2
Analysis of Intact Macromolecular Assemblies On A Bench Top Orbitrap MS System
Overview
Purpose:
Measure proteins in native like state to study
the biologically active conformations.
Methods:
Protein samples were measured using a
Thermo Scientific™ Exactive Plus EMR™ mass
spectrometer with chip based static nanospray using an
Advion™ TriVersa™ Nanomate™.
Results:
It could be shown that for proteins of various
molecular weight clear separation of isotope resp. isoform
signals could be achieved due to full desolvation even
under native conditions.
Introduction
Intact proteins are routinely measured using ESI-MS
instrumentation under acidic, denaturing conditions,
destroying large, non covalent protein assemblies and
substrate bound complexes. Under native conditions, fully
active protein assemblies can be studied, but these
experiments are challenging due to the limited surface
area of protein complexes for protonation at physiological
pH. Ion signals are shifted to higher
m/z
values, which
until recently, only TOF instruments were capable of
detecting. TOF measurements have limited achievable
resolution, making it difficult to resolve specific isoforms or
substrate complexes. With an Exactive Plus EMR bench
top Orbitrap instrument we were able to detect ion signals
up to
m/z
20,000 with high mass resolving power thus
achieving remarkable signal distribution and precision for
various large protein assemblies.
Results
Carbonic Anhydrase
Smaller proteins can easily
spectrometry, but under nati
quite different behavior. The
for example, as member of t
reduced to two major charge
deconvolution based on char
Due to the high resolution of
system, this 29 kDa protein
resolved (see fig. 2).
Herceptin
Herceptin is a therapeutic an
The determination of the gly
for characterization and quali
could achieve a clear baselin
FIGURE 2. Spectrum of car
state. Isotopic resolution i
carbonic_140k_inf_003
#
18-26
RT:
0.54-0.78
AV:
9
NL:
9.09E4
T:
FTMS+pNSIsid=1.00 Fullms21000.00@hcd100.00 [500.00-20000.00]
2600
2700
2800
2900
3000
3100
320
0
5
10
15
20
25
30
35
40
45
50
55
60
65
70
75
80
85
90
95
100
RelativeAbundance
2909.7758
32
2931.3498
2739.4557
3074.6448
2623.7380
2883.1644
Here the +9 charge state de
29069.6107 Da. The active p
molecule has a theoretical m
a mass accuracy of 0.25 pp
