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1 The world leader in serving science
Julie A. Horner 2014 03 04
Recent Advances in Mass Spectrometry - Introducing the Unique Orbitrap Fusion and Quantiva Triple Quadrupole Mass Spectrometers
2
The Industry’s Leading Portfolio of MS Solutions
HR/AM
MS, MSn
App
lied
Mar
kets
Research M
arkets
Ion Traps Triple Quads
Tribrid Orbitrap MS
Non-targeted Analysis
Targeted Analysis
Quantitative Qualitative
• Biomarker Discovery • Proteomics • Metabolism
• Metabolomics • Proteomics • Bioanalysis
• Food Safety • Environmental • Clinical/Toxicology
• Metabolomics • PTM Analysis • Lipidomics
Transform Your Science
Exactive Series MS
3
Thermo Scientific™ Orbitrap Fusion™ Tribrid™ MS
Thermo Scientific™ TSQ Endura™ MS
Thermo Scientific™ TSQ Quantiva™ MS
Transform Your Science We have re-thought every aspect of the design to…
4
Advances in Mass Spectrometry in Brief
Common, Modular Mechanical Components
Common, Easy to Use, Customer Accessible Parts
Common, Modern Electrical Design
Common, Modern Ease to use Software
New, Flagship CMD Industrial Design
Common, High performance, Robust Ion Optics
Improved Usability = Get up and running fast!
Increased Sensitivity & Robustness = More productive lab!
Ease of Use = Get answers quickly!
Higher Quality Data = More Signal & Less Noise !
Compact , Smart Modules = Faster Service & Less Downtime !
High Quality Look & Feel = Pride in Ownership!
The TNG Project Initially focused on developing two key goals 1. Improve customer experience: Ease of Use, Ease of Maintenance, High Learn ability 2. High System Performance: Sensitivity, Robustness, Data Speed
5 The world leader in serving science
Julie Horner
Transforming Quantitative Analysis with TSQ Quantiva and TSQ Endura Triple Quad LC/MS
6
Challenges in Quantitative Mass Spectrometry
Complex Matrices
Small Sample Amounts
Large Sample Numbers
Low Concentration
Triple Quads
7
Next Generation Triple Quadrupole MS
PER
FOR
MA
NC
E USA
BILITY
Sensitive, robust,
ion optics
Intuitive, easy to use software
Fast intelligent
electronics
Robust, easy to maintain
hardware
8
TSQ Quantiva MS Extreme Quantitative Performance • Attogram sensitivity
• Unprecedented usability
• Ultimate robustness
TSQ Endura MS and TSQ Quantiva MS
TSQ Endura MS Extreme Quantitative Value • Best-in-class sensitivity and speed
• Unprecedented usability
• Ultimate robustness
9
Systematic optimization of all electric fields, in concert, to produce breakthrough performance.
Active Ion Management (AIM)
TSQ Quantiva MS—Powered by AIM Technology
10
TSQ Quantiva Triple Quadrupole Mass Spectrometer
High-capacity transfer tube (HCTT)
Active collision cell (Q2)
Electrodynamic ion funnel (EDIF)
Ion beam guide with neutral blocker Asymetric RF drive
HyperQuad quadrupole mass filter (Q1) Dual-mode discrete-dynode detector
11
Active Ion Management: Precision design of all electric fields, optimized in concert, to produce maximum signal and prevent contamination.
TSQ Quantiva MS—Powered by AIM Technology
Active collision cell (Q2) Electrodynamic ion funnel Ion beam guide with neutral blocker HyperQuad quadrupole
mass filter with Asymmetric RF drive
1 3 4 2
1
2
3
4
12
TSQ Quantiva MS—Powered by AIM Technology
Active collision cell (Q2) Electrodynamic ion funnel Ion beam guide with neutral blocker HyperQuad quadrupole
mass filter with Asymmetric RF drive
3 4 1 2
13
TSQ Quantiva MS—Powered by AIM Technology
Active collision cell (Q2) Electrodynamic ion funnel Ion beam guide with neutral blocker HyperQuad quadrupole
mass filter with Asymmetric RF drive
1 2 3 4
14
Increase in Signal 40%
Active collision cell (Q2) Electrodynamic ion funnel Ion beam guide with neutral blocker HyperQuad quadrupole
mass filter with Asymmetric RF drive
TSQ Quantiva MS—Powered by AIM Technology
Equal Amplitude (Balanced)
- +
+ -
10% Bias Amplitude (Asymmetric)
- +
+ -
10
20
30
40
50
60
70
80
90
0 0.5 1 1.5 2 2.5 3 3.5 4
Rel
ativ
e Tr
ansm
issi
on %
FWMD (Da)
Increased Transmission at Every Resolution
Asymmetric RF
Balanced RF
1 3 4 2
500 504 508 516 518 m/z
0
10
20
30
40
50
60
70
80
90
100
Rel
ativ
e A
bund
ance
508.19
509.19
499.41
Resolution; FWHM 0.7 Da FWHM 0.4 Da FWHM 0.2 Da FWHM
15
Active collision cell (Q2) Electrodynamic ion funnel Ion beam guide with neutral blocker HyperQuad quadrupole
mass filter with Asymmetric RF drive
TSQ Quantiva MS—Powered by AIM Technology
1 3 4 2
16
TSQ Quantiva—Powered by AIM Technology
Together this technology can solve your toughest analytical challenges.
