[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