
m
The Code:
Accurate Mass
20
2
1
H
Nuclide
H
13
Mass Defect (mDa)
12
0
Atomic
Mass Defects
(All Different)
C
14
C N 15
N
17
16
O
O 18O 19F
-20
31
-40
-60
P
32
S 33S
34
35
S Cl36
S 37Cl
Mass Defect =
Atom Exact Mass – Nearest Integer
-80
-100
Every CcHhNnOoSs mass
is unique!
79
Br 81Br
127
I
Phosphorylation
vs. Sulfation
[DY[SO3H]MGWMDF-NH2 - 2H]2Theoretical:
1,140.33618 Da
Experimental: 1,140.33614 Da
Error: 0.032 ppm
[DY[PO3H2]MGWMDF-NH2 - 2H]2Theoretical:
1,140.34570 Da
Experimental: 1,140.34576 Da
Error: 0.053 ppm
0.0095 Da
m/Dm50% = 552,000
m2/(m2-m1) = 118,000
Anal. Chem. 2002,
74, 1674-1679
1140.306
1140.326
1140.346
1140.366
Mass (Da)
1140.386
1140.406
Peptide Sequencing
Labile Bonds Cleaved;
X-P Cleavage Preferred
y1
ECD/ETD
IRMPD
CAD
R1 O
Labile Bonds Retained;
No X-P Cleavage
z1·
Rn-1O
Rn
[M+nH]n+
O
H2N C C ... N C C N C C
OH
cn-1
bn-1
Zubarev, R. A.; Horn, D. M.; Fridriksson, E. K.; Kelleher, N. L.; Kruger, N. A.
Lewis, M. A.; Carpenter, B. K.; McLafferty, F. W. "Electron Capture Dissociation for
Structural Characterization of Multiply Charged Protein Cations", Anal. Chem. 2000, 72,
Valence Parity: (H + N) = Even or Odd?
100
c'/z● Overlap (%)
Mass Error
± 1.0 ppm
80
± 0.8 ppm
± 0.6 ppm
60
All Possible
Amino Acid
Combinations
± 0.4 ppm
± 0.2 ppm
± 0.1 ppm
40
20
Anal. Chem. 2011, 83, 8024-8028
0
0
200 400 600 800 1000 1200 1400 1600 1800 2000
Nominal Mass (Da)
…KATEEQLK…
Lys-N
Lys-C
Digestion
KATEEQL
ATEEQLK
Fragmentation
y6’ y5’ y4’ y3’ y2’ y1’
y6 y5 y4 y3 y2 y1
K A T E E Q L
b1 b2 b3 b4 b5 b6
-128 Da
vs.
A T E E Q L K
b1’ b2’ b3’ b4’ b5’ b6’
+128 Da
y ions RCMS. 2015, 29, 659-666 b ions
Bottom-Up MS/MS
Protein
Identification
Phosphorylation
n2 F K G P G D T S N F D D Y E E E E I R V S I N E K
~ x 30
[M + HPO3 + 2H]2+
[M + HPO3
+ 3H]3+
Finding the Phosphate
in a Protein:
One Step Electron
Capture Dissociation
MS/MS
c242+
c8
c5
z
z4
3
z10 z11
z
c65
z
6
c4
z
z
8c c11
1
z12
0
7
z
c1
9
c7
4
z14
c1
5
c1
z15 7
c2
0
1000
1500
c2
z17 z19
c1
8
500
z20
2000
1
c2
2
2500 m/z
Glycosylation
H
O
H
CH3
H
Glycan structure for
E. corallodendron
OH
Man
H
Fucose
Xyl Man Man
CH2OH
HOCH2
O
OH
Fuc GlcNAc
H
H
OH
OH
H
OH
Xylose
O
H
H
H
-N-
OH
OH
OH
GlcNAc
H
OH
OH
OH
H
H
H
Mannose
Lectin from Erythrina corallodendron
ECD Fragmentation Pattern
Håkansson
H. Cooper
Emmett
Costello
Nilsson
= GlcNAc
= Fuc
= Man
= Xyl
c4 c5 c6 c7 c8 c9 c10
2+
c15 c2+
16
SKPAQGYGYLGIFNNSK
2+
z16•
z3
Lectin from Erythrina corallodendron
IRMPD Fragmentation pattern
Håkansson
H. Cooper
Emmett
Costello
Nilsson
= GlcNAc
= Fuc
= Man
= Xyl
SKPAQGYGYLGIFNNSK
Figuring Out the Structure...