Active collision cell (Q2) Electrodynamic ion funnel Ion beam guide with neutral blocker HyperQuad quadrupole
mass filter with Asymmetric RF drive
17
TSQ Quantiva MS: Sensitivity
Challenge TSQ Quantiva MS
Demanding assays which require the absolute lowest limit of detection.
The TSQ Quantiva MS, powered by AIM technology is the world’s most sensitive Triple Quadrupole.
18
TSQ Quantiva MS Sensitivity Study: Pesticides Limits of Quantitation (ppb)
and %RSD (no IS)
Compound Name TSQ Vantage MS TSQ Quantiva MS LOQ Decrease
Oxamyl 20.0 (3%) 0.5 (3%) 40 Methomyl 5.0 (11%) 0.5 (4%) 10
Carbendazim 5.0 (5%) 0.5 (7%) 10 Thiabendazole 1.0 (6%) 0.5 (5%) 2
Aldicarb 20.0 (10%) 0.5 (15%) 40 Simazine 1.0 (14%) 0.5 (4%) 2 Atrazine 1.0 (11%) 0.1 (11%) 10
Azoxystrobin 0.5 (10%) 0.1 (3%) 5 Propanil 100.0 (13%) 5.0 (7%) 20
Teflubenzuron 200.0 (12%) 10.0 (3%) 20 Calculated Value +/- 20% Average
Average Improvement 16x with Excellent Precision
19
Y = 0.000151016*X R^2 = 0.9985 W: Equal
0 2000 4000 6000 8000 10000 12000 fg on Column
0.0
0.5
1.0
1.5
2.0
Are
a R
atio
Y = -0.0153593+0.000148287*X R^2 = 0.9984 W: Equal
0 2000 4000 6000 8000 10000 12000 fg on Column
0.0
0.5
1.0
1.5
2.0
Are
a R
atio
TSQ Quantiva MS Sensitivity Study: Testosterone
TSQ Vantage MS
TSQ Quantiva MS
TestosteroneY = 0.000151016*X R^2 = 0.9985 W: Equal
0 50 100 150 200 250fg on colyumn
0.00
0.01
0.02
0.03
0.04
0.05
Are
a R
atio
fg on Column
Are
a R
atio
TestosteroneY = -0.0153593+0.000148287*X R^2 = 0.9984 W: Equal
0 50 100 150 200 250fg on Column
0.00
0.01
0.02
0.03
0.04
0.05
Are
a R
atio
fg on Column
Are
a R
atio
20
TSQ Quantiva MS TSQ Vantage MS
TSQ Quantiva MS Sensitivity Study: Testosterone
125 fg on Column
2.6 2.7 2.8 2.9 3.0 3.1 3.2 3.3 3.4 3.5 Time (min)
0
100
Rel
ativ
e A
bund
ance
3.25
2.52 3.22 2.59 2.64
3.05
2.82 2.56 2.99 2.74 3.52
3.39 3.14 2.68 3.33 3.18
3.10 2.77 3.42 2.96 2.90 3.50
3.46
RT: 2.51 - 3.53
2.6 2.7 2.8 2.9 3.0 3.1 3.2 3.3 3.4 3.5Time (min)
0
5
10
15
20
25
30
35
40
45
50
55
60
65
70
75
80
85
90
95
100
Re
lativ
e A
bu
nd
an
ce
RT: 3.07MA: 3215
2.66
2.522.63
2.78
2.822.562.73
2.90
3.012.992.95
3.413.13
3.15 3.363.18 3.26
3.333.49
21
LVNELTEFAK (BSA) spiked in E.Coli matrices 1 attamoles on column
NVNDVIAPAFVK (Enolase) spiked in E.Coli matrices 1 attamoles on column
S/N = 50
S/N = 12
TSQ Quantiva MS Sensitivity Study: Peptides
0
2000
4000
6000
8000
10000
12000
1 attomol 10 attomol
Run 1 Run 2 Run 3
0
1000
2000
3000
4000
5000
6000
1 attomol 10 attomol
Run 1 Run 2 Run 3
6.0% CV
5.6% CV
4.9% CV
6.2% CV
1 attomole on column!
