Applications on the Analysis of Intact Proteins

Agilent 6510 Q-TOF

Christine Miller
Application Scientist
August 14, 2006

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Experimental Parameters

• LC Parameters:
– Poroshell C3 1.0 x 75 mm column at 80°C
– Flow rate 0.15 ml/min
– 0.1% formic acid in water (A) or in 90% acetonitrile in water (B)
– Gradient: 5 to 100% B in 10 minutes, hold until 15 minutes
– Stop time 15 min and post time 5 min
• MS Parameters
– Standard autotune conditions
– MS scan range 300 – 5000 at 1 scan/sec acquisition rate

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MS Spectrum and Deconvolved Spectrum of
Bovine Ribonuclease A x10 7 + Scan (5.956-6.267 min, 16 scans) 4000-200-RNaseA.d Deconv oluted (1)
1.35
x10 5 + Scan (5.956-6.267 min, 16 scans) 4000-200-RNaseA.d 13682.1590
1.3
7.25
7 1521.2108
1.25
1.2
Deconvolved
6.75
6.5
1.15
1.1 Spectrum
6.25
6 MS 1.05
1
5.75 0.95
5.5 0.9
5.25 0.85
1369.1876
5
4.75
0.8
0.75
Theoretical: 13682.3898
4.5
4.25
1141.1603 0.7
0.65
Observed: 13682.1590
4
3.75
1244.8072 0.6
0.55
Error: -0.23 Da
3.5
3.25
0.5
0.45
-16.9 ppm
3 0.4
2.75 0.35
2.5 0.3
2.25 0.25
2 0.2
1.75 0.15
1.5 0.1
13780.1916
1.25 0.05
1711.2177 13564.5454 13833.4847
1 0
13500 13600 13700 13800 13900 14000 14100
0.75
Abundance vs. Mass (amu)
0.5
1955.5242
0.25 2281.2875
0
800 1000 1200 1400 1600 1800 2000 2200 2400
Abundance vs. Mass-to-Charge (m/z)

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MS Spectrum and Deconvolved Spectrum of Horse
Myoglobin x10 6 + Scan (474.4-483.1 sec, 8 scans) 4000-200myo.d Deconv oluted (1)

2.2
16951.6584
x10 5

1.05
+ Scan (7.949-8.052 min, 6 scans) 4000-200myo.d 2.1

2
Deconvolved
893.1872
1
1.9
1.8 Spectrum
0.95

0.9
MS 1.7
1.6

0.85 1.5

1.4
0.8
1.3
0.75

0.7
1.2

1.1
Theoretical: 16951.44
0.65 1 Observed: 16951.66
Error: -0.22 Da
0.6 0.9

0.55 0.8

0.5
0.7

0.6
-12.9 ppm
0.45
0.5
0.4
0.4
0.35 0.3

0.3 1211.8108 0.2

0.1 17050.3704
0.25 1304.9478 16781.0598 17218.1251
0
0.2 16750 16800 16850 16900 16950 17000 17050 17100 17150 17200 17250 17300 17350
1413.6178 Abundance vs. Mass (amu)
0.15

0.1 1696.1247

0.05

0
500 600 700 800 900 1000 1100 1200 1300 1400 1500 1600 1700 1800 1900
Abundance vs. Mass-to-Charge (m/z)

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MS Spectrum and Deconvolved Spectrum of
Bovine Beta-casein: Peak 1 + Scan (7.569-7.756 min, 10 scans) 4000-200-bcas.d Deconvoluted (1)
x10 5
2.7
2.6
2.5
2.4
25226.1352

25306.2197
Deconvolved
2.3
2.2
2.1
Spectrum
2
x10 4 + Scan (7.569-7.756 min, 10 scans) 4000-200-bcas.d
1.9
1.8
901.9396
1.4 1.7

MS
1.6
1.3 1.5
1.4
1.2 1.3
1.2
1.1
1.1
1
25386.0686
0.9
1
0.8
0.7 25146.5330
0.9 0.6
0.5
0.8 0.4
0.3 25468.1032 25549.7534
0.7 25630.4213
0.2
0.1 25689.9710
0.6 0
25100 25150 25200 25250 25300 25350 25400 25450 25500 25550 25600 25650 25700
0.5 Abundance vs. Mass (amu)

0.4
1206.0067
1266.3020
∆80 is a phospho group
0.3
1406.8876
0.2

0.1
1577.6069
Beta-casein has isoforms
0 that differ in degree of
700 800 900 1000 1100 1200 1300
Abundance vs. Mass-to-Charge (m/z)
1400 1500 1600
phosphorylation
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Page 5 Month ##, 200X
 MS Spectrum and Deconvolved Spectrum of
Bovine Beta-casein: Peak 2 + Scan (8.377-8.543 min, 9 scans) 4000-200-bcas.d Deconv oluted (1)
x10 6