(Core is known)
-GlcNAc-Gal-NeuAc
or -GlcNAc-Gal antennae
are known
HexNAc loss
HexHexNAc loss
HexNAcNeuAc loss
HexHexNAcFuc loss
HexHexNAcNeuAc loss
(Hex)2HexNAcNeuAc loss
(Hex)5(HexNAc)4NeuAc loss
(Hex)5(HexNAc)4NeuAcFuc loss
C. Nilsson
NeuAc
Gal
Gal
GlcNAc
GlcNAc
Man
GlcNAc
Man
Man
GlcNAc
GlcNAc
Fuc
Glycoprotein Stain
– Pro-Q Emerald
Alzheimer’s
Disease
Patient
Control
Individual
Total Protein Stain –
Sypro Ruby
-1-Glycoprotein
-1Antitrypsin
7 1 2 3 4 8 9 10
Apolipoprotein J
Apolipoprotein E
32
-Trace
5
6
Glycoforms identified by GlycoMod
Spot C5: 1-antitrypsin
NeuAc
NeuAc
NeuAc
Gal
Gal
GlcNAc
GlcNAc
GlcNAc
NeuAc
GlcNAc
Gal
GlcNAc
GlcNAc
Man
NeuAc
Gal
Gal
GlcNAc
Man
GlcNAc
NeuAc
Gal
GlcNAc
GlcNAc
Man
Man
Man
NeuAc
Gal
Gal
Man
Man
NeuAc
Man
GlcNAc
GlcNAc
Man
Man
NeuAc
GlcNAc
Gal
GlcNAc
GlcNAc
Man
Man
Gal
GlcNAc
Man
Man
Man
GlcNAc
GlcNAc
GlcNAc
Fuc
Asp-46
GlcNAc
Asp-83
Asp-247
Alzheimer’s Disease
Biomarker from
Cerebrospinal Fluid
Top-Down MS/MS
Proteomics
100%
Sequence
Coverage
ECD of Ubiquitin: Probe-Mounted Gun
67 of 75 Bonds Cleaved (51 ms Irradiation, 40 scans)
H-MQIFVKTLTGKTITLEVEPSc and z• ions
DTIENVKAKIQDKEGIPPDQ-
QRLIFAGKQLEDGRTLSDYN-
IQKESTLHLVLRLRGG-OH
a• and y ions
Proteomics
Sequence Variants
PTMs
Proteoforms
4
Top-Down Proteomics
Post-Translational Modifications
+
Or?
+
Methylation
Acetylation
Tetrahymena Histone H2B
-NH3
-NMe3 = C3H6
9.4 T ESI FT-ICR MS
R-H
R-Ac = COCH2
Net Difference = CH4 vs. O
(0.0364 Da)
Me3 or Ac?
Me Me2
m/z
Mass
Mol. Cell. Proteomics
2004, 3, 872-886
Lys-111
Lys-41
Lys-4
Lys-3
Ala-1
Methylation/Acetylation of Tetrahymena Histone H2B
N
C
Me Me
Me2 Me2
Ac
Me3 Me3 Ac
Mol. Cell. Proteomics 2004, 3, 872-886
Me
Me2
Me3/Ac
Top-Down Proteomics:
Site-Specific Cys Redox
Potentials
Cellular Redox Environment
Cysteine
Cysteine
+
Disulfide
Reduction
Cystine
Nernst Equation
E′ = E°′ −
RT
nF
ln
R = Gas Constant
n = No. of Electrons
If
[Electron Donor]
[Electron Acceptor]
T = Temperature
F = Faraday Constant
[Electron Donor]
[Electron Acceptor]
= 1,
E′ = E°′
Determination of Disulfide Bond E°′
Protein–S2 + R(SH)2
Protein–(SH)2 + RS2
1) Equilibrate with excess reference redox couple
at a series of known ratios, R(SH2)/RS2
to set ambient reduction potential, E′
E′ = E°′(reference) −
RT
nF
ln
[R(SH)2]
[RS2]
2) Measure ratio of reduced/oxidized protein
3) At midpoint, [Protein-(SH)2 = Protein-S2
E′ = E°′(protein) −
RT
nF
ln
[Protein–(SH)2]
[Protein–S2]
Alkylation of a Protein by N-ethylmaleimide
Protein Mass Increase = 125 Da
Experiment
1. Equilibrate with Excess Reference Redox Couple
Dihydrolipoic Acid / Lipoic Acid; E°′ = −290 mV
2. Derivatize with NEM “Light” (+125 Da)
3. Reduce & Derivatize w. D5-NEM “Heavy” (+130 Da)
H3
H2
D3
D2
Top-Down 14.5 T FT-ICR MS
• CID of Dialkylated (2 NEM) E. coli Thioredoxin
• 35% Peptide Bonds Cleaved
b
108
90
72
54
36
18
S
A
M
V
R
V
D
D
I
A
G
G
K
G
A
K
I
A
I
A
P
L
P
L
I
I
I
N
T
S
H
L
L
I
L
K
L
V
D
D
L
G
T
D
E
Q
L
Q
D
F
I
N
F
L
D
W
A
P
K
K
S
A
D
G
N
E
F
E
E
T
G
F
D
W
Y
A
E
L
T
C
Q
P
V
D
D
G
G
K
A
A
V
P
K
Y
A
N
L
C
L
G
T
L
K
K
T
I
K
A
y
Cys-NEM
b ion
y ion
18
36
54
72
90
108
Determination of E°′ for E. coli Thioredoxin
(4 Different b-Ions)
E°′ = −280.1 ± 0.7 mV
Literature E°′:
−270 mV
−285 mV
Chromophore O.D.
