Bruker Daltonics
Application Note # LCMS-52
Top-Down Proteomics with ETD/PTR:
Essential Data Quality Improvement by Increased
Resolution and Scan Speed of the amaZon Ion Trap
Abstract
Complete protein characterization becomes a major
challenge in both general proteomics as well as quality
control of recombinant proteins. ETD/PTR is a valid
method to generate sequence information in top-down
experiments. The high sensitivity, the perfect combination
of scan speed and resolution as well as robust and fast
ETD/PTR make the amaZonTM the instrument of choice for
those applications.
Introduction
The direct analysis of intact proteins gains highly
increased interest because of the possibility of a
complete protein characterization including termini
and modifications – in particular in combination with
bottom-up approaches. As well, protein systems like
histones where modifications on highly conserved amino
acid sequences decide about biological functions are
analyzed at best on the intact species. Collision induced
dissociation (CID) has drawbacks in terms of a limited
applicable molecular weight and the dependence of
the fragmentation efficiency on the individual bond
strength and amino acid sequence particularly for the
fragmentation of highly charged precursor ions. Electron
transfer dissociation (ETD) and its related proton transfer
reaction (PTR) are alternative ways of fragmenting peptides
and proteins. ETD became the preferred method for
the analysis of post-translational modifications (PTM) in
proteins since it preserves the bonds of modifications. PTR
is a most useful addition for the fragmentation of larger
peptides or even small proteins in the top-down approach.
Intact proteins can be identified and/or sequenced without
any prior enzymatic digestion. While ETD is still used for
the dissociation itself, the charge stripping with PTR anions
lowers the charge states of the highly charged product ions
towards a higher m/z. The resulting PTR-product is easier
to resolve and much more suitable for the detection with
an ion trap. However, this limitation is the main reason for
the sequence read-out in ion traps so far, because a highly
charged ETD fragment ion can be shifted out of the m/z
range of the mass detector when to many PTR-steps are
involved. Therefore, to increase sequence coverage and
reachable fragment mass range, a high resolution is in
general required to observe “less stripped” higher fragment
charge states. This should be achieved, however, at no cost
of time, i.e. at a still highest possible scan speed.
The amaZon ion trap is equipped with the latest ETD/PTR
setup. Its improved electronics and trap design allows
a truly high resolution at a scan speed of still 4,600 u/s.
Presented here are applications of this new design on the
MS/MS analysis of various intact proteins.
Mass resolution even of intact proteins
Fig. 1: Spectrum extension with the 3+
charge state of intact Ubiquitin (MW 8565
Da). The mass resolution here is more than
20,000, allowing to observe the full isotope
pattern of this protein.
ETD/PTR fragmentation
Intens.
Intens.
x10
x10
5
5
x10
5+
'1713.23
5+
'1713.23
Intens.
5+
'1713.23
5
4+
'2141.27
1.5
4+
'2141.27
1.5
1.5
1.0
0.5
1+
273.12
1+
273.12
1.0
1+ 0.5
390.21
1+
537.33
0.0
Intens.
x104
8
500
6
2+ 6+
718.00
'1427.87
2+
960.64 3+
1+
'1151.37
537.33
2+
1+
2+
960.64
3+
390.21
960.64 3+
1+
'1151.37
'1151.37
537.33
1+
390.21
0.5
500
1000
Intens.
4
6+
x10
0.0 8
1000
1500
500 1427.88
1000
Intens.
Intens.
6
4
6+
x10
6+ 4
x10
8
4
1427.88
1427.88
3
6
2
0
4
4
x10 8
2
4
6+
'1427.87
3+
'2854.82
4+
'1857.60
4+
'1857.60
3+
3+
'2854.82
3+
'2854.82
'2476.47
4+
'1857.60
2000 3+
1500
2500
m/z
3+
Intens.
'2476.47
'2476.47
4+
4
5+
x10
1661.98
3+
2000
25001658.41
m/z
1500
2000
2500
m/z
3 Intens.
1655.34
4+
4+
4
5+
5+
1661.98
3+
1658.41
1661.98
3+ 2 x10 1658.41
1655.34
1
2
0
1
3
Fitted isotopic pattern
2
6
0
4
4x10 8
0
4
x10 8
3
1655.34
2
1
Fitted isotopic pattern
0
Fitted isotopic
0 pattern 2
Fitted isotopic pattern
Fitted isotopic pattern
Fitted3 isotopic pattern
6
4
2
6
0
4
1426
2
2
0
6+
'1427.87
2+
1+
718.00273.12
1.0
0.0
2+
718.00
4+
'2141.27
0
1426
1427
1428
3
1427
1426
1429
1428
1427
1429
1428
1430 m/z
1
2
0 1654
1
1430
2 m/z
1
1429
1430 m/z
0 1654
1656
1656
0 1654
1658
1658
1656
1660
1660
1658
1662
1662
1660
1664
1664
1662
m/z
m/z
1664
m/z
Fig. 2: ETD/PTR fragment spectrum of Ubiquitin. Up to 6+ charge states can be resolved in the complete MS/MS spectrum.