23983.1267
8

7.5

7
Deconvolved 24023.0403

6.5

6
Spectrum
5.5
x10 5 + Scan (8.377-8.543 min, 9 scans) 4000-200-bcas.d
5
2.6 1045.4829
2.5
2.4
2.3
MS 4.5

2.2 3.5
2.1 1411.7554
3
2
1.9 2.5
1.8 1999.5665
2 24004.7456
1.7 23964.7771
1.5
1.6
24092.0787
1.5 1
1.4 23903.3983
0.5 23944.2876 24074.9659
1.3 23865.8726 24112.4370
1599.8529
1.2 0
1.1 23860 23880 23900 23920 23940 23960 23980 24000 24020 24040 24060 24080 24100 24120 24140
1845.8275 Abundance vs. Mass (amu)
1 1714.0549 2181.2459
0.9
0.8
0.7
0.6
Theoretical (with 5 phospho
0.5
0.4 groups) = 23983.3828
0.3
0.2 2399.2759 Deconvolved mass = 23983.1267
0.1
0 Error = -0.26 Da
700 800 900 1000 1100 1200 1300 1400 1500 1600 1700 1800 1900 2000 2100 2200 2300 2400 2500
Abundance vs. Mass-to-Charge (m/z)
-10.7 ppm
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MS Spectrum and Deconvolved Spectrum of
Bovine Carbonic Anhydrase
x10 7 + Scan (8.195-8.423 min, 12 scans) 4000-200-CA.d Deconv oluted (1)

x10 5 + Scan (8.195-8.423 min, 12 scans) 4000-200-CA.d

1.1
1.05
Deconvolved 29024.9810

Spectrum
3.6
880.5393 1
3.4 0.95

3.2 MS 0.9
0.85
3
0.8
2.8 0.75

2.6 0.7
0.65
2.4
0.6
2.2 0.55 Theoretical: 29024.8457
2 0.5

1.8
0.45 Observed: 29024.9810
1.6
0.4
0.35
Error: 0.13 Da
1.4 0.3

0.25
4.7 ppm
1.2
0.2
1
0.15 28435.3434
0.8 0.1
0.05 28212.2496 28595.3591
0.6
0
0.4 28000 28200 28400 28600 28800 29000 29200 29400
1935.9562 Abundance vs. Mass (amu)
0.2 2233.6375

0
600 800 1000 1200 1400 1600 1800 2000 2200 2400
Abundance vs. Mass-to-Charge (m/z)

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MS Spectrum and Deconvolved Spectrum of
Bovine Serum Albumin x10 6 + Scan (8.046-8.253 min, 11 scans) 4000-200-BSA.d Deconv oluted (1)

1.7
66429.8960
1.65
+ Scan (8.046-8.253 min, 11 scans) 4000-200-BSA.d 1.6
x10 5 1.55
1.3
1.25 1254.4450
1.5
1.45
1.4
Deconvolved
1.2
1.15 MS 1.35
1.3
1.25
Spectrum
1.1 1.2
1.05 1.15
1.1 66548.6223
1
1.05
0.95 1
0.9 0.95
0.9
0.85 0.85
0.8 0.8
0.75
0.75 0.7
0.7 0.65
0.6
0.65 0.55
0.6 0.5
0.45
0.55
0.4
0.5 0.35
0.3
0.45
0.25
0.4 0.2
0.35 0.15
0.1
0.3 0.05
66246.0112
0.25 0
66200 66300 66400 66500 66600 66700 66800 66900 67000 67100 67200
0.2 Abundance vs. Mass (amu)
0.15
0.1
0.05
0
900 1000 1100 1200 1300 1400 1500 1600 1700 1800 1900 2000 2100 2200 2300 2400
Abundance vs. Mass-to-Charge (m/z)

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Deconvolved Spectrum of Horseradish Peroxidase
x10 6 + Scan (8.228-8.311 min, 5 scans) 4000-200-peroxidase.d Deconv oluted (1)

