Pre-Pittcon Sales Training Webinars

The world leader in serving science
LTQ Velos a jeho kombinace s
FT-MS Orbitrap
Je něco převratně nového v
iontových pastech?
Petr Verner
ThermoFisher Scientific, Praha
LTQ Velos
 Co je nového?
• Hardware
• HESI II probe standard
• S-Lens ion optics
• Dual Cell Linear Trap
• Prakticky?
• 2x Rychlejší skenování
• Zvýšená citlivost
• Lepší statistika
• Vyšší rozlišení
2
Customer value
Hardware features
Benefit
Customer Value
Dual Cell Linear Trap
1.Higher Isolation efficiency
2.Higher CID efficiency
3.Faster scan speed
4.Higher resolution
increased sample IDs
S-Lens
Increased ion flux
Higher sensitivity
Lower fill time
Low-level analyte
detection
HESI II probe
Better sample desolvation
Higher sensitivity
Less solvent noise
Increased Robustness
Low-level analyte
detection
The increased sensitivity and decreased cycle time of the LTQ Velos allow greater depth of sample
analysis resulting in increased protein coverage and enhanced access to MSn structural elucidation
on a chromatographic time scale.
3
LTQ Velos – What’s new?
 Source changes
• HESI II standard
• Nový design sweep cap
• Stejné jako na TSQ Vantage
4
Dual Cell Linear Ion Trap Configuration
No Tube Lens or Skimmer
Front
Lens
Center
Lens
Detector
1
Octopole
HPT
HPC
LPC
Detector
2
5
Back
Lens
LTQ Velos
 S-Lens – podobné jako na TSQ Vantage
6
LTQ Velos – What’s new?
 Zahnutý Q0
7
LTQ Velos
 It has 2 linear traps
8
High Pressure
Low Pressure
High Trapping Efficiency
High Dissociation Efficiency
High Isolation Efficiency
5.0 x10-3 Torr He
Square
Quadrupoles
Electrospray
Source
Ion Transfer
Tube
Higher Resolution/Scan Rate
3.5x10-4 Torr He
Front
Lens
Center
Lens
Detector
1
Octopole
HPT
HPC
SRIG
Inter- multipole
Lens 1
Split Gate
Lens
9
LPC
Detector
2
Back
Lens
n
Ion Trap Scan Function - MS
Ion
Injection
Ion
Isolation
Ion
Dissociation
n-1
Mass
Analysis
1
10
Motivation 1: Trapping Efficiency
He Pressure Effects - TIC
2000
Standard
Pressure
~60% Trap Eff.
TIC
1600
Higher
Pressure
>95% Trap Eff.
1200
800
400
0
0
1
2
3
4
He Pressure (mtorr)
11
5
6
Motivation 2: CID Efficiency
He Pressure Effects – CID Efficiency
for MRFA (m/z 524, 30 ms)
100
CID Efficiency
80
Higher Pressure
>80% CID Eff.
60
Standard Pressure
~68% CID Eff.
40
20
0
2
4
6
He Pressure (mtorr)
12
8
10
Motivation 3: Resolution/Scan Rate
He Pressure Effects - Peak Width at Scan
Rate 16,667 amu/sec
Peak Width (amu)
1.8
1.6
1.4
Standard Pressure
0.55 amu FWHM
1.2
1
0.8
0.6
0.4
Lower Pressure
<0.4 amu FWHM
0.2
0
0
1
2
3
4
He Pressure (mtorr)
13
5
6
Motivation 3: Resolution/Scan Rate
Effect of Scan Rate on Peak Width
(Res. Ej. Optimized)
0.7
FWHM (amu)
0.6
0.5
2x Current
Scan Rate
0.4
0.3
Current
Scan Rate
0.2
Scan Rate Range 1
Scan Rate Range 2
0.1
Scan Rate Range 3
0
0
5000
10000
15000
20000
Scan Rate (amu/sec)
14
25000
30000
35000
LTQ Velos – dobrá, ale ještě něco nového?