With excellent precision
22
Ultimate Sensitivity on the TSQ Quantiva MS 3.7% RSD for
Triplicate Injections Blank RT: 0.06 - 1.51
0.2 0.4 0.6 0.8 1.0 1.2 1.4Time (min)
0
20
40
60
80
100
Rel
ativ
e A
bund
ance
0
20
40
60
80
100
Rel
ativ
e A
bund
ance
0
20
40
60
80
100
Rel
ativ
e A
bund
ance
RT: 0.77 A: 241
RT: 0.77 A: 254
RT: 0.77 A: 259
RT: 0.06 - 1.50
0.2 0.4 0.6 0.8 1.0 1.2 1.4Time (min)
0
20
40
60
80
100
Rel
ativ
e Ab
unda
nce
Rep 1
Rep 2
Rep 3
70ag Verapamil in
plasma, on column
92K Molecules Verapamil in
plasma, on column
23
TSQ Quantiva MS and TSQ Endura MS: Speed
Challenge TSQ Quantiva MS
Need the ability to analyze more compounds in very short run times.
The AIM technology includes an ultra fast collision cell designed for fast SRMs with no loss.
24
0
2000000
4000000
6000000
8000000
10000000
12000000
14000000
9.9 SRM/sec
47 SRM/sec
166 SRM/sec
333 SRMs/sec
500 SRMS/Sec
Peak
Are
a
0
1000000
2000000
3000000
4000000
5000000
6000000
1.75 1.85 1.95 2.05
Sign
al
Time (minutes)
250 Transitions Monitored Simultaneously
167 SRMS/second 500 SRMS/second
Ultimate SRM Speed on the TSQ Quantiva MS
3 points across the peak, per transition
10 points across the peak, per transition
Traditional Triple Quads
High Sensitivity at Every Speed
25
1ppb %RSDAsulam 4.88Flonicamid 2.67AzinophosMethyloxon 5.93Fenamiphos 1.94Norflurazon 3.36Tebuthiuron 7.71
Triplicate injections- No Internal Standard Automatic Integration
500 SRMs/Second in Onion Matrix
0
5
10
15
20
25
30
35
40
45
50
2 4 6 8 10 12 14 16 18 20 22 24 Time (min)
26
960 1200 1440 1680 1920 0.00E+000
2.70E+007
5.40E+007
8.10E+007
1.08E+008
1.35E+008 Signal Switch
Sign
al
Time (mSec)
Positive Mode
Negative Mode
Switch
Experimental
• 100 Scans in each mode • SIM Mode with width of 0.7 • m/z 508.2 in positive • n/z 506.2 in negative • Points spaced by ~5 ms
Fast Polarity Switching
20 milliseconds for complete
signal stability
1440 1460 1480 1500 1520 15400
3000000
6000000
9000000
12000000
15000000 Signal Switch
Sig
nal
Time (mSec)
Stabilized Signal ∼20 mSec
Signal Switch
Sign
al
Time (mSec)
27
TSQ Quantiva MS: Ease of Use
Challenge TSQ Quantiva MS
Analysts don’t have time to waste learning new software or optimizing methods.
The entire software workflow has been redesigned to remove un-needed parameters.
28
TSQ Quantiva MS—Easy to Clean
No tools required for removal of
ion optics
29
Ultimate Usability of the TSQ Quantiva MS
The new TSQ software is designed to allow all users to rapidly build new methods.
30
TSQ Quantiva MS and TSQ Endura MS
TSQ Endura MS Extreme Quantitative Value • Designed for non-stop operation.
• For scientists who need to run routine samples day in and day out.
TSQ Quantiva MS Extreme Quantitative Performance • Designed for the most
challenging assays.
• For scientists needing to stay at the forefront of analytical technology. Thermo Scientific™
TSQ Quantiva™ MS Thermo Scientific™ TSQ Endura™ MS
Mass Range 10-1850 10-3400
Max SRM Number 30,000 SRMs 30,000 SRMs
SRM/Sec 500 SRMs/sec 500 SRMs/sec
Ion Optics Active Ion Management (AIM)
• Ion Max NG source • Electrodynamic
ion funnel • ion beam guide with
neutral blocker • 6 mm HyperQuad
quadrupoles with asymmetric RF drive
S-LENS with Beam Blocker Technology
Quadrupole Design 4mm Quadrupoles with Asymetric RF
Reserpine Specification
100,000 : 1 S/N for 1 pg Reserpine
10,000 : 1 S/N for 1 pg Reserpine
31 The world leader in serving science
Vlad Zabrouskov Shannon Eliuk
Orbitrap Fusion Tribrid MS
32
Challenges in Life Science Mass Spectrometry
Complex Matrices
Small Sample Amounts
Large Sample Numbers
Low Concentration
Thermo Scientific Orbitrap Fusion Tribrid MS
Difficult to Operate Mass Spectrometers
33
PER
FOR
MA
NC
E USA
BILITY
Revolutionary Architecture
Intuitive, easy to use Software
Three high-performance Analyzers
Dynamic Scan Management for operational
efficiency
34
Orbitrap Fusion
Unmatched Analytical Performance
Revolutionary performance
Exceptional versatility
Unprecedented usability
Orbitrap Fusion Tribrid Mass Spectrometer
Tribrid (trī-brǝd) n. three Mass Analyzers working together to produce unmatched analytical results
1
2
3
35
Reduced noise and increased robustness Active beam guide prevents neutrals from entering the quadrupole and improves robustness.