1.6
1.55 43174.0600
1.5
1.45

x10 5 + Scan (8.228-8.311 min, 5 scans) 4000-200-peroxidase.d

1.4
1.35
1.3
Deconvolved
Spectrum
1.15 1799.9365 1.25

MS
1.2
1.1 1.15
1.05 1.1
1.05
1 1
0.95 0.95
0.9
0.9 0.85
0.8
0.85
0.75
0.8 0.7
0.65
0.75
0.6 42352.2618
0.7 0.55
0.5
0.65
0.45
0.6 0.4
0.35
0.55 0.3
0.5 0.25
0.2
0.45 0.15
2879.2704 0.1
44199.0765
0.4
2399.5749 0.05
0.35 0
2699.3842 42000 42200 42400 42600 42800 43000 43200 43400 43600 43800 44000 44200 44400 4460
0.3
Abundance vs. Mass (amu)
0.25
0.2
0.15 3084.8454
0.1
0.05
0
1400 1600 1800 2000 2200 2400 2600 2800 3000 320
Abundance vs. Mass-to-Charge (m/z)

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Separation of Intact and Reduced IgG2 Lambda

x10 7 + TIC Scan 5000-250-IgG-3400fmol.d

1 1
4
3.5 Intact IgG2 Lambda
3
2.5
2
1.5
1
0.5

x10 7 + TIC Scan 5000-250-IgG-reduced-5ul.d

Reduced IgG2 Lambda Heavy chain

2.75 1 1
2.5
2.25
2
1.75 Light chain
1.5
1.25
1
0.75
0.5
0.25

0.5 1 1.5 2 2.5 3 3.5 4 4.5 5 5.5 6 6.5 7 7.5 8 8.5 9 9.5 10 10.5 11 11.5 12 12.5 13 13.5 14 14.5
Abundance vs. Acquisition Time (min)

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 Mass Spectrum of IgG2 Light Chain
x10 5 + Scan (7.547-7.609 min, 4 scans) 5000-250-IgG-reduced-5ul.d Dec

4 22717.2700
3.8
3.6

Deconvolved
3.4
3.2
x10 4 + Scan (7.547-7.609 min, 4 scans) 5000-250-IgG-reduced-5ul.d
3

Spectrum
1.7
1033.5970 2.8
1.6 2.6
2.4
∆162 indicates
1.5

1.4 MS 2.2
2
1.3 1.8 glycoforms differ
1.2
1.6
1.4
by a hexose
1.1 1.2 22879.1740
1
1
0.8
0.9 2066.1782 0.6

0.4
0.8 1337.2999 0.2 23041.4756
1420.8084 1894.0735 22611.3672
0.7 0
22500 22600 22700 22800 22900 23000 23100 2320
0.6 Abundance vs. Mass (amu)
1515.4612
0.5

0.4
1748.4545
0.3

0.2
2272.6545
0.1

0
700 800 900 1000 1100 1200 1300 1400 1500 1600 1700 1800 1900 2000 2100 2200 2300
Abundance vs. Mass-to-Charge (m/z)

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Mass Spectrum of IgG2 Heavy Chain

x10 4 + Scan (8.437-8.520 min, 5 scans) 5000-250-IgG-reduced-5ul.d Deconv oluted (1)

5.5
51119.0849
Deconvolved
5

4.5
51282.0500
Spectrum
x10 3 + Scan (8.437-8.520 min, 5 scans) 5000-250-IgG-reduced-5ul.d 4

∆162 indicates
8.5
922.0110 3.5
8
7.5 MS 3

2.5
50956.7698
glycoforms differ
7
2
by a hexose
6.5
1.5 51443.2834
6 51575.2391
5.5 1088.6468 1

5 0.5 50809.6898
51659.9338
4.5 0
50800 50900 51000 51100 51200 51300 51400 51500 51600 51700 51800
4 Abundance vs. Mass (amu)
1279.0132
3.5
3
2.5
2
663.4571
1.5
783.4951
1 1710.2570
0.5
0
600 700 800 900 1000 1100 1200 1300 1400 1500 1600 1700 1800
Abundance vs. Mass-to-Charge (m/z)

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Mass Spectrum of Intact Human IgG2 Lambda

x10 4 + Scan (8.017-8.203 min, 10 scans) 5000-250-IgG-3400fmol.d

1.05 2792.2253
1 2740.5660
2690.6806

x10 4 + Scan (8.017-8.203 min, 10 scans) 5000-250-IgG-3400fmol.d 0.95

0.9
0.85
0.8

1.05 2792.2253 0.75

0.7

1 0.65

2466.4330 0.6
0.55
0.95 0.5
0.45 2801.3374
2699.8149
0.9 0.4
0.35
2749.2891

0.3
0.85 0.25
0.2

0.8 0.15
0.1

0.75
0.05

2670 2680 2690 2700 2710 2720 2730 2740 2750 2760 2770 2780 2790 2800 2810 2820
Abundance vs. Mass-to-Charge (m/z)
0.7
0.65
0.6
0.55
0.5
0.45
0.4
0.35 2114.0342
0.3
0.25
0.2
0.15
3216.9395
0.1
3289.0190
0.05