Experimental
Results
ETD/PTR-MS/MS was performed on the amaZon ETD ion
trap. The highly improved control of the non-linear ejection
process and the further developments in the trap enable a
fast scan mode with 4,600 u/s speed at peak width of
< 0.1u for fragments. Performing ETD and PTR is particularily easy in the amaZon setup since only a single reservoir
filled with the neutral ETD/PTR-reagent compound is
needed. Switching between ETD and PTR is just a matter
of nCI lens parameters and can be done within a few msec.
The high mass resolution of the amaZon allows for a
mass resolution even of intact proteins. Figure 1 shows
a spectrum extension with the 3+ charge state of intact
Ubiquitin (MW 8565 Da). The mass resolution here is more
than 20,000 FWHM, allowing to observe the full isotope
pattern of this protein. The scan speed of 4,600 u/s enables
the spectra acquisition in a seamless scan across the entire
m/z range of 50 – 3,000 in a reasonable time. Using ETD/
PTR to fragment Ubiquitin results in data presented in figure
2. Up to 6+ charge states can be resolved in the complete
MS/MS spectrum. The Bruker patented peak recognition
software SNAP IITM picks the peaks within the isotopic
patterns and determines the monoisotopic fragment masses
with high confidence. The subsequent database search
Zwith MASCOT returns a complete sequence coverage of
the intact protein from the full mass of 8,565 Da down to diand tri-peptide fragments (Fig. 3).
The purified proteins were injected into the ion trap by
offline nanospray (Triversa Nanomate). ETD/PTR of the
isolated protein is performed with reagent anions dedicated
for either ETD or PTR. The formation of the required
different reagent anions is accomplished from only one
neutral compound by altering the voltage settings of the
negative chemical ionization source. For the protein marker
identification and sequence characterization, the tissue
lysate was ultra-sonificated in an ice bath. The extract was
centrifuged with a Vivaspin 5000 ultra-filtration unit. The
supernatant was collected and separated with an Agilent
mRP protein column into a 96-well plate.
Fractions were measured with MALDI-TOF MS for
peak localization, and the respective protein fractions
subsequently measured with the amaZon ETD coupled to a
TriVersa Nanomate.
Figure 4 shows the same for an even higher mass protein,
Myoglobin. Here, 200 fmol/uL were injected by the Triversa
nanomate at a flow rate of ca. 50 nL/min. The MASCOT
result show the sequence assignment up to ca. 11,000 Da
MW with an remarkable average mass accuracy for an ion
trap of 23 ppm.
Complete sequence coverage of intact protein
Fig. 3: Result of the database search by MASCOT revealing the complete sequence of Ubiquitin.
Complete sequence coverage with remarkable mass accuracy
Intens .
Intens .
1000
Fitted
isotopic
pattern
500
2000
3+
c63 4+z* 52c40 2+z* 26
5+
1432.95
1436.99 1439.39
0
1442.76
Fitted
isotopic
pattern
800
600
400
m/z
200
1000
1500
1428
1432
2000
1436
1440
1444
2327.50 4+
2264.17 2+
2225.82 3+
2163.97 5+
2092.58 2+
2054.71 5+
1046.79 4+
'985.53 3+
1584.32 4+
1910.486 4+
1878.245 4+
0
2421.09 5+
986.0
1142.11 2+
984.0
1509.77 3+
1092.17 5+
1395.37 3+
1364.94 4+
1335.88 5+
391.28 1+
865.70 4+
447.22 1+
c39
2500
m/z
1957.83 5+
3000
0
3+
1429.06
500
0
1669.57 4+
1000
0
1500
1000
527.29 2+
2000
2000
500
371.30 1+
275.15 1+
804.44 3+
924.82 3+
3000
562.80 2+
a)
4000
z* 27
1000
762.08 3+
712.39 2+
654.88 2+
615.83 2+
5000
c44
2568.21 4+
1500
3+
5+
985.53
984.32
1637.583 4+
2825.08 6+
2000
1790.65 4+
1726.61 4+
Intens.
m/z
b)
Fig 4.: ETD/PTR of intact
Myoglobin (200 fmol/µl);
m/z= 653 [M+26H]26+
a) ETD MS/MS -> PTR; data
processing with SNAP IITM.
c)
RMS error 23.35 ppm
b) BioTools annotation of TD
Mascot search result (score =
393; Table 1) of the processed
spectrum.
c) RMS mass error 23 ppm from
the Mascot data base search
(Fig. 1d).
Table 1 shows typical MASCOT scores for ETD/PTR on
highly charged protein species with the amaZon. Even with
an isolated charge state of 26+ for Myoglobin, ETD and
effective PTR in combination with the high mass resolution
of the amaZon generate a Top-Down MASCOT score of
nearly 400 for the protein identification.
An excellent sequence coverage is obtained for both the
C- and N-terminus. The confirmed sequence range
comprises 30 resp. 25 amino acids. Disulfide bridges are
not cleaved in ETD, therefore no fragments are obtained
beyond the cysteines. This fact can be used to localize the
crosslink, here between C31 and C141.
