q exactivetm: a true qual-quanapps.thermoscientific.com/media/sid/lsms/pdf/...exactive q-exactive...
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Q ExactiveTM: A True Qual-Quan HR/AM Mass Spectrometer for
Routine Proteomics Applications
Yi Zhang, Ph.D. ThermoFisher Scientific
2
Outline
• Introduction of Q Exactive
• Performance in Discovery Proteomics
• Flexible Targeted Quantification
• Mass Measurement of Intact mAb
• Summary
3
Q ExactiveTM - Innovations and Features
First Benchtop Quadrupole-Orbitrap Mass Spectrometer
ORBITRAP Resolution: 140,000
Mass Accuracy: < 2 ppm
QUADRUPOLE MASS FILTER Mass Range: 50 – 4000 amu Prec Isolation: 0.4 – 10 amu
HCD/C-Trap Combo Cell
ION SOURCE S-Lens
4
High Sensitivity with Quadrupole-based SIM Scan
0
1000
2000
3000
4000
5000
6000
195.082 195.084 195.086 195.088 195.09 195.092 195.094
S/N
(spe
ctru
m)
S/N (FMS) S/N (SIM10)
Gain in sensitivity (7x)
Caffeine
0
20
40
60
80
100
Rel
ativ
e A
bund
ance
0
20
40
60
80
100
195.0876 N=248402.81
195.0877 N=20741.58
Full MS S/N = 745
SIM (10amu) S/N = 5400
Sensitivity Gain: 5 – 10 folds
5
Improved Resolution – Advanced Signal Processing
0
20000
40000
60000
80000
100000
120000
140000
160000
0 5 10 15
Reso
luti
on
Scan Speed [Hz]
Exactive
Q-Exactive
Resolution at m/z 200
Resolution at m/z 400
Max. scan speed (Hz)
17,500 12,500 12 35,000 25,000 7 70,000 50,000 3 140,000 100,000 1.5
• Increases resolution by 2X at the same transient length
6
Scan Innovations
• Parallel Filling and Detection
• Spectrum Multiplexing
7
Outline
• Introduction of Q Exactive
• Performance in Discovery Proteomics
• Peptide/Protein Identification
• TMT based Quantification
• Flexible Targeted Quantification
• Mass Measurement of Intact mAb
• Summary
8
Discovery Proteomics – 10Hz HCD Scan
256ms 64ms
• Parallel Filling and Detection • Fast Orbitrap Scanning • pAGC
10 HR/AM HCD scans in 1s
9
1 Full MS +10 HR/AM HCD Scans in One Second
1 2
3 7
5 6
4 8
9
10
1 2 5 6 7 8 9 10 4 3
∆ = 0.0181 min = 1.086 sec
Ecoli_50min_newcolumn_Top10_15cm_reje... 2/17/2011 3:39:42 PM
RT: 15.8279 - 51.4773
16 18 20 22 24 26 28 30 32 34 36 38 40 42 44 46 48 50 Time (min)
0
20
40
60
80
100
Rel
ativ
e A
bund
ance
41.5779 41.6221
25.9001 16.0095 33.0433 47.5177 27.4920 30.4806 17.9024 25.7411 22.3863 27.1739
39.1805 35.5786 30.9771 38.7325
23.9072 28.4175 49.3818 20.3567 18.7207 41.1998 37.5942 34.8784 25.4406 43.4203 21.9561 34.7425 41.0942 46.5441 43.5925 49.1418
49.6742
RT: 32.2438 - 32.2701
32.244 32.246 32.248 32.250 32.252 32.254 32.256 32.258 32.260 32.262 32.264 32.266 32.268 32.270 Time (min)
0.0
0.1
0.2
0.3
0.0
0.1
0.2
0.3
Rel
ativ
e A
bund
ance
32.2464 32.2645
32.2538 32.2564 32.2577 32.2551 32.2590 32.2603 32.2629 32.2694 32.2525 32.2512 32.2616 32.2447
Ecoli_50min_newcolumn_Top10_15cm_reject_1 # 12365 RT: 32.25 AV: 1 NL: 1.58E8 T: FTMS + p NSI Full ms [300.00-2000.00]
350 400 450 500 550 600 650 700 750 800 850 900 950 1000 1050 1100 1150 m/z
0
20
40
60
80
100
Rel
ativ
e A
bund
ance
536.7949 z=2
575.8452 z=2
664.3292 z=2
392.7555 z=2
484.2459 z=3
382.9046 z=3
581.9907 z=3
711.3712 z=3
445.7585 z=2
505.2733 z=2 419.2133
z=3
329.2184 z=2
1150.6826 z=1
551.8184 z=2
950.9638 z=2
784.5037 z=?