 Nový RF system
 Nový Digital board
 Nový Source Board
 Nová geometrie lineárních pastí
 Nová Transfer ion optics
 Krátce: zvenku může připomínat staré dobré LTQ, ale uvnitř je
mnoho novinek
15
LTQ Velos
 Co tedy přináší nového uživateli?
• Dvojnásobnou rychlost skenování (vyjma TURBO a
UltraZoom Skenu)
• Lepší rozlišení (i v Ultra Zoom skenu)
• Výrazně zkrácený tzv. Ion injection time
• Zvýšená citlivost 4-5X
16
Scan Speed vs Resolution
LCQ Fleet
LTQ XL
LTQ Velos
TurboScan
80,000 amu/s
@ 3 amu FWHM
125,000 amu/s
@ 3 amu FWHM
125,000 amu/s
@ 3 amu FWHM
Normal
12,500 amu/s
@ 0.7 amu FWHM
16,700 amu/s
@ 0.7 amu FWHM
33,300 amu/s
@ 0.6 amu FWHM
Enhanced
5000 amu/s
@ 0.35 amu FWHM
5000 amu/s
@ 0.45 amu FWHM
10,000 amu/s
@ 0.35 amu FWHM
ZoomScan
1100 amu/s
@ 0.25 amu FWHM
1100 amu/s
@ 0.3 amu FWHM
2200 amu/s
@ 0.25 amu FWHM
Ultra ZoomScan
N/A
27 amu/s
@ 0.15 amu FWHM
27 amu/s
@ 0.1 amu FWHM
17
Installation Specs
LCQ Fleet
LTQ XL
LTQ Velos
Reserpine
Concentration
2 uL 1 pg/uL
2 uL 125 fg/uL
2 uL 50 fg/uL
Signal to Noise
100:1
100:1
100:1
18
Co bude s LXQ?
 LXQ bude nadále vyráběno – jako součást ToxSpec analyzátoru
(klinická biochemie)
• Náhrada HPLC systému Remedy
 Jinak LCQ Fleet, LTQ XL, LTQ Velos
19
LCQ Fleet
• Exceptional Analytical Value
• Routine analysis of complex samples
• Proven MSn performance for compound
identification and structural elucidation
• Familiar platform - provides ease of use
Challenges
• Sample/Process
•
•
•
•
control
Reliability
Non MS operators
Complex Matrices
Multi-Component
Samples
Tools





Customer Benefits
LCQ Fleet
Accela
Hypersil Gold Columns
Metworks
Mass Frontier





20
Established reputation
Structural elucidation with MSn
Ease of use
Greater sensitivity than UV/Single
Quad
Greater Specificity
LTQ XL
•Exceptional coverage at breakthrough
speed
•Fast cycle time, high sensitivity
•Unmatched MSn spectral quality
•Platform for hybridization
Challenges
• Post Translational
•
•
•
Modification
Identification
Rapid gradients
Complex samples
Biological matrices
Tools
Customer Benefits
LTQ XL
 ETD
 MALDI
 Accela
 Hypersil Gold Columns
 Discoverer
 Metworks
 Mass Frontier


21





Identify the site of PTMs
Multiple fragmentation techniques
for maximum information
Tissue Imaging Solution
Excellent quality MSn spectra for
library searching
Upgradeable to Orbitrap
Upgradeable to ETD
LTQ Velos
• Balanced 2D linear trap
• Rapid reliable structural information
• Ultra-Fast cycle time – up to 10Hz
• The most sensitive full scan MS
• Standard with HESI
Challenges
• Complex matrices
• Co-Eluting
•
•
compounds
Post Translation
Modification
Early phase
metabolite ID and
Quan
Tools
Customer Benefits
LTQ Velos
 ETD
 Dual Pump
 Metworks
 Mass Frontier
 Discoverer







22
Fast cycle time for identifying coeluters
Simultaneous Quan and Qual
Excellent quality MSn spectra on
a u-HPLC time scale
Data Dependant Decision Tree
provides optimum fragmentation
Upgradeable to Orbitrap Velos
Upgradeable to ETD
MALDI v kombinaci s LTQ
 MALDI bude dostupné pouze s LTQ XL
• We will continue to work on the software, both control and data
processing to improve this product further.
