[application note]
T O P- DOW N S EQU EN C ING US ING S YNA P T H IG H D E F IN IT IO N MA S S S P EC T ROM E T RY (H DMS)
Thérèse McKenna, Christopher J Hughes, and James I Langridge
INT RODUCT ION
Top-down proteomics can be described as the identification and
characterization of intact proteins from tandem mass spectrometry
experiments, enabling the identification of Post Translational
Modifications (PTMs). The top-down approach provides direct
measurement of the intact mass of the protein, as well as fragment
ion information relating to the amino acid sequence. This crucial
information may be lost in a “bottom-up” proteomics strategy, for
example, when a tryptic peptide on which a PTM resides does not
ionize well. However, even with a small to medium-sized protein,
the large number of ions and the range of charge states produced
during fragmentation can lead to a spectrum that can be extremely
complex, challenging to interpret , and, therefore, time-consuming.
The Waters® Syanpt™ High Definition Mass Spectrometry™
(HDMS™) System, which couples high-efficiency ion mobility
separations (IMS) with time-of-flight (TOF) mass analysis, provides
an additional dimension of separation aiding the interpretation of
these complex, multiply-charged datasets.
In this application note, we separate protein mixtures by nano-scale
liquid chromatography using UltraPerformance LC® (UPLC®)
technology, and collect fractions for analysis. For each protein
fraction, we select and fragment different charge states using the
HDMS mode of analysis to separate these fragments by their differ-
ing mobility, due to their size, shape, and charge. Post-processing
of these data produces simplified top-down fragment ion spectra,
containing ions of mainly one charge state.
This ion mobility approach reveals low abundance fragments that
would otherwise be masked by more intense species in a conven-
tional MS/MS spectrum, and leads to enhanced sequence coverage
of the protein studied. Here we present data using ubiquitin, a highly
conserved small regulatory protein, separated from a protein
mixture, to demonstrate the advantages of coupling ion mobility-
based separation with mass analysis.
EX PERIMENTAL
Samples
The protein mixture contained 3 pmol/µL each of ubiquitin ribonu-
clease, cytochrome C, and beta lactoglobulin.
LC, fraction collection, and infusion
Intact protein mixtures were separated using reversed-phase liquid
chromatography. The gradient used was 20 to 40% of 0.1% formic
acid in acetonitrile (solvent B) in 10 minutes, then ramped to 95%
at 12 minutes. Solvent A was aqueous 0.1% formic acid. One-
minute wide fractions were collected.
The column eluent was split inside the Triversa Nanomate (Advion
BioSciences) between the LC coupler and fraction collection into
a 384-well plate. 5 µL acetonitrile was added to each protein-
containing well and a sample volume of 1 µL was aspirated. The
infused flow rate was 20 to 60 nL/min.
Mass spectrometer
The Synapt HDMS System with Triwave™ technology, Figure 1, was
used in these studies. Ions produced during electrospray ionization
are sampled by a ZSpray™ source and pass through a quadrupole,
which may be set to transmit a wide mass range, or to isolate ions
Figure 1. Schematic of the Synapt HDMS System, which incorporates Triwave technology.
[application note]
of a specific m/z. High-efficiency ion-mobility separation is then
performed in the Triwave device.
The Triwave consists of three travelling wave (T-Wave™) ion guides.1
The first, the Trap T-Wave, traps and accumulates ions, in this case
for up to 51 msec, then these stored ions are gated into the IMS
T-Wave in which the ion mobility separation occurs.
The Transfer T-Wave is then used to transport the separated ions
into the orthoganol acceleration time-of-flight (oa-TOF) mass
spectrometer for subsequent mass analysis. The pressure in the Trap
and Transfer T-Wave regions was about 10-2 mbar of Argon and
the pressure in the IMS T-Wave was 0.5 mbar of Nitrogen. Ions may
be fragmented on entrance to the Trap T-Wave and/or the Transfer
T-Wave. The IMS T-Wave pulse velocity and voltage may be tuned to
provide the optimal ion mobility separation.
RESULTS
The five proteins were resolved chromatographically over the
15 minute separation.
By combining across the individual chromatographic peaks, TOF-
mass spectra can be obtained for each of the intact protein species,
as shown for ubiquitin in Figure 2. The multiply-charged envelope
for each protein covers a wide m/z range and, using the different
m/z values from the series, an accurate molecular weight of the
protein can be calculated.