1072.5819 z=1
630.3730 z=2
725.8647 z=2 700.3623
z=2 902.8879
z=2 1008.5358
z=1 871.9796
z=2 835.3868
z=2 1096.3795
z=? 1182.6012
z=?
x2 x5
10
High Quality HCD Spectra
y₅⁺589.29150
b₂⁺247.14401
y₁⁺175.11888
y₆⁺688.36139
a₂⁺219.14905
y₂⁺288.20270
a₁⁺120.08099
100 200 300 400 500 600 700 800 900
m/z
0
2
4
6
8
10
12
Inte
nsity
[cou
nts]
(10^
6)
Extracted from: C:\Xcalibur\data\Kai\Ecoli_50min_newcolumn_Top10_15cm_reject_1.raw #12410 RT: 32.33 FTMS, HCD, z=+2, Mono m/z=467.75339 Da, MH+=934.49950 Da, Match Tol.=20 mmu
y₂⁺260.19653
y₄⁺388.25467
y₁⁺-H₂O129.10219
a₁⁺120.08088
100 200 300 400 500 600 700
m/z
0
1
2
3
4
Inte
nsity
[cou
nts]
(10^
6)
Extracted from: C:\Xcalibur\data\Kai\Ecoli_50min_newcolumn_Top10_15cm_reject_1.raw #12411 RT: 32.33 FTMS, HCD, z=+2, Mono m/z=341.69992 Da, MH+=682.39256 Da, Match Tol.=20 mmu
y₅⁺475.28632
y₁₀⁺931.52252
y₄⁺388.25351
y₆⁺532.31097
y₁₀⁺-NH₃914.49854
y₈⁺702.41449
y₉⁺803.45911
a₉²⁺-H₂O398.24179b₃⁺
272.15991
y₇⁺589.32959
b₂⁺171.11258
b₃⁺-H₂O254.14955
200 400 600 800 1000 1200
m/z
0
1
2
3
4
5
Inte
nsity
[cou
nts]
(10^
6)
Extracted from: C:\Xcalibur\data\Kai\Ecoli_50min_newcolumn_Top10_15cm_reject_1.raw #12412 RT: 32.33 FTMS, HCD, z=+2, Mono m/z=658.38232 Da, MH+=1315.75737 Da, Match Tol.=20 mmu
a₅⁺438.30667 y₇⁺
685.38513
a₇³⁺-NH₃197.12845 y₆⁺
588.33417
b₃⁺, a₆²⁺268.16522
y₅⁺517.29755
b₅⁺466.30200
b₄⁺367.23297
y₁⁺-H₂O129.10220
y₂⁺246.18097
200 400 600 800 1000 1200
m/z
0.0
0.5
1.0
1.5
2.0
2.5
3.0
3.5
Inte
nsity
[cou
nts]
(10^
6)
Extracted from: C:\Xcalibur\data\Kai\Ecoli_50min_newcolumn_Top10_15cm_reject_1.raw #12413 RT: 32.34 FTMS, HCD, z=+3, Mono m/z=384.23242 Da, MH+=1150.68271 Da, Match Tol.=20 mmu
a₁₀⁺-H₂O, y₉⁺929.44843
y₁₀⁺1044.47485
y₄⁺440.26059
y₈⁺800.40826
y₇⁺729.36896
y₅⁺527.29431
a₆³⁺, b₂⁺, a₄²⁺201.12317
y₆⁺598.33002
a₂⁺173.12833
200 400 600 800 1000 1200 1400
m/z
0.0
0.5
1.0
1.5
2.0
2.5
Inte
nsity
[cou
nts]
(10^
6)
Extracted from: C:\Xcalibur\data\Kai\Ecoli_50min_newcolumn_Top10_15cm_reject_1.raw #12414 RT: 32.34 FTMS, HCD, z=+3, Mono m/z=453.23071 Da, MH+=1357.67759 Da, Match Tol.=20 mmu
b₇⁺692.37195
a₇²⁺332.69171
y₄⁺430.23016
y₆⁺676.36572
y₁₁²⁺592.30835
y₅⁺577.29816
y₈⁺846.47083
y₉²⁺492.26865
y₂⁺244.16534
a₂⁺157.13342
200 400 600 800 1000 1200 1400
m/z
0
1
2
3
4
Inte
nsity
[cou
nts]
(10^
6)
Extracted from: C:\Xcalibur\data\Kai\Ecoli_50min_newcolumn_Top10_15cm_reject_1.raw #12415 RT: 32.34 FTMS, HCD, z=+3, Mono m/z=456.58206 Da, MH+=1367.73163 Da, Match Tol.=20 mmu