Compact ETD source uses Townsend discharge, making it more reliable and easier to use.
Excellent sensitivity and selectivity Quadrupole precursor selection at isolation widths down to 0.4 amu improves sensitivity and selectivity.
Maximum throughput by massive parallelization Ion-routing multipole facilitates parallel analysis and performs HCD at any fragmentation stage.
MSn and sensitive mass analysis Dual-pressure linear ion trap provides MSn CID and ETD
fragmentation and fast, sensitive mass analysis.
Unsurpassed resolution and speed Ultra-high-field Orbitrap mass analyzer
offers resolution exceeding 450,000 and scan speeds up to 15 Hz.
Active beam guide (ABG)
Ultra-high-field Orbitrap mass analyzer
Dual-pressure linear ion trap
Ion-routing multipole
Quadrupole mass filter
EASY-ETD ion source
Orbitrap Fusion Tribrid Mass Spectrometer
36
Orbitrap Fusion Tribrid Mass Spectrometer
Unmatched Analytical Performance 450,000 Resolution to remove spectral interferences
CID/HCD/ETD detected by the Ion Trap or Orbitrap at any level of MSn for maximum experimental flexibility
Powered by the new Dynamic Scan Management architecture that ensures efficient operation of the mass spectrometer
Scan rate OTMS2 15 Hz
Scan rate ITMS2 20 Hz
Max resolution 450K at m/z 195
Quad isolation down to 0.4 amu
Ion trap isolation down to 0.2 amu
Mass Accuracy 3 ppm ext, 1 ppm int
Dissociation CID, HCD, ETD
MSn Up to MS10 in ion trap or Orbitrap
Analyzers Q, OTMS, ITMS
Detectors Ion Trap, Orbitrap
Compact 1186 x 674 x 650 mm (w, d, h)
37
>450,000 Resolution on the Orbitrap Fusion MS
O N N S
N O
N H O H
310 315 320 325 330m/z
0
10
20
30
40
50
60
70
80
90
100
Rel
ativ
e A
bund
ance
317.1642
318.1691
319.1736333.1586
Timolol: glaucoma drug, beta blocker 0.3 ppm
318.
31
60,000 Resolution 120,000 Resolution 240,000 Resolution > 450,000 Resolution
Cocaethylene: Ethanol + Cocaine 1.6 ppm
O
O
N O
O
318.10 318.15 318.20 318.25 0
100 318.1689 318.1692
318.2401 318.1181 318.2275 318.1806
318.1696
318.1675
318.1611 318.2400
318.1694
318.1676
318.1610
m/z
This resolution is an order of
magnitude better than any
competitive instrument
38
Orbitrap Fusion Tribrid Mass Spectrometer
Challenge Orbitrap Fusion MS Samples with high complexity, wide dynamic range, and need for low level sensitivity.
Flexible instrument architecture lets you analyze low concentration species in the ultra-sensitive ion trap yet also flexible enough to analyze any MSn product in the Orbitrap analyzer.