The top-down analysis provides in many cases
complementary information to bottom-up fragmentation.
One example shown here is the sequence analysis
of recombinant interferon beta (Fig. 5). Bottom-up
fragmentation could confirm large parts of the core
sequence, but failed to provide information on both termini.
In order to confirm the termini the intact protein was
analysed by ETD-PTR and the generated spectrum was
matched against the theoretical sequence in BioToolsTM.
Another interesting example for the usefulness of ETD/
PTR for top-down sequencing is the histone system whose
biological function strongly depends on the attached
modification. Histons are highly basic lysine and argininerich DNA-binding proteins. Conventional bottom-up
proteomic analysis including CID MS/MS and trypsin
hydrolysis is less promising for a complete characterization
of histones including their multiple modifications. Figure
6 shows part of the processed ETD/PTR MS/MS data
of a human histon ([M+16H]16+; m/z 709) after charge
deconvolution. Biotools reveals two sequence series: one
including H18 being phosphorylated, the other one nonphosphorylated – indicating ca. 50% phosphorylation on
H18.
MALDI Imaging is a rather new technology where
molecular information is retrieved directly from tissue
sections by matrix-assisted laser desorption ionization
(MALDI) time-of-flight (TOF) mass spectrometry. It allows
histology researchers to measure spatially resolved peptide,
protein and lipid profiles in tissue sections. Tissue-type
specific molecular signatures (e.g. from tumors) can be
generated and used for biomarker discovery and molecular
histology. After potential biomarker candidates are detected
on the tissue, they need to be identified. Shown in figure 7
is a potential protein marker discovered by a MALDI-TOF
MS Imaging experiment of breast cancer tissue (Fig 3a).
The tissue was then extracted and purified. ETD/PTR of the
isolated intact multiply charged protein lead to successful
biomarker identification with a Mascot Score of 126.
Table 1: ETD/PTR MS/MS applying the new maximum resolution scan modedata processing with SNAP IITM
Protein
MWmono
m/z (precursor)
charge state
Mascot Score
Ubiquitin
8560
714
12
375
RNAse A*
13682
978
14
194
Lysozym C*
14303
842
17 207 Myoglobin**
16941
653
26
393 *treatment with DTT for the reductively opening of the cystein SS-bridges
**data base search with Mascot TD, surpass Mascot´s precursor mass limit > 16kDA
Confirmation of intact protein termini
c
c
c
Fig. 5: ETD/PTR fragment spectrum of Interferon beta with complete sequence information on both termini.
ETD/PTR indicates partial phosphorylation of histone
Fig. 6: Part of the ETD/PTR spectrum of Histone pH4. Sequence coverage with either phosphorylated (top, significant fragments marked
with red stars) or non-phosphorylated H18 (bottom, fragments marked with green stars). Both fragment ion series are present, indicating ca.
50% phosphorylation.
Conclusion
The amaZon´s maximum resolution scan mode (4,600 u/
sec) allows the seamless and fast detection of a plethora of
multiply charged ETD-fragments up to 6+. Data processing
with SNAP IITM generates a list of monoisotopic ETDfragment masses, enabling the ID of the intact protein
by a Mascot data base search. This enables a complete
characterization of proteins up to MW of 20 kDa including
post-translational modifications. ETD/PTR with the amaZon
ion trap reaches new levels for Top-Down proteomics and
is ready to become a powerful tool for characterization of
intact protein biomarkers.
Ion trap analyzer.
Fig. 7 (following page):
a)MALDI Imaging results of a breast cancer tissue; HER2
positive/negative cells
b)workflow for sample processing
i) Lysate of entire tissue cells, ii) LC-separation and fractionation, iii) biomarker identification, iv) sequence characterization
with ETD MS/MS
c)ETD/PTR spectrum of an intact protein from a cell lysate (top);
maximum resolutionscan mode (4,800 u/sec, resolution up to 5+ ions)
data processing with SNAP IITM
d)BioTools annotation of Mascot search result of the processed spectrum
Top-Down ID and Sequence characterization of an intact biomarker protein
a)
HER 2 negative
HER2 positive
4000
6000
8000
10000
b)
MALDI TOF MS Autoflex
to identify fractions
including biomarker
Cap LC
i)
Tissue lysis
(0.1% TFA)
ultrasonic
centrifugation
dilution
ii) LC separation
iii)
Agilent
mRP column
5um 0,5x100mm,
12 uL/min
Fractionating
96 well plate
4,0 ul/vial (2x)
iv)
ETD/PTR
of 3 combined fractions
offline nanospray
amaZon ETD
Maximum Resolution
Intens.
c)
ETD/PTR
3000
2000
1000
0
250
d)
500
750
1000
1250
1500
1750
2000
2250
2500
m/z
Authors
Andrea, Schneider, Christian Albers, Andreas Brekenfeld,
Christoph Gebhardt, Eckhard Schwabe, Ralf Hartmer, Arnd
Ingendoh; Bruker Daltonik, Bremen, Germany
Keywords
Instrumentation & Software
ETD
amaZon series
PTR
intact protein
PTM
high resolution
high scan speed
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