b₆²⁺, b₃⁺342.20215
y₅⁺618.30170
y₆⁺731.38538
y₇⁺860.42780
b₂⁺213.15939
y₄⁺487.26202
a₂⁺185.16463
100 200 300 400 500 600 700 800 900 1000 1100
m/z
0
10
20
30
40
Inte
nsity
[cou
nts]
(10^
6)
Extracted from: C:\Xcalibur\data\Kai\Ecoli_50min_newcolumn_Top10_15cm_reject_1.raw #12416 RT: 32.34 FTMS, HCD, z=+2, Mono m/z=536.79449 Da, MH+=1072.58171 Da, Match Tol.=20 mmu
y₁⁺175.11887 y₁₁⁺
1132.57153y₅⁺
620.31427y₉⁺
934.47302
y₆⁺707.34601
a₁⁺120.08097
200 400 600 800 1000 1200 1400
m/z
0.0
0.2
0.4
0.6
0.8
1.0
1.2
1.4
Inte
nsity
[cou
nts]
(10^
6)
Extracted from: C:\Xcalibur\data\Kai\Ecoli_50min_newcolumn_Top10_15cm_reject_1.raw #12417 RT: 32.34 FTMS, HCD, z=+2, Mono m/z=720.34052 Da, MH+=1439.67375 Da, Match Tol.=20 mmu
b₅⁺620.26434
y₁₁⁺1165.69214
y₁⁺147.11258
b₄⁺521.19879
y₇⁺755.46484
b₃⁺393.13989
a₂⁺250.11826
200 400 600 800 1000 1200 1400 1600 1800
m/z
0.0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
1.0
Inte
nsity
[cou
nts]
(10^
6)
Extracted from: C:\Xcalibur\data\Kai\Ecoli_50min_newcolumn_Top10_15cm_reject_1.raw #12418 RT: 32.34 FTMS, HCD, z=+2, Mono m/z=892.97919 Da, MH+=1784.95110 Da, Match Tol.=20 mmu
b₅⁺-H₂O528.19189
b₄⁺-H₂O457.15570
y₈⁺909.54944
y₃³⁺129.10216
y₂⁺272.17108
b₃⁺-H₂O328.11536
b₂⁺-H₂O241.08124
a₂⁺231.09689
y₁₇²⁺926.00580
y₁₀²⁺568.84778
y₁₄²⁺782.45026
y₁₂²⁺653.90033
y₁₃²⁺717.92920
200 400 600 800 1000 1200
m/z
0
50
100
150
200
250
300
Inte
nsity
[cou
nts]
(10^
3)
Extracted from: C:\Xcalibur\data\Kai\Ecoli_50min_newcolumn_Top10_15cm_reject_1.raw #12419 RT: 32.35 FTMS, HCD, z=+3, Mono m/z=703.70172 Da, MH+=2109.09061 Da, Match Tol.=20 mmu
11
Peptide/Protein Identification
*About 4500 proteins are expressed in yeast during log-phase growth
2452
6975
8353
1361
3539
4767
0
2000
4000
6000
8000
10000
10ng 100ng 1000ng
Q Exactive TripTOF 5600
691
1321 1395
357
724
1017
0
250
500
750
1000
1250
1500
10ng 100ng 1000ng
Number of Unique Peptides Number of Unique Protein Groups
Yeast Tryptic Digest (10, 100, 1000ng)
Column: C18, 75µmx15cm LC: 350nL/min, 90min
Q Exactive: Top10
TripleTOF 5600: Top20
12
0
20
40
60
80
100
120
140
160 Q Exactive
TripleTOF 5600
Peptide/Protein Identification
Protein identified from 10 ng yeast digest
*Yeast cellular protein copy numbers are from Weissman and co-workers, Nature, 2003, 16, 737-7341.