39
Ion Routing Multipole
C-Trap
Ion Trap
HPC
LPC
MP0 Q1
Full Orbitrap MS scan
1st parent ion isolation in Q1
1st MS2 scan: CID in the Ion Trap 2nd parent ion isolation in Q1
Orbitrap
2nd MS2 scan: CID in the Ion Trap
Full Orbitrap MS scan
m/z
Full FTMS Scan m/z
1st MS2 CID Scan
m/z
2nd MS2 CID Scan
Data Dependent Experiment: OTMS> CID ITMS2
Dynamic Scan Management Ensures Efficiency
40
Full Scan, 240K, 0.5s 1 2 3 4 5 6 7 8 9 10
1 2 3 4 5 6 7 8 9 10
450 500 550 600 650 700 750 800 850m/z
654.31
654.98
583.73 655.31473.31
557.61 620.38557.94 644.84584.23446.21
790.43668.81512.77446.54 488.27
558.28 669.31469.75 733.46 760.88517.28493.31 621.38416.25 717.84 849.40791.43
537.30
584.74
440.22
446.88 669.82 835.91710.83 856.34761.89416.75 468.26
611.64
809.44 877
Ion Trafficking and Dynamic Scan Management
Parent Ion Q1 isolation
HPC
LPC
Ion Injection
Full OTMS Scan
Ion Trap Detection of the Fragments
Full Scan, 240K, 0.5s Full Scan, 240K, 0.5s 1 2 3
41
Speed = Many More Points Across LC Peak 1 ug HeLa, 140 min run
16
245
76
424
MS MS/MS
Scans in 40s OT Elite Orbitrap Fusion
10 15 20 25 30 35 40 45 50 55 60 65 70 Time (min)
0
100
Rel
ativ
e A
bund
ance
26.58 576.29 67.72
652.36 31.39 508.74 19.59
599.77 6.41 533.27 12.84 408.73
56.07 590.81
39.23 566.77
51.94 581.31 60.33
472.77 65.77 706.40
10 15 20 25 30 35 40 45 50 55 60 65 70
Time (min)
0
100
Rel
ativ
e A
bund
ance
26.58 576.29 67.72
652.36 31.39 508.74
19.59 599.77 6.41
533.27 12.84 408.73
56.07 590.81
39.23 566.77
51.94 581.31 60.33
472.77 65.77 706.40
Top Speed
27.0 27.1 27.2 27.3 27.4 27.5 27.6 27.7 27.8 27.9 28.0 0
100
27.45 508.75
27.51 508.75
27.44 508.75
27.56 508.74 27.38
508.74 27.68 576.28 27.26 472.76 27.75 576.28 27.20 472.76
28.00 485.28
27.07 812.41
27.89 624.80
Time (min)
Rel
ativ
e A
bund
ance
Orbitrap Fusion MS
Top N
~40s 31.0 31.1 31.2 31.3 31.4 31.5 31.6 31.7 31.8 31.9
Time (min)
0
100
Rel
ativ
e A
bund
ance
31.39 508.74 31.35
508.74 31.53
576.28 31.31
508.74 31.62
576.28 31.26 508.74 31.80 403.22
31.00 640.30 31.09 572.31
Orbitrap Elite MS
42
3604
2861
4996
3939
140 Min 70 Min
Protein Groups
Orbitrap Elite MS Orbitrap Fusion MS
Orbitrap Fusion MS outperforms
Orbitrap Elite MS in half the time
1 ug HeLa
43
Orbitrap Fusion MS Detects More Low-Abundance Proteins
363%
198% 172%
154% 137% 127% 116% 119% 116% 121%
0.0
2.0
4.0
1.5
2.0
2.5
3.0
3.5
4.0
4.5
5.0
5.5
6.0
Low Level Detection on Orbitrap Fusion MS
29
500
1226
3576
4445
3119
2208
732
181 47 0
1250
2500
3750
5000
1.5
2.0
2.5
3.0
3.5
4.0
4.5
5.0
5.5
6.0
Protein Molecules Per Cell, Log 10
Unique Peptides as a function of the protein cellular abundance Orbitrap Elite MS Q Exactive MS Orbitrap Fusion MS
Pept
ides
Det
ecte
d Fo
ld Im
prov
emen
t
Detection of low-abundance proteins is key for understanding biological systems
Ghaemmaghami, S.; Huh, W. K.; Bower, K.; Howson, R. W.; Belle, A.; Dephoure, N.; O’Shea, E. K.; Weissman, J. S. Nature 2003, 16, 737–7341.
Transcription Factors
44
Orbitrap Fusion Tribrid Mass Spectrometer
Challenge Orbitrap Fusion MS As proteomics becomes more quantitative, the ability to perform relative quantitation of many samples accurately is critical.
New capabilities driven by the unique hardware architecture have produced a breakthrough with Tandem Mass Tag multiplexing performance.
