Recent Advances in Mass Spectrometry - Thermo Fisherapps.thermoscientific.com/media/cmd/hypersite-events... · 2014. 2. 27. · TSQ Endura MS . Extreme Quantitative Value • Designed

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


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.


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




3 4 1 2

13




1 2 3 4

14

Increase in Signal 40%




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




1 3 4 2

16

TSQ Quantiva—Powered by AIM Technology

Together this technology can solve your toughest analytical challenges.



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


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


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


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


36


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


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


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


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


C-Trap

Ion Trap

HPC

LPC

MP0 Q1


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


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


54


55


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


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


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


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


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


C-Trap

Ion Trap

HPC

LPC

Orbitrap

MP0 Q1


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 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

Documents

Recent Advances in Mass Spectrometry - Thermo Fisherapps.thermoscientific.com/media/cmd/hypersite-events... · 2014. 2. 27. · TSQ Endura MS . Extreme Quantitative Value • Designed