above & beyond your expectations - thermo...
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ABOVE & BEYOND your expectations Introducing the AB SCIEX 5800 TOF/TOF™
System
Fadi
A. AbdiTOFTOF Sr. Product Manager
2 ASMS 2009 © 2009 Applied Biosystems Inc. and MDS Inc. Joint Owners ABOVE & BEYOND
AB SCIEX Multigenerational MALDI TOF/TOF Technology
2001:
AB 4700 MALDI TOFTOF Analyzer
–
High Quality MS and true MSMS on the same platform
–
Speed for high throughput proteomics and LC -
MALDI–
Flexibility
modes and mass range–
Quantitation
using MALDI MS
DE MALDI source
250 L/s 250 L/s 250 L/s
Target gas
2nd source (DE-accelerated
fragment ions)
TIS
Detectors
Reflectron
Collision cellSolid StateNano Laser
Retarding lens
Beam 1 lens & x/y deflectors
Beam 2 lens, metastable suppressor & x/y deflectors
200 Hz
3 ASMS 2009 © 2009 Applied Biosystems Inc. and MDS Inc. Joint Owners ABOVE & BEYOND
AB SCIEX Multigenerational MALDI TOF/TOF Technology
2005:
AB 4800 MALDI TOFTOF Analyzer
–
OptiBeam™
On-Axis Laser •
10x improvement in MS and MS/MS sensitivity–
QuanTIS™
Precursor Selector•
Precursor Selection equivalent that of quads and traps
–
Large μ-titer plate format•
More sample capacity for higher throughput applications like LC MALDI
–
Unique dual plate loading chamber•
Enables 24/7 operation
Laser Path
Laser Path
Aperture to TOF
Aperture to TOF
4 ASMS 2009 © 2009 Applied Biosystems Inc. and MDS Inc. Joint Owners ABOVE & BEYOND
The AB SCIEX TOF/TOFTM
5800 System The latest innovation in MALDI TOFTOF Technology
●
The fastest and most sensitive
MALDI system ever developed with over 10 times faster
than any MALDI existing technology ●
The 5800 System extends protein identification
and accelerate throughput,
and yet is configured to provide an "Easy Access" walkup solution
for multi-user laboratories for faster results
●
For MALDI imaging the 5800 System will accelerate throughput
and eliminates matrix contaminations
that force time-
consuming cleaning between samples acquisitions
5 ASMS 2009 © 2009 Applied Biosystems Inc. and MDS Inc. Joint Owners ABOVE & BEYOND
The AB SCIEX TOF/TOFTM
5800 System Technology and Hardware Improvements
Heated
laser mirror1kHz laser
New heated source New Detector
New Reflectron Assembly
High resolution QuanTIS
On Axis Ionization
6 ASMS 2009 © 2009 Applied Biosystems Inc. and MDS Inc. Joint Owners ABOVE & BEYOND
HeatingNo Heating
What Changes Have We
Made to Hardware… Re-engineered Source One and Laser Mirror
●
Heating of laser mirror–
continuous–
physical cleaning of mirror not necessary
No Bake-out
●
Bake-out–
Total automation–
End of run, operation stopped, source temperature raised
After Bake-out
7 ASMS 2009 © 2009 Applied Biosystems Inc. and MDS Inc. Joint Owners ABOVE & BEYOND
The AB SCIEX TOF/TOF™
5800 System Unmatched Sensitivity on any MALDI Platform
1668 1670 1672 1674 1676 1678Mass (m/z)
3843.5
0102030405060708090
100
Final -
Shots 2000 -
250amol/ul neurotensin
1672.23(R24682, S593)
1668 1670 1672 1674 1676 1678Mass (m/z)
3419.0
0102030405060708090
100
Final -
Shots 2000 -
200amol/ul neurotensin1672.22(R23907,S492)
1668 1670 1672 1674 1676 1678Mass (m/z)
2442.9
0102030405060708090
100
Final -
Shots 2000 -
150amol/ul neurotensin1672.23(R22907,S347)
1668 1670 1672 1674 1676 1678Mass (m/z)
1460.8
0102030405060708090
100
Final -
Shots 2000 -