45
Proteomics in Space and Time
Wild Type, 8 runs
Mutant, 8 runs
X3 = 24
X3 = 24 TMT 6 labeling technology lets you multiplex 6 runs and
reduce your experiments from 144 h to 24 h
46
Isobaric Labeling: Challenges and Resolutions
Peptides Labeled Peptides
Full MS
One Signal 6 Reporter Ions m/z m/z
MS/MS
LC-MS/MS
HCD FRAGMENTATION
1 2
3 4
5 6
47
MS2 Isolation
Reference: Ting et al., Nature Methods, Vol 8, No 11, 2011
Interference
Precursor
Spectral Noise Level
Interference Compresses TMT Ratios
0
4
8
12 Ratios for Yeast Peptides
Expected FTMS2 OT Elite
0
4
8
12
126 127 128 129 130 131
Human
0
4
8
12
126 127 128 129 130 131
Yeast
TMT Ratios 128/126 127/126 126/131
48
HCD MS3, OT
MS3 TMT Workflow for Accurate Quantitation
Source: Data courtesy Graeme McAllister, Steve Gygi Lab, Harvard University
Precursor Ion
CID MS2, Ion Trap
0
2
4
6
8
10 Expected
FTMS2 OT Elite
FTMS3 OT Elite
TMT Ratios 128/126 127/126 126/131
0
500
1000
1500
2000
2500
Unique Peptides Quantified Unique Peptides
Loss of Sensitivity
49
HCD MS3, OT
Synchronous Precursor Selection Allows for Accurate and Sensitive Quantitation
Source: Data courtesy Graeme McAllister
Precursor Ion
Synchronous Precursor Selection CID MS2, Ion Trap
0
2
4
6
8
10
128/126 127/126 126/131 TMT Ratio
Expected
FTMS3 OT Elite
SPS FTMS3 OT Fusion
50
Ion Routing Multipole
C-Trap
Ion Trap
HPC
LPC
MP0 Q1
Full Orbitrap MS scan
Parent ion isolation in Q1
MS2 scan: CID in the Ion Trap
SPS HCD FTMS3 scan Synchronous Precursor Selection in the Ion Trap
HCD MS3
FTMS detection of MS3 fragments
OTMS> CID ITMS2> SPS HCD OTMS3 Orbitrap
m/z
Full OTMS Scan m/z
MS2 CID Scan
m/z
HCD MS3 OTMS scan
TMT3 Experiment, Powered by Synchronous Precursor Selection
51
Orbitrap Fusion Tribrid Mass Spectrometer
Challenge Orbitrap Fusion MS
Mass spectrometers grew more powerful, sophisticated and ….difficult to use. Many labs demand that new analytical methods be created quickly and easily.
Revolutionary new software allows for drag-and-drop method creation and editing.
52
Drag-n-drop Method Editor is Easy to Use
53
Drag-n-drop Method Editor is Easy to Use
54
Drag-n-drop Method Editor is Easy to Use
55
Orbitrap Fusion Tribrid Mass Spectrometer
Challenge Orbitrap Fusion MS Peptide and protein characterization with mapping sequence alteration, labile PTMs and increasing amino acid sequence coverage.
EASY ETD: new ETD source, anion filtering and dynamic scan management significantly improves quality of ETD spectra.
56
Reagent/Internal Calibrant Source
Tube lens is replaced with discharge ion source
Before Now
57
Discharge Ion Source Detail
Reagent Entry Aperture
Electron Entry Aperture
Anion Exit Aperture
Ion Axis
58
Compact Townsend Discharge Ionization,
No Filament
Stable Reagent Ion Flux
New Front Reagent Source: ETD and Internal Calibration
59
ETD: Intelligent Precursor Ion Prioritization
SAX enriched human serum glycopeptides, 120 min C18 LC
345
826
OT Elite OT Fusion
Identified Glycopeptides
Orbitrap ETD Spectrum
60
1 2 3 4 5 6 7 8 9 0
100
R e l
a t i v
e I n
t e n s
i t y
Time (min)
362.11690 m/z
N
N H O
O S N
O
= O
H+
Internal Calibration: LC/MS of Omeprazole Metabolites
61
3.7 3.8 3.9 4.0 4.1 4.2 4.3 4.4 4.5 4.6 4.7 4.8 4.9 Time (min)
0.55 ppm
0.19 ppm
0.36 ppm
0.28 ppm
0.47 ppm
0.35 ppm
0.55 ppm
0.28 ppm
0.28 ppm
0.03 ppm
-0.06 ppm
0.11 ppm 0.19 ppm
0.02 ppm
0.47 ppm
0.55 ppm 0.55 ppm
0.55 ppm
0.47 ppm
0.55 ppm
0.19 ppm
0.19 ppm
-0.14 ppm 0.19 ppm
Internal Calibration: LC/MS of Omeprazole Metabolites
62
3.7 3.8 3.9 4.0 4.1 4.2 4.3 4.4 4.5 4.6 4.7 4.8 4.9 Time (min)