For ubiquitin, the calculated MW was 8565 Da. Conventional TOF
MS/MS data was acquired by isolating a particular charge state of
each protein in the quadrupole and fragmenting that species in the
Trap T-Wave. The MS/MS spectrum obtained following the isolation
and fragmentation of the 9+ ion, m/z 943 of ubiquitin is shown in
Figure 2. Upper panel shows typical TOF-MS data from LC separated ubiquitin, showing the multiply charged ion series from which the molecular weight is calculated. The lower panel shows a conventional TOF-MS/MS spectrum obtained by fragmenting the 9+ charge ion of ubiquitin, which includes ions of many different charge states.
m/z500 600 700 800 900 1000 1100 1200 1300 1400 1500 1600 1700
%
0
100 779.57
714.68
659.79
602.72
857.42
817.38
952.58
903.10
1071.54
1017.68
1224.46
1428.55
19-Apr-2007 14:59:02UAA031B3 after 5uL acetonitrile MSMS 953
m/z100 200 300 400 500 600 700 800 900 1000 1100 1200 1300 1400 1500 1600 1700 1800 1900 2000 2100 2200 2300
%
0
100
190407_034 85 (2.990) Sm (SG, 2x6.00); Cm (15:87) TOF MS ES+ 256
86.10
1141.96790.05104.06
260.13129.12
726.00
373.23
300.15
661.47
401.21
520.31
512.26
501.35
443.30
619.40
521.32
1141.71790.54
854.08
854.59
913.77
913.971137.72
914.17 1117.70
1064.68
1142.22
1142.47
1142.72
1184.75
1216.77
1217.27
1248.80
1372.83
1373.34
1414.94
Mass Spectrum of Ubiquitin
Deconvoluted Mass 8565Da
Fragment ion (MS/MS) spectrum of
953 (9+) ion from Ubiquitin
[application note]
the lower panel of Figure 2. It can clearly be seen that fragmenta-
tion of the highly-charged precursor ion results in a complex
MS/MS spectrum, containing fragments with a variety of different
charge states. This can be challenging to interpret, due to the
overlapping fragments of different charge states.
The Synapt HDMS System provides the ability to reduce spectral
complexity, in this case based upon separating fragment ions by
their charge state. Ion mobility separations were performed whereby
the MS/MS fragments were separated in the IMS T-Wave, producing
data that is more easily interpreted and related to the protein
sequence. Three precursor ions per protein were fragmented in
the experiments and the fragmentation spectra were then used to
generate sequence information.
Data Processing
DriftScope™ Software, used to view HDMS data, displays the drift
time vs. the m/z for each ion present in the data. Regions can be
selected to produce new data files containing simplified spectra,
mainly containing just one charge state. Maximum entropy algo-
rithms are then used to deconvolute the spectra and fragment ion
stretches, which can then be interpreted to determine the amino acid
sequence of the protein.
The data presented in Figure 3 shows the DriftScope plot obtained
from the Trap T-Wave fragmentation of the 9+ precursor ion from
ubiquitin. The bands of color represent the intensity of the ions, with
the boxed areas showing the charge state separation. The distinct
separation of the singly-charged ions is typical for this type of
analysis. DriftScope Software enables the user to select plot regions
of interest to interrogate corresponding mass spectra. The different
bands are found to correspond to different charge states or classes
of ions. Selecting the entire plot produces data equivalent to that
acquired without ion mobility separation, and hence allows a com-
parison of data produced by conventional MS/MS with ion mobility
separated MS/MS (HDMS).
A comparison is shown in Figure 4 from the fragmentation of the
m/z 952.5 ion from ubiquitin. Selection of the quadruply charged
(4+) region from the analysis in HDMS mode produces an increase
in the signal-to-noise level of the spectrum when compared to
the conventional MS/MS data. This allows clear assignment of
amino acid sequence to the fragment ions, and provides increased
sequence coverage.
Figure 3. Drift time plots vs. m/z for the Trap T-wave fragmentation of one of the multiply charged precursor ions from ubiquitin, showing separation into distinct charge state bands.
[application note]
Figure 5 shows data selected from the singly-charged region of the
HDMS data. It is interesting to note that the singly-charged b11 ion
observed in the HDMS mode is masked by the more intense y444+
ion in the conventional TOF-MS/MS data. In all cases, examination
of the ion mobility separated fragments reveals information that
was not observed, or was hidden by more intense species in the
conventional TOF-MS/MS acquisition.
Figure 4. Improved signal-to-noise level observed with HDMS mode for the quadruply charged y’’ series fragment ions from ubiquitin.
Figure 5. Selection of the 1+ region reveals the b11 ion, lower trace, which is masked by the more intense y44
4+ ion in the conventional TOF-MS/MS data shown in the upper trace.
Sequence coverage for proteins of different molecular weight
The sequence information (b and y’’ ions) obtained from the
four different proteins analysed in TOF-MS and HDMS mode was
investigated. This information is summarized in Figure 6, where
the percentage increase in sequence coverage for the HDMS data is
plotted for each of the proteins. Sequence coverage was increased in
all experiments where ion mobility was employed as an additional
separation dimension.
Figure 6. Percentage increase in b and y ions when HDMS data is compared with conventional TOF-MS/MS data.