Trp Fluorescence
HPLC
Electrochem
UV-VIS NADPH
Non-Protein
Biomarkers
OH
OH
O
OH
OH
O
OH
O
OH
O
OH
O
O
NHAc
O
OH
HO
O
HN
OH
OH
O
HO
O
OH
OH
OH
COOH
NHAc O
GM1a
OH
NeuAc =
N-Acetylneuraminic Acid
(Sialic Acid)
HexNAc =
N-Acetylhexosamine
Hex = Hexose
GM1b
HO
HO
OH
NHAc O
OH
OH
COOH
OH
OH
O
OH
O
O
O
OH
O
O
NHAc
O
O
OH
OH
OH
O
OH
OH
O
HN
OH
OH
JASMS 2005,
16, 752-762
M-
400
600
2n
M-(
’)
800 m/z 1000
M-(
M-(
’)
)
[M+2H]2+
** * *
*
1200
1400
[M+H]+& [M+2H]+•
O
[(M-H2O)+H]+
o
[(M-COCH3-H)]
’
~x6
’)
**
o
0,2)
M-(
OH
o
M-
M-(
)
’)
M-(
’
)
M-(
’
*
Ceramide
Ceramide’
Ceramide’’
Fatty Acid+NH2
~x2
**
o
M-( ’) (from
’
’
’’
0,2)
O
O
HN
NH Ac
200
NH Ac
’(from
Sphingosine’’
o
o *
*o
o
ECD of (+) GM1
O
HN
OH
CcHhNnOoSs
DBE = c - h/2 + n/2 + 1
McLafferty & Turecek
Interpretation of Mass Spectra
1993
0
Anal.Chem. 2007, 79, 8423-8430
GD1 (d18:1/24:1)
GD1 (d18:1/24:0)
GD1 (d18:1/23:1)
GD1 (d18:1/23:0)
GD1 (d18:1/22:0)
GD1 (d18:1/16:0)
2000
asialo-GM1 (d18:1/16:0)
OH
GM1b (d18:1/24:1)+O
HN
GM1b (d18:1/24:1)
GM1b (d18:1/24:0)
GM1b (d18:1/23:0)
4000
GM1b (d18:1/22:0)
S/N Ratio
GM1b (d18:1/17:0)
GM1b (d18:1/16:0)+O
GM1b (d18:1/16:0)
GM1 (18:1/18:0)
GM2α (d18:1/24:1)
O
GM2α (d18:1/24:0)
3000
GM2α (d18:1/22:0)
GM2α (d18:1/16:0)
GM3 (d18:1/24:1)
GM3 (d18:1/24:0)
GM3 (d18:1/23:0)
GM3 (d18:1/22:1)
GM3 (d18:1/22:0)
GM3 (d18:1/18:1)
GM3 (d18:1/18:0)
GM3 (d18:1/17:0)
GM3 (d18:1/16:1)
GM3 (d18:1/16:0)+O
GM3 (d18:1/16:0)
GM3 (d18:1/15:0)
GM3 (d18:1/14:0)
Gangliosides
LTQ 14.5 T FT-ICR MS
U87 + DI312/24 hr + SN38/24 hr
U87 + P53/24 hr + SN38/24 hr
O
Glucose
Galactose
GalNac
Neuraminic acid
1000
0
(32:0)
(32:1)
(34:1)
(34:1)+O
(34:2)
(34:2)+O
(34:3)
(35:1)
(35:2)
(36:1)
(36:1)+O
(36:2)
(36:2)+O
(36:3)
(36:3)+O
(36:4)
(36:4)+O
(36:5)
(37:2)
(37:3)
(37:4)
(37:5)
(38:2)
(38:2)+O
(38:3)
(38:3)+O
(38:3)+2O
(38:3)+3O
(38:4)
(38:4)+O
(38:4)+2O
(38:4)+3O
(38:4)+4O
(38:5)
(38:5)+O
(38:5)+2O
(38:5)+3O
(38:6)
(38:6)+O
(39:3)
(39:4)
(39:4)+O
(39:4)+2O
(39:4)+3O
(39:5)
(40:3)
(40:4)
(40:4) +O
(40:4)+2O
(40:5)
(40:5)+O
(40:5)+2O
(40:6)
(40:6)+O
(40:7)
Phosphatidylinositols
LTQ 14.5 T FT-ICR MS
S/N Ratio
~X4
8000
U87 +DI312/24 hr + SN38/24 hr
U87 + P53/24 hr + SN38/24 hr
6000
4000
2000
PI (18:1(9Z)/18:1(9Z))