0.55 ppm
0.19 ppm
0.36 ppm
0.28 ppm
0.47 ppm
0.35 ppm
0.55 ppm
0.28 ppm
0.28 ppm
0.03 ppm
-0.06 ppm
0.11 ppm 0.19 ppm
0.02 ppm
0.47 ppm
0.55 ppm 0.55 ppm
0.55 ppm
0.47 ppm
0.55 ppm
0.19 ppm
0.19 ppm
-0.14 ppm 0.19 ppm
Internal Calibration: LC/MS of Omeprazole Metabolites
63
3.7 3.8 3.9 4.0 4.1 4.2 4.3 4.4 4.5 4.6 4.7 4.8 4.9 Time (min)
0.55 ppm
0.19 ppm
0.36 ppm
0.28 ppm
0.47 ppm
0.35 ppm
0.55 ppm
0.28 ppm
0.28 ppm
0.03 ppm
-0.06 ppm
0.11 ppm 0.19 ppm
0.02 ppm
0.47 ppm
0.55 ppm 0.55 ppm
0.55 ppm
0.47 ppm
0.55 ppm
0.19 ppm
0.19 ppm
-0.14 ppm 0.19 ppm
Internal Calibration: LC/MS of Omeprazole Metabolites
64
Waters mAb Standard: Mass Analysis of Large Proteins Observed Average
Mass Expected
Mass 148220.8 148220.4 148382.8 148382.5 148545.2 148544.6 148708.3 148706.7 148868.1 148868.8
65
Top Down MSn of Carbonic Anhydrase
36+
ETD MS2
ETD MS3
7+
HCD MS2
66
Combined Sequence Coverage
ETD HCD ETD MS3 (only unique z fragments are shown)
67
Isotopic Resolution on 47+ of Bovine Enolase
R=240,0000 M 46,641.39 Da
68
Orbitrap Fusion Tribrid Mass Spectrometer
Challenge Orbitrap Fusion MS A lot of molecules require special fragmentation conditions to reveal their structural information.
Instrument architecture allows for full flexibility of MSn experiments for structural analysis including novel workflows such as HCD3 and MS2 HCD followed by MS3 CID.
69
Ion Routing Multipole
C-Trap
Ion Trap
HPC
LPC
Orbitrap
MP0 Q1
Full Orbitrap MS scan
Parent ion isolation in Q1 and HCD MS2> OTMS detection of MS2 fragments HCD fragment isolation in the ion trap and HCD MS3
Orbitrap MS detection of HCD MS3 fragments
OTMS> HCD OTMS2> HCD OTMS3
m/z
Full FTMS Scan
m/z
HCD FTMS2 Scan
m/z
HCD FTMS3 Scan
HCD MSn is Unique to Orbitrap Fusion MS
70
Propranolol Top 3 HCD MS3
Mass accuracy for all fragments
< 2.5 ppm
MS2 R
elat
ive
Abun
danc
e
MS3 +
50 60 70 80 90 100 110 120 130 140 150 160 170 180 190 m/z
0
100
Rel
ativ
e Ab
unda
nce
155.0858
165.0701
141.0702 173.5394
183.0803 125.4088 48.7723 74.0841 62.5255 80.3531
+ +
N H O H
+
O
O
O N 3 H O H
+
100 110 120 130 140 150 160 170 180 190 200 210 220 230 240 250 260 270 m/z
183.0807
260.1647 116.1073
157.0650
165.0701 141.0701 129.0702 173.5344 218.1178
114.0915 242.1538 225.1275
152.0627
187.3135 0
100
71
HCD as MS2 Event in an Ion Tree Experiment
• In order to get diagnostic fragmentation for structure ID we build a fragmentation tree.
• We have to go down far enough to get rich fragmentation; from there we can branch wide and learn how fragments connect together.
• Using HCD as a “full mass range MS2 scan” for fragment selection lets us branch sooner and get faster data.
HCD MS2
MS3 MS3 MS3 MS3
CID MS2
CID MS3
MS4 MS4 MS4 Extra MSn stage = lost time and signal
72
100 200 300 400 500m/z
0
20
40
60
80
100
Rel
ativ
e Ab
unda
nce
171.1167
466.2732
131.0855
262.158591.0543233.1164 298.1796
HCD MS2
3
1 2
Fexofenadine: HCD or CID MS2
CID MS3
50 100 150m/z
0
50
100143.0
129.1
156.0115.091.1
2 1
CID MS2
150 200 250 300 350 400 450 500 m/z 0
20
40
60
80
100 466.2721
503.2963
CID MS3
200 300 400 m/z 0
20
40
60
80
100 171.12
298.18 262.16
288.17 200 400
m/z
0
50
100466.2
298.1171.0379.1
2
50 100m/z
0
50
10091.0
116.052.7
3
1
73
Orbitrap Fusion Tribrid Mass Spectrometer
Orbitrap Fusion MS
Orbitrap Fusion Tribrid MS is the ultimate research tool for those facing the most difficult analytical challenges in cell biology, proteomics, and structural analysis.
SOLUTION
Up to 450,000 resolution available to separate chemical noise from real analyte.