1700 1800 1900 2000 2100 2200 2300 2400 2500 2600 2700 2800 2900 3000 3100 3200 3300 3400
Abundance vs. Mass-to-Charge (m/z)

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 Deconvolved Spectrum of Human IgG Lambda
x10 4 + Scan (8.017-8.203 min, 10 scans) 5000-250-IgG-3400fmol.d Deconv oluted (1)
6.4

6.2
6 ∆162 indicates that 147796.8259
147956.2420

some glycoforms differ

5.8
5.6
5.4

5.2
by a hexose
5
147634.7540
4.8
4.6
4.4
4.2 148116.7796

4
3.8

3.6
3.4
3.2
3
148280.6912
2.8 147475.2733
2.6

2.4
2.2
2

1.8 148414.8277

1.6
1.4

1.2 148588.1250
1
147321.9628
0.8
0.6
0.4
147172.3284 149058.5439
0.2
0
147000 147100 147200 147300 147400 147500 147600 147700 147800 147900 148000 148100 148200 148300 148400 148500 148600 148700 148800 148900 149000 149100 149200
Abundance vs. Mass (amu)

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Sensitivity of IgG2 Lambda Analysis

x10 2 + Scan (8.356-8.791 min, 22 scans) 5000-250-IgG-170fmol.d

6 2506.0212
2792.7107 170 fmol on-column
5
4
3
2 2142.9382
1
0
x10 3 + Scan (8.258-8.362 min, 6 scans) 5000-250-IgG-340fmol.d

2 2789.7659

1.5
2508.8408 340 fmol on-column
1
2176.7198
0.5
3448.4758
0
x10 3 + Scan (8.189-8.251 min, 4 scans) 5000-250-IgG-680fmol.d

4 2508.3429
2791.3202 680 fmol on-column
3
2 2240.2250
1
3369.9148
0
x10 4 + Scan (8.017-8.203 min, 10 scans) 5000-250-IgG-3400fmol.d

1
0.8
2466.4330 2792.2253
3.4 pmol on-column
0.6
0.4 2114.0342
0.2 3216.9395
0
1900 2000 2100 2200 2300 2400 2500 2600 2700 2800 2900 3000 3100 3200 3300 3400 3500
Abundance vs. Mass-to-Charge (m/z)

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 Applications on the Proteomics Applications
Agilent 6510 Q-TOF

Christine Miller
Ning Tang
Application Scientists
August 14, 2006

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Page 16 Month ##, 200X
De Novo Sequencing of Q-TOF Spectra

• Excellent match between database search

and de novo result
• Peptide fragments cover entire backbone
• Immonium ions confirm N-termini

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Ion Trap MS/MS of a Tetraphosphopeptide
x106 b8
983.8

6 CID

4 y172+
[y18-H3PO4]2+ b-ions: 6
y-ions: 10
1071.0
y162+
2 [M+3H-H3PO4]3+
[b11-17]2+
y4 [y10-H3PO4]2+ b7 y9 b
476.4 625.5 884.8 b10-17 y11-H3PO4 13y11
807.7 1120.2 1235.6 b12
1417.6 1515.6 y12-18 y13 y16-H3PO4
0
400 600 800 1000 1200 1400 1600 1800 m/z
3+
x105 [M+3H]
1042.2

1.0 [M+2H]2+
1563.0
ETD
0.8

0.6
L s s s E E SI T
T I S E s
0.4
[M+4H]4+ C 2+ C242+
z4 781.8
C192+ 20 C212+ 1484.4
460.4
z5 z6 C172+ 1203.7
1268.8
0.2 z2 z3 589.5
718.6
C 2+
260.1 373.3 z7 C182+ C22 2+ 23
2+
C15884.8
C162+ z18
643.0 1681.4 2140.9
0.0
200 400 600 800 1000 1200 1400 1600 1800 2000 m/z

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QTOF MS/MS of a Beta-Casein Tetraphosphopeptide
RELEELNVPGEIVEsLsssEESITR

x10 4 + Product Ion (4.070-4.121 min, 2 scans) (1561.6532 -> **) tetra-msms-06.d
3 1366.1905 [M+2H-4H3PO4]2+
2.8
2.6
b8
2.4 [M+2H-3H3PO4]2+
983.5193
2.2
2