Anal.Chem. 2007, 79, 8423-8430
Sulfatides
LTQ 14.5 T FT-ICR MS
S/N Ratio
U87 DI312/24 hr + SN38/24 hr
500
U87 P53/24 hr + SN38/24 hr
400
300
200
100
0
(34:1)
(34:1)+O
(34:2)
(34:2)+O
O
Sulfatide (34:1)
O
-
4OS
HN
OH
Anal.Chem. 2007, 79, 8423-8430
Galactose
(42:2)
Isotopes
and
Charge
State
CO+
28 29
C70+
840
Bovine Ubiquitin
842
844
(M+10H)10+
C378H629N105O118S
8,559.6 Da
857.0
m/z
858.0
Isotopic Depletion
FK506-Binding Protein
C527H830N146O155S3
Cys-22
(M+10H)10+
Ala-22
Monoisotopic
(0.65%)
Natural
Abundance
Monoisotopic
11,780 Dalton
13C,15N
Depleted
1179.0
Marshall, A.G., Senko, M.W., Li, W.,
Li, M., Dillon, S., Guan, S., Logan, T.M.,
JACS 1997, 119, 433-434.
1179.4
1179.8
m/z
1180.2
Monoclonal Antibody – RAS-111 (IgG1) Glycoforms
Pfizer (Wyeth/Ayerst)
147,756 Da
C6528H10088N1728O2098S44
Proteins as Drugs: $80B/Year
16 Disulfide Bonds
1325 Amino Acids
Plus Glycans
(+) ESI 9.4 T FT-ICR MS
Anal. Chem. 2013, 85, 4239-4246
Calculated: 147,757.5 Da
Experimental: 147,755.5 Da
55+
53+
57+
Quadrupole
Isolation
51+
IgG1k
Monoclonal
Antibody
59+
49+
61+
67+
2,200
65+
63+
47+
2,400
2,600
m/z
2,800
3,000
3,200
Quadrupole-Isolated Monoclonal IgG1k Antibody
57+ + K
+ Phosphate
ESI 9.4 T FT-ICR MS
14 s Transient
125 Scans
2.7 s
6 Beats
0.0 3.4 6.8 10.2 13.6
Time (sec)
2,591
2,592
2,593
2,594
2,595
1/57 Da
2,592.85
2,592.93
2,593.01
2,593.09
m/z
2,593.17
2,596
2,597
Magnitude-Mode
RP ~ 330,000
2,593.26
2,593.34
z1148+
8+
c886+ c118
c1037+
c1178+
Electron Capture Dissociation of IgG1k
z997+
z906+
6+
88
c896+
c1057+
c543+
8+
1620
1,620
5+
c906+
c1067+z119
c916+
c1047+
c1218+
z896+
c1077+
z1057+
c926+
c1238+
7+
y1057+
z 895+
z 1066+
c915+
z 90 5+ 6+
z 108
c92 5+
c463+
1600
1,600
91
z1027+
8+
c1047+ z117 z
z1007+
z 1096+z
1640
1,640
1660
1,660
1680
1,680
1700
1,700
1990
1,720
2000
2010
2020
2030
2040
2050
Heavy Chain Fragments (Blue)
Light Chain Fragments (Red)
500
1000
1500
2000m/z 2500
3000
3500
4000
ECD MS/MS Antibody Light Chain Fragment Map
(118 c-ions; 8 z-ions; 11 y-ions; 70 unique cleavages)
c
Anal. Chem. 2013, 85, 4239-4246
z
y
ECD MS/MS Antibody Heavy Chain Fragment Map
(77 c-ions; 204 z-ions; 41 y-ions; 154 unique cleavages)
Protein Complexes
H/D Exchange
Engen, J. Anal. Chem. 2009, 81(19), 7870.
