6
Analysis of Intact Macromolecular Assemblies On A Bench Top Orbitrap MS System
For small proteins up to 35 k
achieved for determination of
Larger proteins show clear se
and conjugates. Acquisition ti
rang of seconds.
Sample introduction with the
proved to be easy to handle,
stable spray conditions for be
with minimum time consumpti
data acquisition for maximum
Acknowledgemen
We would like to thank Profes
from the University of Uthrec
supplying samples of E. coli
etermination of
carried out using
.
Advion, TriVersa and NanoMate are trademarks of Advion I
of Bio-Rad Laboratories, Inc, Hercules, CA, USA. All other t
its subsidiaries.
This information is not intended to encourage use of these
property rights of others.
Presented at HUPO 2013
D Gas Control via
trol SW
Applying
in-source
dissociation
energy
xactive Plus EMR
Pyruvate Kinase
In native state Pyruvate kinase is a tetrameric protein
assembly of intermediate size. The full protein assembly
appeared as a strong signal in the spectrum and due to full
desolvation of the molecules in the mass spectrometer the
full pattern of isoform combinations is visible (see fig. 4).
Upon slight application for fragmentation energy the
monomeric subunits with their isoform pattern are visible
together with the full assembly. The mass difference of 324
amu is clearly visible in the deconvoluted spectra of the
subunit as well as the multitude of isoform combinations for
the tetrameric complex.
y mass
s even these show
rbonic anhydrase
f proteins, is
king a classical
pattern difficult.
p detection
isotopically
GroEL
The chaperone protein GroEL from E. coli belongs to the
group large proteins with a mass of roughly 800 kDa of the
fulllyactive 14-mer complex. With proteins of this size, the
resolving power of the MS instrumentation becomes a
minor part for successful resolution of isoforms and
conjugates, but desolvation capabilities are the limiting
factor. With GroEL we could achieve full desolvation
resulting in sharp baseline separation of the different
charge states (see fig. 5).
s On A Bench Top Orbitrap MS
karov
1
, Maciej Bromirski
1
ydrase in native
hieved.
3500
3600
3700
3800
3900
3636.9632
3674.8277
3581.9352
3850.5058
3514.7714
3722.6411
V:
18
NL:
9.56E4
100.00 [500.00-20000.00]
2909.4
2909.6
2909.8
2910.0
2910.2
2910.4
2910.6
2910.8
2911.0
m/z
2909.7735
2909.6750
2909.8761
2909.5724
2910.0743
2909.4731
2909.3744
2910.1769
2910.2755
2716
2910.3762
2910.5759
2910.6776
2910.8742
2910.9758
FIGURE 4. Experimental and deconvoluted spectra of
Pyruvate Kinase showing the resolution of the
isoforms with bound 2,5-anhydro-D-glucitol 1,6-
bisphosphate (GBP)
PK-qb
#
1
RT:
3.76
AV:
1
NL:
2.00E6
T:
FTMS+ pNSI sid=100.00 Fullms [350.00-15000.00]
2000
2500
3000
3500
4000
4500
5000
5500
6000
6500
7000
7500
8000
m/z
0
20
40
60
80
100
RelativeAbundance
7077.2434
7298.7102
6868.7773
2534.2444
7534.6141
2649.3563
6672.1657
7797.1159
2331.6150
6005.5679
5620.3099
3237.8960
3907.0561
4911.2229
4483.0348
GBP
GBP
GBP
GBP
GBP
GBP
GBP
GBP
GBP
GBP GBP
monomer
tetramer
deconvoluted spectrum
deconvoluted spectrum
tetramer
PK-qb
#
1
RT:
3.76
AV:
1
NL:
2.00E6
T:
FTMS+ pNSI sid=100.00 Fullms [350.00-15000.00]
6800
6900
7000
7100
7200
7300
7400
m/z
0
20
40
60
80
100
RelativeAbundance
7077.2434
7298.7102
7106.9044
6868.7773
7339.7162
6907.3192
7037.9373
7258.2765
7208.9077
GBP
PK-qb
#
1
RT:
3.76
AV:
1
NL:
3.99E5
T:
FTMS+ pNSI sid=100.00 Fullms [350.00-15000.00]
2000
2200
2400
2600
2800
3000
m/z
0
20
40
60
80
100
RelativeAbundance
2534.2444
2428.6827
2649.3563
2331.6150
2242.0009
2775.4958 2914.3670
2081.5859
monomer
GBP