 MALDI will NOT be available on the LTQ Velos
• Most of the LTQ Velos improvement are due to the source and
optics which are removed for MALDI
23
Alprazolam in Plasma
The world leader in serving science
Alprazolam LTQ Velos – Plasma with IS
5 orders of Dynamic range – LOQ 40fg on column
Alprazolam
Y = 0.000554475+0.0347005*X R^2 = 0.9963 W: 1/X
140
120
Alprazolam
Y = 0.000556373+0.0347005*X R^2 = 0.9963 W: 1/X
100
0.010
80
Area Ratio
0.008
60
0.006
0.004
40
0.002
20
0.000
0.00
0.05
0
0
1000
2000
3000
pg on column
25
4000
0.10
0.15
pg on column
0.20
0.25
Alprazolam LTQ Velos – Plasma with IS
pg on column
LTQ XL (%RSD)
LTQ Velos (%RSD)
0.04
12.84
0.1
9.44
0.2
4.60
0.4
7.48
5.79
1
20.82
3.28
2
5.81
3.52
4
4.21
6.10
10
5.56
3.13
20
5.08
3.85
40
3.82
2.20
100
2.05
3.45
200
0.86
6.26
400
2.02
4.32
1000
4.21
3.05
2000
1.60
3.94
4000
1.42
26
LTQ XL vs. LTQ Velos – 400fg on column
Relative Abundance
RT: 0.70 - 1.30 SM: 7G
100
95
90
85
80
75
70
65
60
55
50
45
40
35
30
25
20
15
10
5
0
0.8
0.9
SM: 7G
1.0
Time (min)
1.1
1.2
30 scans across the peak
Relative Abundance
RT: 0.70 - 1.30
100
95
90
85
80
75
70
65
60
55
50
45
40
35
30
25
20
15
10
5
0
10 scans across the peak
0.8
0.9
1.0
Time (min)
27
1.1
1.2
1.3
The world leader in serving science
Multi Component
Quantification
Amphetamine
Y = -0.00047078+0.00210668*X R^2 = 0.9969 W: 1/X
Codeine
Y = 0.000139457+0.00158557*X R^2 = 0.9968 W: 1/X
4.5
4.0
3.0
3.5
2.5
3.0
Area Ratio
2.0
1.5
2.5
2.0
1.5
1.0
1.0
0.5
0.5
0.0
0
0.0
500
1000
1500
2000
pg on column
Methamphetamine
Y = 0.000175724+0.00417322*X R^2 = 0.9946 W: 1/X
0
500
1000
1500
pg on column
2000
1000
pg on Column
2000
Lidocaine
Y = 0.000192073+0.00421984*X R^2 = 0.9966 W: 1/X
9
9
8
8
7
7
6
Area Ratio
Area Ratio
4 Compound Quan LTQ Velos – IS (5 scan events)
5
4
6
5
4
3
3
2
2
1
1
0
0
0
500
1000
pg on Column
1500
2000
0
29
500
1500
4 Compound Quan LTQ Velos – IS (5 scan events)
Level
pg on Column
Codeine
%RSD
Lidocaine
%RSD
Amphetamine
%RSD
Methamphetamine
%RSD
0.1
24.6
3.0
34.2
0.2
4.1
8.1
13.1
0.4
12.9
10.5
3.5
9.0
1
11.7
3.6
2.9
4.2
2
6.0
6.0
5.8
4.8
4
4.1
2.8
4.8
3.0
10
5.4
3.8
8.6
4.9
20
2.9
2.9
4.8
5.5
40
3.3
4.7
2.3
2.8
30
4 Compound Quan LTQ XL – IS (5 scan events)
Level
pg on Column
Codeine
%RSD
Lidocaine
%RSD
Amphetamine
%RSD
Methamphetamine
%RSD
0.1
0.2
0.4
1
40.7
2
63.2
17.9
4
13.7
8.3
12.8
9.0
10
10.6
13.9
11.0
3.2
20
13.8
5.3
8.9
8.2
40
4.7
2.2
13.2
10.7
31
15.91
4 Compound Quan LTQ Velos – IS (5 scan events)
4pg on column - No Smoothing
RT:
0.00 - 1.40