Citations
Publications
5000
175
4500
150
4000
3500
125
3000
100
2500
75
2000
50
1500
1000
25
0
1990
500
1995
2000
Year
2005
0
2010
Amide backbone Hydrogen/Deuterium Exchange
Marcsisin, S. R.; Engen, J. R. Anal Bioanal Chem 2010, 397, 967-972
SLOW and FAST Exchanging Hydrogens
Marcsisin, S. R.; Engen, J. R. Anal Bioanal Chem 2010, 397, 967-972
H/D Exchange Monitored by High-Resolution MS
Apoprotein or
protein in complex
D
H
Dilute 10 fold
in D2O buffer
D
D
H
D
D
D
Quench pD/pH
2.3 ~ 2.5
Temp ~1-2 ºC
D
H/D Exchange
Time (min) Peptide from Protein in Complex
Peptide from Apoprotein
Low-pH Active
Enzyme
0.0
ESI FT-ICR MS
0.5
Peptide Separation
2.0
Fast, to Minimize
Back- Exchange
of DH
60
240
Jasco HPLC System
480
m/z
Blank control
(No D present)
*
Δ
Δ
Δ
*
ESI FT-ICR MS of Two Myoglobin Fragments with
Protease Type XIII Digestion (ProZap C18 Separation)
*
Δ
*
Δ
Δ Fragment 114-135 (4+)
Δ
30 s HDX
*
*
Δ*
2 min HDX
*
Δ
*
*
Δ
*
Δ
Δ
*
Δ
*
Δ
*
Δ*
*
*
*
563.5
*
Δ
564.5
Δ
*
*
*
Δ
Δ
*
*
Δ
Δ
*
565.5
Δ
Δ
*
Δ
m/z
*
Δ
Δ
Δ
Δ
Δ
Δ
*
Δ
30 min HDX
*
*
Δ
*
Δ
Δ
Δ
*
Δ
Δ
< 15 mDa
Δ
Δ
*
*
8 min HDX
562.5
Δ
*
Δ
Δ
Δ
Δ
Δ
Δ
*Δ
Fragment 119-135 (3+)
*
Δ
*
566.5
Δ
*
Δ
Δ
567.5
Zhang, H.-M. et al. Anal. Chem., 80, 9034-9041 (2008)
Bottom of Helix III Protected upon Assembly
Centroid
Mass
1498
CA
1497
1496
1495
1494
1493
1492
Assembled CA
1491
1490
1
10
100
Time (min)
1000
Helices VI and VII Not Protected upon Assembly
Centroid
Mass
1941
1940
CA
1939
Assembled CA
1938
1937
1936
1935
1934
1
10
100 1000
Time (min)
Deuterium Uptake
Automated Screening of Deuterium
Incorporation Profiles
Time (s)
Analysis in less than 10 min!
Volume 325
Number 4
24 January 2003
BioMedNet reports: “U.S.
scientists … use mass
spectrometry and
chemical cross-linking to
identify…surface of the
HIV capsid protein…This
represents…new
technologies to address
an important and current
issue in virology.” 1/23/03
HIV
Immature
SU
Mature
MA
CA
TM
RNA
NC
Gag
Gag (55 kDa)
MA
CA
p2
NC
p1
p6
24 kDa
J. Lanman/P. R. Prevelige, Jr. (U. Alabama Birmingham)
CA Monomers form a Hexamer Lattice
From Li et al, Nature 407:409 (2000)
Cyclophilin Loop
Unassembled CA
H/D Exchange
Rate Constant
< 0.001 min-1
I
IV
0.001-0.01
0.01-0.1
0.1-1.0
IX
1-10
> 10
I
IV
IX
IV
I
Change in H/D
Exchange Rate
on Assembly
IX
Faster
Unchanged
Slower
K70
K182
Lanman et al. J. Mol. Biol.