Flexible instrument architecture lets you analyze low concentration species in the ultra-sensitive ion trap yet also flexible enough to analyze any MSn product in the Orbitrap analyzer.
New software powered by Dynamic Scan Management tools automatically adjusts scan parameters on the fly to maximize analytical results.
74
The Industry’s Leading Portfolio of MS Solutions
HR/AM
MS, MSn
App
lied
Mar
kets
Research M
arkets
Non-targeted Analysis
Targeted Analysis
Quantitative Qualitative Transform
Your Science
Ion Traps Triple Quads
Tribrid Orbitrap MS
Exactive Series MS
• Biomarker Discovery • Proteomics • Metabolism
• Metabolomics • Proteomics • Bioanalysis
• Food Safety • Environmental • Clinical/Toxicology
• Metabolomics • PTM Analysis • Lipidomics
75
Transform Your Science
Recent Advances in Mass Spectrometry - Introducing the Unique Orbitrap Fusion and Quantiva Triple Quadrupole Mass SpectrometersSlide Number 2Slide Number 3Advances in Mass Spectrometry in BriefTransforming Quantitative Analysis with TSQ Quantiva and TSQ Endura Triple Quad LC/MSChallenges in Quantitative Mass SpectrometryNext Generation Triple Quadrupole MSTSQ Endura MS and TSQ Quantiva MSTSQ Quantiva MS—Powered by AIM TechnologyTSQ Quantiva Triple Quadrupole Mass SpectrometerTSQ Quantiva MS—Powered by AIM TechnologyTSQ Quantiva MS—Powered by AIM TechnologyTSQ Quantiva MS—Powered by AIM TechnologyTSQ Quantiva MS—Powered by AIM TechnologyTSQ Quantiva MS—Powered by AIM TechnologyTSQ Quantiva—Powered by AIM TechnologyTSQ Quantiva MS: SensitivityTSQ Quantiva MS Sensitivity Study: PesticidesTSQ Quantiva MS Sensitivity Study: TestosteroneTSQ Quantiva MS Sensitivity Study: TestosteroneTSQ Quantiva MS Sensitivity Study: PeptidesUltimate Sensitivity on the TSQ Quantiva MSTSQ Quantiva MS and TSQ Endura MS: SpeedUltimate SRM Speed on the TSQ Quantiva MS500 SRMs/Second in Onion MatrixFast Polarity SwitchingTSQ Quantiva MS: Ease of UseTSQ Quantiva MS—Easy to CleanUltimate Usability of the TSQ Quantiva MSTSQ Quantiva MS and TSQ Endura MSOrbitrap Fusion Tribrid MSChallenges in Life Science Mass SpectrometrySlide Number 33Orbitrap Fusion Tribrid Mass SpectrometerOrbitrap Fusion Tribrid Mass SpectrometerOrbitrap Fusion Tribrid Mass Spectrometer>450,000 Resolution on the Orbitrap Fusion MSOrbitrap Fusion Tribrid Mass SpectrometerSlide Number 39Ion Trafficking and Dynamic Scan ManagementSpeed = Many More Points Across LC PeakProtein GroupsLow Level Detection on Orbitrap Fusion MSOrbitrap Fusion Tribrid Mass SpectrometerProteomics in Space and TimeIsobaric Labeling: Challenges and ResolutionsInterference Compresses TMT RatiosMS3 TMT Workflow for Accurate QuantitationSynchronous Precursor Selection Allows for �Accurate and Sensitive QuantitationTMT3 Experiment, Powered by Synchronous Precursor SelectionOrbitrap Fusion Tribrid Mass SpectrometerDrag-n-drop Method Editor is Easy to UseDrag-n-drop Method Editor is Easy to UseDrag-n-drop Method Editor is Easy to UseOrbitrap Fusion Tribrid Mass SpectrometerReagent/Internal Calibrant SourceDischarge Ion Source DetailNew Front Reagent Source: ETD and Internal CalibrationETD: Intelligent Precursor Ion Prioritization Internal Calibration: LC/MS of Omeprazole Metabolites Internal Calibration: LC/MS of Omeprazole Metabolites Internal Calibration: LC/MS of Omeprazole Metabolites Internal Calibration: LC/MS of Omeprazole Metabolites Waters mAb Standard: Mass Analysis of Large ProteinsTop Down MSn of Carbonic AnhydraseCombined Sequence CoverageIsotopic Resolution on 47+ of Bovine EnolaseOrbitrap Fusion Tribrid Mass SpectrometerHCD MSn is Unique to Orbitrap Fusion MSPropranolol Top 3 HCD MS3HCD as MS2 Event in an Ion Tree ExperimentFexofenadine: HCD or CID MS2Orbitrap Fusion Tribrid Mass SpectrometerSlide Number 74Slide Number 75