1.8
y4
1.6
476.2846
1.4 y2 a8 [M+2H-2H3PO4]2+
1.2 276.1669 b7
y1
1 y5 884.4540
y3
0.8 605.3256 b6 [M+2H-H3PO4]2+
389.2520 b4 b5 b11 b14 b -H PO y -H PO
0.6 b2 770.4063 1607.8407 16 3 4 16 3 4
R y6 1266.6345
b3 y y18-H3PO4
0.4 1789.9330 1944.8196
R/P 16 2140.7659
0.2
0
100 200 300 400 500 600 700 800 900 1000 1100 1200 1300 1400 1500 1600 1700 1800 1900 2000 2100 2200
Abundance vs. Mass-to-Charge (m/z)

b-ions: 11
y-ions: 9
**Data collected without reference mass correction

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Relative Quantitation Using iTRAQ Labeling
x10 3 + Product Ion (6.657-6.702 min, 3 scans) (542.77997, 542.77820, 542.78015 -> **) texas_hrt_frac4.d
3.2 542.78473
3
2.8
2.6
2.4
iTRAQ
2.2
2
reporter ions
1.8 115.10764
1.6 328.86191
940.45287
1.4 362.19061
1.2
1
175.11882
0.8 803.38893
690.30354
0.6
0.4 72.07319 282.16707 401.24341
493.28054
0.2 601.34811 724.37695 848.32010
0
50 100 150 200 250 300 350 400 450 500 550 600 650 700 750 800 850 900 950 1000
Abundance vs. Mass-to-Charge (m/z)

Zoom view of iTRAQ reporter ions

x10 3 + Product Ion (6.657-6.702 min, 3 scans) (542.77997, 542.77820, 542.78015 -> **) texas_hrt_frac4.d

2 iTRAQ basics:
0.3 ppm 115.10764 -5.7 ppm
1.8
1.6 114.11123
• Used for differential expression
1.4 -1.5 ppm • Each sample type reacted with a
116.11178
1.2
3.8 ppm different tag (up to 4 types)
1 117.11545
0.8
• Reacted samples are mixed and
0.6
0.4 analyzed in a single run
0.2
112.09223 113.07852 118.12111 • MS/MS generates “reporter” ions
0
112 113 114 115 116 117
Abundance vs. Mass-to-Charge (m/z)
118 119 showing amount in each sample
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Sensitivity With Fast Data Dependent MS/MS
Analysis of 1 fmol BSA Digest; 5 scan/sec acquisition rate

x10 6 + TIC MS(all) 1fmolBSA-03.d

4 Total ion chromatogram, MS and MS/MS

1 1

3.5
3
2.5
2
1.5
1
0.5

x10 4 + BPC(400.00000-1200.00000) Scan 1fmolBSA-03.d

3
1
Base peak chromatogram, MS 1

2.5

1.5

0.5

x10 4 + EIC(653.36170) Scan 1fmolBSA-03.d

Extracted ion chromatogram

2.4 1 1
2.2
2
1.8
1.6
1.4
1.2
m/z 653.3617
1
0.8
0.6
0.4
0.2
0
0.2 0.4 0.6 0.8 1 1.2 1.4 1.6 1.8 2 2.2 2.4 2.6 2.8 3 3.2 3.4 3.6 3.8 4 4.2 4.4 4.6 4.8 5 5.2 5.4 5.6 5.8 6 6.2 6.4 6.6 6.8 7 7.2 7.4 7.6 7.8 8 8.2 8.4 8.6 8.8
Abundance vs. Acquisition Time (min)

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MS and MS/MS Spectrum of m/z 653.3617
1 fmol BSA digest at 5 scan/sec acquisition
+ Scan (3.457 min) 1fmolBSA-03.d x10 2 + Product Ion (3.462-3.473 min, 2 scans) (653.36755, 653.36487 -> **) 1fmolBSA-03.d
x10 3
653.36194 9.5
5.4 653.36195
5.2 9
5 0.4 ppm 8.5 [M+2H]+2
4.8
4.6
8
0.4 ppm
4.4 7.5
4.2
4
7
b2
3.8 6.5
251.14807
3.6 6
3.4
3.2 5.5
3 5 H
2.8
2.6 4.5
110.07192
2.4
2.2
4
IL y9
2
3.5
b8++ y8 1055.57465
1.8 3
1.6 465.25324
2.5
1.4 332.21091 b5 y6
956.49626

1.2 2
1 1.5 842.49407 y10
0.8
1
0.6
0.4
y1 1168.63194
651.51520 656.38389 0.5
0.2
0
0
100 200 300 400 500 600 700 800 900 1000 1100 1200
650 651 652 653 654 655 656 657 658
Abundance vs. Mass-to-Charge (m/z)
Abundance vs. Mass-to-Charge (m/z)

Group/Presentation Title
Agilent Restricted
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