19-Apr-2007 14:59:02UAA031B3 after 5uL acetonitrile MSMS 953
m/z1280 1300 1320 1340 1360 1380 1400 1420 1440 1460 1480 1500 1520 1540 1560
%
0
100
%
0
100 1372.83
1287.81
1286.90
1309.81
1309.56
1287.97
1303.93
1296.87
1310.07
1372.31
1337.861310.32
1324.89
1323.821337.59
1351.89
1338.87 1352.89
1355.87
1373.34
1414.94
1373.84 1387.88
1387.39 1388.13
1407.891388.39
1415.961433.98
1419.92
1432.98
1453.98
1434.99
1437.93
1522.951454.94
1478.911462.01
1463.95
1516.94
1505.931492.91
1539.97
1523.99
1546.08
1571.971547.09
1387.88
1387.391309.81
1309.57
1309.33
1305.30
1305.05
1277.561304.79
1301.311278.38
1369.86
1337.831310.07
1337.61
1333.321310.57
1369.62
1338.33
1338.601369.37
1338.871365.64
1340.86
1370.84
1383.33
1419.911388.13
1419.66
1388.39
1419.39
1388.67
1415.401388.87
1415.19
1400.20
1420.16
1420.39
1473.201420.87
1444.511422.12 1469.19
1505.191501.191473.69
1474.43
1505.941533.66
1507.96 1559.271534.46 1573.04
Conventional TofMS/MS data
y46
y47y48
y49 y50
y51y52 y53
y54
4+ region from HDMS data
19-Apr-2007 14:59:02UAA031B3 after 5uL acetonitrile MSMS 953
m/z1280 1300 1320 1340 1360 1380 1400 1420 1440 1460 1480 1500 1520 1540 1560
%
0
100
%
0
100 1372.83
1287.81
1286.90
1309.81
1309.56
1287.97
1303.93
1296.87
1310.07
1372.31
1337.861310.32
1324.89
1323.821337.59
1351.89
1338.87 1352.89
1355.87
1373.34
1414.94
1373.84 1387.88
1387.39 1388.13
1407.891388.39
1415.961433.98
1419.92
1432.98
1453.98
1434.99
1437.93
1522.951454.94
1478.911462.01
1463.95
1516.94
1505.931492.91
1539.97
1523.99
1546.08
1571.971547.09
1387.88
1387.391309.81
1309.57
1309.33
1305.30
1305.05
1277.561304.79
1301.311278.38
1369.86
1337.831310.07
1337.61
1333.321310.57
1369.62
1338.33
1338.601369.37
1338.871365.64
1340.86
1370.84
1383.33
1419.911388.13
1419.66
1388.39
1419.39
1388.67
1415.401388.87
1415.19
1400.20
1420.16
1420.39
1473.201420.87
1444.511422.12 1469.19
1505.191501.191473.69
1474.43
1505.941533.66
1507.96 1559.271534.46 1573.04
Conventional TofMS/MS data
y46
y47y48
y49 y50
y51y52 y53
y54
4+ region from HDMS data
m/z1232 1234 1236 1238 1240 1242 1244 1246 1248 1250 1252 1254 1256
%
0
100
%
0
100 1248.74
1247.741244.45
1243.761233.681231.81 1235.70 1237.75 1240.82 1254.711252.74
1247.82
1234.851232.83
1233.801240.81
1236.88 1242.78
1249.82
1255.831250.87
Conventional TofMS/MS data
1+region from HDMSdata
Y444+
b11
m/z1232 1234 1236 1238 1240 1242 1244 1246 1248 1250 1252 1254 1256
%
0
100
%
0
100 1248.74
1247.741244.45
1243.761233.681231.81 1235.70 1237.75 1240.82 1254.711252.74
1247.82
1234.851232.83
1233.801240.81
1236.88 1242.78
1249.82
1255.831250.87
Conventional TofMS/MS data
1+region from HDMSdata
Y444+
b11
[application note]
Waters Corporation 34 Maple Street Milford, MA 01757 U.S.A. T: 1 508 478 2000 F: 1 508 872 1990 www.waters.com
Summary
• Forthesmalltomediumsizedproteinsanalyzed,theHDMS
data produces an increase in sequence coverage resulting
in more complete protein characterization compared to the
conventional TOF-MS/MS experiment.
• SimplifiedmassspectraaregeneratedintheHDMSanalysis
where fragment ions, produced from highly charged precursor
ions in the TRAP T-Wave, are separated in the IMS T-Wave.
• Lowintensityspecieswhicharenotobservedinaconventional
TOF-MS/MS analysis could be identified and characterized.
The travelling wave device described here is similar to that
described by Kirchner in US Patent 5,206,506 (1993).
Waters and UPLC are registered trademarks of Waters Corporation. The Science of What’s Possible, Synapt, High Definition Mass Spectrometry, HDMS, Triwave, T-Wave, ZSpray, and Driftscope are trademarks of Waters Corporation. All other trademarks are property of their respective owners.
©2007 Waters Corporation Printed in the USA September 2007 720002342EN AG-PDF