100
Amphetamine
80
60
40
20
100
0
80
Amphetamine-d6
60
40
20
100
0
80
Methamphetamine
60
40
20
100
0
80
60
Lidocaine
40
20
100
0
80
60
40
Codeine
20
0
0.0
0.2
0.4
0.6
0.8
Time (min)
32
1.0
1.2
1.4
4 Compound Quan LTQ XL – IS (5 scan events)
4 pg on column - No Smoothing
RT: 0.00 - 1.41
100
Amphetamine
80
60
40
20
0
100
80
Amphetamine-d6
60
40
20
Relative Abundance
0
100
80
Methamphetamine
60
40
20
0
100
80
Lidocaine
60
40
20
0
100
80
Codeine
60
40
20
0
0.0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
Time (min)
33
0.8
0.9
1.0
1.1
1.2
1.3
1.4
Source Ruggedness
The world leader in serving science
LTQ Velos Ruggedness – 600 injections singly crashed plasma
20 pg on Column of Alprazolam in Crashed Plasma
400000
350000
1.9% RSD with no internal standard
300000
Peak Area
250000
200000
150000
100000
50000
0
0
100
200
300
Number of injections
35
400
500
600
LTQ Velos Malathion
The world leader in serving science
LTQ Velos Full Scan MS2 – 10ppb Malathion
The Full Scan Solution to Chemical Noise
XIC m/z 99 Fragment
XIC m/z 285 Fragment
RT: 2.30 - 3.16 SM: 7G
RT: 2.30 - 3.16 SM: 7G
85
NL: 4.78E1
100
m/z= 98.50-99.50
F: ITMS
+ c ESI Full ms2
[email protected]
95
[90.00-350.00] MS
Genesis 90
MalathionQuan_039
85
80
80
75
75
70
70
65
65
60
60
100
95
Relative Abundance
90
55
50
45
50
45
40
35
35
30
30
25
25
20
20
15
15
10
10
5
5
2.4
2.5
2.6
2.7
2.8
Time (min)
2.9
3.0
NL: 4.1
m/z= 2
ITMS +
331.10
[90.00
Genes
Malath
55
40
0
2.3
AH: 3977
SN: 44
0
2.3
3.1
37
2.4
2.5
2.6
2.7
2.8
Time (min)
2.9
3.0
3.1
Calibration Curve 0.1 to 20 ppb Malathion
Malathion
Y = 1030.8+27661.5*X R^2 = 0.9904
W: 1/X
600000
500000
Area
400000
300000
200000
100000
0
0
5
10
ppb
38
15
20
0.1 ppb Malathion in 50X Diluted QuEChERS Extract
S/N 634
2.0E3
39
Proteomics
The world leader in serving science
Increase in Identification for the most complex mixtures
Increase in Protein ID
500 ng
Increase in Unique Peptide ID
500 ng
Increase in Protein ID for LTQ Vs. Velos
(500 ng)
1400
1200
64%
Increase in Unique Peptide ID for LTQ Vs. Velos
(500 ng)
55%
53%
3500
3000
77%
2500
1000
2000
800
LTQ
LTQ Velos
LTQ
LTQ Velos
600
1500
400
1000
200
500
0
0
'60 min'
'180 min'
'60 min'
41
'180 min'
Sensitivity for Low Level Injections (20 ng)
Increase in ID LTQ Vs. LTQ Velos
20 ng (C. Elegans)
500
400
1200
1000
104%
124%
800
300
Series1
LTQ XL
LTQ Velos
600
Series2
200
400
100
200
0
0
Unique Peptides
Proteins
42
Metabolism
The world leader in serving science
Maropitant Dog Sample
60 min incubation on LTQ XL