Same sample, Same LC column, Same gradient, 1% FDR
Num
ber o
f Pro
tein
s Id
entif
ied
Cellular copy number
4x more ID
13
Peptide/Protein Identification - Larger Dynamic Range on Q Exactive
Same Sample, Same LC column, Same Gradient, 1% FDR
Most Abundant Least Abundant
10 ng load
1000 ng load
Precursor intensity of identified peptides (normalized to the most intense peak)
TripleTof 5600
Q Exactive
14
Peptide of YOR020C, 149 copy number, identified from 10 ng yeast digest
b₅⁺-NH₃498.17874
y₆⁺-H₂O758.45410
y₉⁺1120.55273
y₂⁺322.18576
y₈⁺1006.51752
y₆⁺776.46594
y₁₀⁺1177.57886
y₁⁺175.11826
y₇⁺891.48932
y₅⁺647.42157
y₃⁺435.26990
y₄⁺548.35284
200 400 600 800 1000 1200
m/z
0
10
20
30
40
50
Inte
nsity
[cou
nts]
(10^
3)
Extracted from: C:\Xcalibur\data\Zhiqi\ID_QE\20110321_10ngYeastDigest_Top10_140min_01.raw #23048 RT: 77.42 FTMS, HCD, z=+2, Mono m/z=645.83948 Da, MH+=1290.67168 Da, Match Tol.=20 mmuL G N D D E V I L F RL G N D D E V I L F R MASCOT IonScore: 90
Exp Value: 3.8 E-9
Peptide/Protein Identification – High Quality HCD Spectrum
High confidence identification from 10 ng of Yeast Digest
15
Discovery Quan (TMT)
Num
ber o
f pep
tides
at 1
% F
DR
Amount of E. coli digest
2736
3888 4376
5252
6527
3245
4850
5537
7180
9298
0
1000
2000
3000
4000
5000
6000
7000
8000
9000
10000
20ng 40ng 80ng 200ng 500ng
unique peptide total IDed MSMS spectra quantifiable MSMS spectra
TMT 6-plex labeled E. coli digest
Quantification Rate: >97%
16
Discovery Quan (TMT) – Accuracy and Precision
1.04 1.01 1.02 1.05 1.03
0
0.2
0.4
0.6
0.8
1
1.2
1.4
127/126 128/126 129/126 130/126 131/126
observed expected
TMT
repo
rter i
on ra
tio
Result from 80 ng E. Coli digest
Quantification Error <5%, CV<13%
17
Outline
• Introduction of Q Exactive
• Performance in Discovery Proteomics
• Flexible Targeted Quantification
• Targeted msx SIM
• Targeted HCD
• Mass Measurement of Intact mAb
• Summary
18
HR/AM Targeted Quantification on the Q Exactive
HR/AM Targeted Quantification
Quantification
Targeted msx SIM Targeted HCD
LC Peak Area of Precursor
LC Peak Area of MS/MS Fragments
Instrument method
Selectivity, Speed, Sensitivity
19
Quantification with Targeted msx SIM
492.2665
492.2495
492.2634
R: 35K R: 140K
m/z
492.2661
492.2511
R: 70K
30ppm
High Resolution (140K) Accurate Mass (<5ppm) Selectivity
Spectrum Multiplexing Speed
20
Quantification with Targeted msx SIM
Quadrupole-Based SIM Sensitivity
21
17.0 17.2 17.4 17.6 17.8 18.0 18.2 18.4 18.6 Time (min)
0
20
40
60
80
100 0
20
40
60
80
100
Rel
ativ
e A
bund
ance
0
20
40
60
80
100 18.12
18.11
18.10
Quantification with Targeted msx SIM in Medium Complex Background
12 Heavy Peptides (10amol – 100fmol)
In 10ng Yeast Digest
msx tSIM Resolution: 140K SIM: 4amu msx: 4
GISNEGQNASIK 5ppm XIC
100fmol
1fmol
10amol
0.E+00
1.E+09
2.E+09
3.E+09
4.E+09
0 50 100 Sample Amount (fmole)
Pea
k A
rea
SSAAPPPPPR
GISNEGQNASIK
DIPVPKPK
LOD: 10amol LOQ: 50amol (CV <10%) Linear Dynamic Range: 4 orders