2003, 325, 759-772
B
2
A’
1
3
A
B’
B
1
A
A’
2
3
B’
Buckminsterfullerene
(Buckyball)
250 CA Hexamers
12 CA Pentamers
Pornillos et al.,
Cell 2009, 137, 1282-1292
Molecular Mechanism of RNA Packaging
Hexameric
ATPase P4
ssRNA
Portal Vertex
Lam
Emmett
Lisal
Kainov
Tuma
Orientation of P4 within its Procapsid
C-terminus
Capsid
Interior
Translocation Direction
Apical Domain Associated
with Procapsid
P4 Alone
C-terminus
C-terminus
C-Terminus Associated with
Procapsid
Lam
Emmett
Lisal
Kainov
Tuma
P4 in Procapsid
H/D Exchange
Rate
< 0.1 h-1
0.1 h-1 - 1 h-1
1 h-1 - 10 h-1
10 h-1 - 100 h-1
> 100 h-1
Procapsid
ssRNA
Hexameric
NTPase
Lam
Emmett
Tuma
Functional H/D Imaging
during ATP Hydrolysis
Mechanism of RNA Loading during Initiation of Packaging:
5'
RNA-Induced Ring Opening
Protected Exposed
3’
0 sec
676
678
680
682
684
Bimodal Isotopic
Pattern
upon Mixing with
RNA
At 1 hr
Add poly(C)
676
678
680
682
684
1.5 hr
676
678
680
682
684
682
684
2 hr
676
678
680
Affected Peptide
Resides in the
Hydrophobic
Core
P1
P4
Glycyl Transfer-RNA Synthetase
Hot Spots Opened in Five
Charcot-Marie-Tooth-causing Mutants
He, W.; Zhang, H.-M.; Chong, Y. E.; Guo, M.; Marshall, A. G.;
Yang, X.-L. PNAS U.S.A. 2011, 108, 12307-12312
Gastrointestinal Cancer
5000-10,000 new cases/year
in USA
GIST Alliance (www.gistalliance.com)
Median survival:
60 mo. with primary disease
19 months if metastatic
12 months if recurrence
Initial Treatment: Imatinib
If Resistant: Sunitinib
Domain
vs.
Intact Enzyme
Wild Type Receptor Tyrosine Kinase (KIT)
( Kinetic Insertion Domain)
10
20
30
40
50
60
70
80
....|....| ....|....| ....|....| ....|....| ....|....| ....|....| ....|....| ....|....|
GPTYKYLQKP MYEVQWKVVE EINGNNYVYI DPTQLPYDHK WEFPRNRLSF GKTLGAGAFG KVVEATAYGL IKSDAAMTVA
90
100
110
120
130
140
150
160
....|....| ....|....| ....|....| ....|....| ....|....| ....|....| ....|....| ....|....|
VKMLKPSAHL TEREALMSEL KVLSYLGNHM NIVNLLGACT IGGPTLVITE YCCYGDLLNF LRRKRDSFIC SKQEDHAEAA
170
180
190
200
210
220
230
240
....|....| ....|....| ....|....| ....|....| ....|....| ....|....| ....|....| ....|....|
LYKNLLHSKE SSCSDSTNEY MDMKPGVSYV VPTKADKRRS VRIGSYIERD VTPAIMEDDE LALDLEDLLS FSYQVAKGMA
250
260
270
280
290
300
310
320
....|....| ....|....| ....|....| ....|....| ....|....| ....|....| ....|....| ....|....|
FLASKNCIHR DLAARNILLT HGRITKICDF GLARDIKNDS NYVVKGNARL PVKWMAPESI FNCVYTFESD VWSYGIFLWE
330
340
350
360
370
380
390
400
....|....| ....|....| ....|....| ....|....| ....|....| ....|....| ....|....| ....|....|
LFSLGSSPYP GMPVDSKFYK MIKEGFRMLS PEHAPAEMYD IMKTCWDADP LKRPTFKQIV QLIEKQISES TNHIYSNLAN
410
420
430
435
....|....| ....|....| ....|....| ....|
CSPNRQKPVV DHSVRINSVG STASSSQPLL VHDDV
Wild-Type KIT without KID Domain
10
20
30
40
50
60
70
80
....|....| ....|....| ....|....| ....|....| ....|....| ....|....| ....|....| ....|....|
GPTYKYLQKP MYEVQWKVVE EINGNNYVYI DPTQLPYDHK WEFPRNRLSF GKTLGAGAFG KVVEATAYGL IKSDAAMTVA
90
100
110
120
130
140
150
160
....|....| ....|....| ....|....| ....|....| ....|....| ....|....| ....|....| ....|....|
VKMLKPSAHL TEREALMSEL KVLSYLGNHM NIVNLLGACT IGGPTLVITE YCCYGDLLNF LRRKRDSFIC SKTSPAIMED
170
180
190
200
210
220
230
240
....|....| ....|....| ....|....| ....|....| ....|....| ....|....| ....|....| ....|....|
DELALDLEDL LSFSYQVAKG MAFLASKNCI HRDLAARNIL LTHGRITKIC DFGLARDIKN DSNYVVKGNA RLPVKWMAPE
250
260
270
280
290
300
310
320
....|....| ....|....| ....|....| ....|....| ....|....| ....|....| ....|....| ....|....|
SIFNCVYTFE SDVWSYGIFL WELFSLGSSP YPGMPVDSKF YKMIKEGFRM LSPEHAPAEM YDIMKTCWDA DPLKRPTFKQ
330 335
....|....| ....|.