RT: 0.00 - 8.00
NL: 2.21E7
Base Peak F: ITMS + c ESI Full ms
[300.00-700.00] MS
Dog60min2_MS3Top5_LTQ
469.35
100
Base Peak Chromatogram
50
381.13 391.13
0
100
391.17
485.32
485.30
312.47 391.15
399.23
NL: 2.21E7
Base Peak m/z= 468.40-470.40 F:
ITMS + c ESI Full ms [300.00-700.00]
MS Dog60min2_MS3Top5_LTQ
469.35
Parent
50
+O
50
NL: 3.52E6
Base Peak m/z= 484.00-486.00 F:
ITMS + c ESI Full ms [300.00-700.00]
MS Dog60min2_MS3Top5_LTQ
485.31
485.27 484.30
485.95 485.29
484.93
0
100
469.30 469.33
469.31
469.33 469.20 469.32
485.32
485.30
468.61 469.23
0
100
485.36 485.27
NL: 1.63E6
Base Peak m/z= 454.00-456.00 F:
ITMS + c ESI Full ms [300.00-700.00]
MS Dog60min2_MS3Top5_LTQ
455.31
-CH2
50
454.77
0
100
+2O
50
455.29
501.29
501.22
454.02 454.33
501.19
501.29
501.09
500.25 500.21
0
0
1
2
454.39 454.38 455.30 455.60
3
4
Time (min)
501.60 500.26
5
6
44
501.39
7
NL: 2.00E5
Base Peak m/z= 500.00-502.00 F:
ITMS + c ESI Full ms [300.00-700.00]
MS Dog60min2_MS3Top5_LTQ
8
Maropitant Dog Sample
60 min incubation on LTQ Velos ~ 10X Higher signal
RT: 0.00 - 8.00
NL: 2.09E8
Base Peak F: ITMS + c ESI Full ms
[300.00-700.00] MS
Dog60min2_Top5MS3_Velos
469.98
100
Base Peak Chromatogram
50
381.58 413.79
0
100
462.76
485.69
485.64
421.80 398.95 637.59
NL: 2.09E8
Base Peak m/z= 468.40-470.40 F:
ITMS + c ESI Full ms [300.00-700.00]
MS Dog60min2_Top5MS3_Velos
469.98
Parent
50
469.16 469.65
0
100
+O
50
NL: 2.10E7
Base Peak m/z= 484.00-486.00 F:
ITMS + c ESI Full ms [300.00-700.00]
MS Dog60min2_Top5MS3_Velos
485.67
485.69
484.65 485.67
485.18
0
100
469.79 469.87
469.67 469.65 469.74
469.86
485.69
485.64
485.90 485.85
NL: 1.15E7
Base Peak m/z= 454.00-456.00 F:
ITMS + c ESI Full ms [300.00-700.00]
MS Dog60min2_Top5MS3_Velos
455.68
-CH2
50
454.63 454.72 455.79
501.61
455.14
0
100
454.82 455.85 454.87 454.02
NL: 1.57E6
Base Peak m/z= 500.00-502.00 F:
ITMS + c ESI Full ms [300.00-700.00]
MS Dog60min2_Top5MS3_Velos
+2O
50
501.35
0
0
501.69
500.56 501.55
1
2
500.58
3
4
Time (min)
5
501.75 501.85
6
45
7
Souhrn








The LTQ Velos is 5X more sensitive than the LTQ XL
The LTQ Velos is 2X faster than the LTQ XL
The LTQ Velos can scan at 10Hz
At low concentrations the LTQ Velos gets twice the number of
unique peptides and protein identified as an LTQ XL
LTQ Velos can be upgraded to ETD and Orbitrap
Excellent Simultaneous Quan and Qual instrument
High quality data (more samples identified in less time)
Low level compound detection




HESI II
S-Lens
Dual Cell Linear Trap
Reliability
46