R2: 0.9991-0.9999
22
• Full MS/MS Spectra – All Transitions
• Selectivity • MS/MS • High Resolution: 17,500 • Accurate Mass: <5ppm
• Speed
• 12 Hz
• Sensitivity • High quality HCD scan at low amol level
Quantification with Targeted HCD
23
Quantification with Targeted HCD in Complex Matrix RT: 14.97 - 21.09
15.0 15.5 16.0 16.5 17.0 17.5 18.0 18.5 19.0 19.5 20.0 20.5 21.0Time (min)
0
20
40
60
80
100
Relat
ive A
bund
ance
0
20
40
60
80
100
Relat
ive A
bund
ance
0
20
40
60
80
100
Relat
ive A
bund
ance
15.74
15.82
15.7215.67
15.8515.88
15.96
18.6218.67
18.60
18.57
18.6918.55
20.6420.6120.58
20.65
20.5720.67
20.54
GPGEDFR394.193-633.3051
VAHTVAYLGK355.877-324.2350
YFQGYAR457.732-594.2989
11 Heavy Peptides (10amol – 100fmol)
In 250ng CSF Digest
y = 3820.1x + 578349R² = 0.9999
0
50000000
100000000
150000000
200000000
250000000
300000000
350000000
400000000
450000000
0 20000 40000 60000 80000 100000 120000
Peak
are
a
Sample amount (amole)
1000
10000
100000
1000000
10000000
100000000
1E+09
1 10 100 1000 10000 100000
YFQGYAR
tHCD Resolution: 17,500
LOD: 10-100amol Linear Dynamic Range: 3-4 Orders
10amol
24
1600 1800 2000 2200 2400 2600 2800 3000 3200 3400 3600 3800 4000 m/z
0 5
10 15 20 25 30 35 40 45 50 55 60 65 70 75 80 85 90 95
100
Rel
ativ
e Ab
unda
nce
2745.7720
2851.3544
2907.2595 2556.4844
2965.3764 2471.3405
3025.8632 2391.6288
-7 ppm G0F+G1F
G0F+G0F G0F+G2F (or 2G1F)
G1F+G2F G2F+G2F G0+G0F
G0+G0 2xMan5
G1F+G2F+SA
5.0 ppm
-0.7 ppm -8.5 ppm
-0.9 ppm
2680 2700 2720 2740 2760 2780 2800 2820 m/z
0
5
10
15
20
25
30
35
40
45
50
55
60
65
70
75
80
85
90
95
100
Rel
ativ
e A
bund
ance
2745.7720
2797.5697 2695.8919
Mass Measurement of Intact Monoclonal Antibody
Protein Deconvolution
R: 17.5K
25
Summary
• Q Exactive is the most competitive instrument for routine discovery proteomics analysis, with its 10Hz duty cycle, high dynamic range and high quality HCD scans.
• Q Exactive offers flexible HR/AM quantification approaches, targeted msx SIM and targeted HCD, with improved throughput, higher sensitivity and 4-order linear dynamic range.
• Q Exactive enables smooth transition from discovery proteomics to targeted quantification.
• Q Exactive accurately and reproducibly measures the mass of intact antibody to ~7ppm, and provides a robust QC solution for biopharmaceutical products.
26
Acknowledgement
S. Horning I. Mylchreest A. Guiller R.A.Purrmann F. Grosse-Coosmann A. Kuehn T. Rietpietsch E. Denisov R. Malek M. Biel C. Henrich M. Mueller A. Venckus F. Gebrehewit F. Afroz O. Hengelbrock M. Antonczak S. Moehring S. Nimkar M. Kosak K. Scheffler S. Daniels Y. Xun
D. Cho X. He Y. Huang A. Kholomeev K. Persson F. Paffen B. Rose A. Boegehold J. Grote W. Huels A. Schumbera S. Simmel P. Bennett K. Cook A. Huhmer Z. Hao S. Peterman Y. Zhang K. Comstock T. Stratton D. Ghosh C. Yang M. Blackburn
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