IVQLIEKQIS ESTNHI
Deuterium
Uptake
Deuterium
Uptake
7
7
542-548 in JM domain
6
558-568 in JM domain
6
5
5
4
4
3
3
2
2
WT with KID + Sunitinib
1
WT with KID + Sunitinib
WT w/o KID + Sunitinib
1
WT w/o KID + Sunitinib
0
0
7
7
624-636 in C-alpha
6
816-828 in A-loop
6
5
5
4
4
3
3
2
WT with KID + Sunitinib
2
WT with KID + Sunitinib
1
WT w/o KID + Sunitinib
1
WT w/o KID + Sunitinib
0
0
0
1
2
Time (h)
3
4
0
1
2
3
Gajiwala, K.S. et al. PNAS (2009) 106 (5), 1542-1547
4
WT
KIT
+/KID
Domain
after
230
hrmin
Exchange
WT
KIT
+/KID
Domain
after
Exchange
WT KIT +/- KID Domain after 8 min Exchange
KIT Fragments
WT KIT vs. D816H Mutant after 30 min H/D Exchange
KIT Fragments
Drug Design: Tuberculosis
Leucine is an essential amino acid for humans,
but plants and microorganisms make their own.
Rate-Limiting Step for Leucine Synthesis in\
Mycobacterium tuberculosis (Mt) is
α-isopropylmalate synthase (IPMS)
(Obvious Anti-Tuberculosis Drug Target)
Feedback Inhibition by the leucine.
Some IPMS mutants (e.g., Y410F) are resistant to leucine
inhibition—why?
X-ray studies of IPMS failed to identify structural difference
upon leucine binding and in the IPMS mutants
TB IPMS is a Homodimer
Each monomer consists of an N-terminal Catalytic domain, a
Linker Domain and a C-terminal Regulatory Domain
Peter Frantom, John Blanchard
Albert Einstein College of Medicine
Biochemistry 2009, 48, 7457-7464
Active Site Conformational Changes
on Binding of Leucine
Deuterium Uptake
5
Fragment 78-87 in the Active Site
4
3
WT IPMS
2
WT IPMS + Leu
Y410F IPMS
1
Y410F IPMS + Leu
0
0
1
2
3
4
WT Active Site Protected on Leucine Binding
Mutant Active Site Unaffected by Leucine BInding
Comparison of the Conformations of Y410F and WT MtIPMS
Fragment 406-416 in
the Linker Domain
5
4
IPMS
3
Y410F IPMS
Deuterium Uptake
2
1
0
0
2
4
6
8
Fragment 457-462 in
the Linker Domain
5
4
• Fragment in the linker I domain is
much more flexible in Y410F than
in the WT IPMS
3
2
IPMS
1
Y410F IPMS
0
0
2
4
Time (h)
6
8
• Y410F substitution uncouples the
allosteric network from the active
site, which causes the loss of
signal transduction
Top Ring (7*57 kDa)
AP
AP
I
EQ
I
EQ
I
EQ
AP
Bottom Ring
Electron Microscopy
GroEL
GroEL+ATP
Ranson, N. A.; Farr, G. W.; Roseman, A. M.;
Gowen, B.; Fenton, W. A.; Horwich, A. L.;
Saibil, H. R. Cell 2001, 107, 869-879.
GroES Cap (7*10 kDa)
Largest Protein Complex
(868 kDa!) Characterized by
H/D Exchange
AA 263-272 (Strongly affected by
binding of GroES)
GroEL cis ring (7*57 kDa)
Nature Scientific Rpts. 2013, 3, 1247
GroEL trans ring (7*57 kDa)
"Chaperone" Complex:
Folds Proteins to Correct Shape
AA 263-272 (Unaffected by binding
of GroES)
AA117-127
AA422-441
GroEL
GroEL-ATPγs
GroEL-ADPAlFx
GroEL
GroEL-ATPγs
GroEL-ADPAlFx
COPII Complex in Intracellular Trafficking
(Coatomer Protein Complex II)
Nature Reviews Microbiology 6, 363-374 (May 2008)
Coatamer Protein Complex II: 7.7 MDa Cage
Sec13: 34 kDa
Sec31: 127 kDa
+
Edge
Cage
Diameter: ~ 600 Å
Traffic 2010, 11 (3)
Sequence Coverage: Sec13 Alone, 96%
Sequence Coverage: Sec13 in “Edge”, 92%
Shared Fragments (81% Coverage)
Sec13
Sec31
Sec13/31 Edge
Deuterium Uptake Mapped onto Sec13
upon Sec13/31 Edge Formation
0
15
30
40
50
Averaged Relative Difference
Cockroach
m/Δm50% = 200,000 at m/z 400 @ 1 Scan/sec
Neuropeptides
14.5 T LC/FT-ICR MS
1183
1184
1185
*
1175
1250
1275
P. D. Verhaert
Delft U. Technology
*
400
800
m/z
1200
1600
Cockroach
Neuropeptides
(7 T)
...
P. D. Verhaert
Delft U. Technol.
*
*
.
*
*
*
.
.
*
.
*
.
*
*
*
1183
14.5 T
*
..
.
1185
1184
m/z
Bitumen
ESI 9.4 T FT-ICR MS
m/Δm50% = 300,000
Magnitude Mode
m/Δm50% = 119,242
C57H97O3S1
m/Δm50% = 400,000
m/Δm50% = 508,320
3.4 mDa
C60H93O3
Absorption Mode
C57H97O3S1
Anal. Chem. 2010, 82,
8807-8812
861.70
861.75
m/z
861.80
Cryocoolers,
JT fridge for zero loss
2 K Cryostat
Nb3Sn
coils
1026 mm
to field center
123 mm
+/- 5 ppm
100 mm by
100 mm cylinder
4.2 K Vessel
Fringe Field
50 Gauss at
1.6 m from
field center
NbTi
coils
Drift rate
<4 ppb/hr
2 K Vessel with Magnet coils
Bovine Serum Albumin
66,463 Da
21 Tesla
6 s Transient
100 Transients
S/N Ratio > 1,000
[M+48 H]48+
m/Dm50% = = 1,100,000
1384.5 1384.7 1384.9 1385.1 1385.3 1385.5 1385.7 1385.9
m/z
[M+48H]48+
Bovine Serum Albumin
66,433 Da
Single 12 s Transient
S/N Ratio > 500:1
1384
m
= 2,000,000
Dm50%
1385
m/z
1386
[M+48H]48+
Bovine Serum Albumin
66,433 Da
0.38 second
Detection Period
1384
m
= 150,000
Dm50%
1385
m/z
1386
HCD of Carbonic Anhydrase
21 Tesla
4+
16+
958
959
m/z
960
y618+
Carbonic Anhydrase
29,025 Da
100 0.76 s Transients
HCD of [M+36H]36+
y618+
y61-H2
b133-H2O17+
O8+
y537+
b13117+
878
b132
880
882
b13317+
17+
884
886
888
[M+36H]36+
600
700
800
900
m/z
1000
1100
1200
1300
c182+
Carbonic Anhydrase
29,025 Da
c10811+
100 0.76 s Transients
z778+
FETD of [M+34H]34+
z687+
c394+
c697+/z697+
z485+ z889+
c586+
z879+
z899+
z384+
c606+
z495+
c596+
1105
1110
1115
c808+
1120
1125
1130
1135
[M+34H]34+
600
700
800
900
1000 1100 1200 1300 1400 1500
m/z
Ac
Carbonic Anhydrase: FETD and CAD Combined
142/258 = 68% Sequence Coverage
c
b
z
y
Triple
Frequency
Detection!
RF+
+
+
+
-
-
+
+
-
-+
RF-
Mass Spectral Peaks
Peak Ht Ratio = 1:1
Need m/Δm50% = 340,000
580.480
580.500
m/z
580.520
Mass Spectral Peaks
Peak Ht Ratio = 1:1
Need m/Δm50% = 340,000
Peak Ht Ratio = 100:1
Need 10x Higher Resolving Power
m/Δm50% = 3,350,000
580.480
580.500
m/z
580.520
m
 SNR  √ #Pts/Width
s(m)
Dm50%
Noise
m
105,817 Peaks > 6σ
500 < m/z < 2000
De-Asphalted Crude Oil
Positive Ion
Electrospray
9.4 Tesla
Fourier Transform
Ion Cyclotron
Resonance
Mass Spectrum
500
750
1000
1250
m/z
1500
1750
2000
The best reason for higher magnetic field:
Experiments that can be performed
only with heroic difficulty at low field
become routine at high field.
Example: Petroleomics, Proteomics