100amol/ul neurotensin
1672.24(R20294,S196)
1668 1670 1672 1674 1676 1678Mass (m/z)
4166.9
0102030405060708090
100
Final -
Shots 2000 -
50amol/ul neurotensin1672.15(R22641,S117)
1668 1670 1672 1674 1676 1678Mass (m/z)
1771.6
0102030405060708090
100
Final -
Shots 7500 -
25amol/ul neurotensin
1672.17(R18461,S58)
250 amol
150 amol
50 amol
200 amol
100 amol
25 amol
8 ASMS 2009 © 2009 Applied Biosystems Inc. and MDS Inc. Joint Owners ABOVE & BEYOND
799.0 1441.8 2084.6 2727.4 3370.2 4013.0
Mass (m/z)
3478.4
0
10
20
30
40
50
60
70
80
90
100
Final -
Shots 400 -
CAL5; Run #40; Label D22
2465.19(R33215,S2547)
2093.08(R31175,S1756)
904.46(R12885,S821)
1570.67(R26259,S993)
The AB SCIEX TOF/TOF™
5800 System Faster Acquisition with up to 80% better MS Resolution than 4800
2463.0 2464.6 2466.2 2467.8 2469.4 2471.0Mass (m/z)
347
0
10
20
30
40
50
60
70
80
90
100
Final - Shots 400 - CAL5; Run #40; Label D22
2465.18896(R33215,S2611)
> 33K Resolution
9 ASMS 2009 © 2009 Applied Biosystems Inc. and MDS Inc. Joint Owners ABOVE & BEYOND
MSMS 2465 @ 1kHz (500 shots) Faster Acquisition with 40% better MSMS Resolution than 4800
9.0 528.2 1047.4 1566.6 2085.8 2605.0Mass (m/z)
3.7E+
0
10
20
30
40
50
60
70
80
90
100
Final - Shots 500 - MSMS New ETP Grids all Hardware; Run #7; Label K21 Prec = 2465.2000
1326.68909(R4350,S1387)
2319.06348(R7593,S629)
1328.67334(R4367,S249)2301.06519(R7261,S276)1309.62830(R3850,S252)
1743.83423(R5751,S136)1211.65601(R3728,S220) 2303.00562(R8021,S124)857.51349(R2977,S208) 1916.89636(R6266,S88)1311.59192(R3601,S49)580.36438(R3053,S142) 2273.09058(R7234,S69)1918.75830(R3801,S29)840.46417(R2691,S77)237.10611(R2101,S155) 1585.79407(R5430,S39)1166.58569(R3580,S40)584.18719(R3189,S47)
2314.0 2316.2 2318.4 2320.6 2322.8 2325.0Mass (m/z)
1.7
0
10
20
30
40
50
60
70
80
90
100
Final - Shots 500 - MSMS New ETP Grids all Hardware; Run #7; Label K2
2319.06348(R7593,S629)
2318.09229(R6743,S846)
2320.04956(R7892,S419)
2321.02881(R6810,S172)
2321.99341(R6559,S55)2317.07666(R6581,S26)
1325.0 1326.4 1327.8 1329.2 1330.6 1332.0Mass (m/z)
3.7
0
10
20
30
40
50
60
70
80
90
100
Final - Shots 500 - MSMS New ETP Grids all Hardware; Run #7; Label K2
1326.68909(R4350,S1387)
1327.69177(R4232,S654)
1328.67334(R4367,S249)
1329.66089(R4082,S70)
462.0 463.6 465.2 466.8 468.4 470.0Mass (m/z)
274
0
10
20
30
40
50
60
70
80
90
100
Final - Shots 500 - MSMS New ETP Grids all Hardware; Run #7; Label K2
464.25510(R2430,S142)
465.25266(R2362,S39)469.15463(R24
MSMS resolution >7500
10 ASMS 2009 © 2009 Applied Biosystems Inc. and MDS Inc. Joint Owners ABOVE & BEYOND
649.0 1321.8 1994.6 2667.4 3340.2 4013.0Mass (m /z)
5718.8
0
10
20
30
40
50
60
70
80
90
100
% In
tens
ity
TOF/TOF™ Re fle c tor Spe c # 1 M C[BP = 2 4 6 6 .2 , 5 7 1 9 ]
904.4677
1296.6862 2465.2058
1570.6779
1829.9698
2109.07591251.6637 3657.9431
2093.08671324.6852973.5281 2302.1472 2493.2014
resolution 17250
resolution 22550
resolution 25350
resolution 31900
resolution 28700
resolution 25100
# of spectra E3 E4 E5 E6 E7
#1‐20 0,96 0,47 0,53 1,29 0,42
#21‐40 0,27 1,04 0,68 0,56 0,61
#41‐60 ‐0,07 0,51 0,19 ‐0,03 0,15
#61‐80 ‐0,83 ‐0,20 ‐0,90 ‐0,95 0,56
The AB SCIEX TOF/TOF™
5800 System Higher Resolution with sub-ppm
mass accuracy
<1 ppm
mass accuracy
11 ASMS 2009 © 2009 Applied Biosystems Inc. and MDS Inc. Joint Owners ABOVE & BEYOND
Intelligent MS/MS Acquisition Dynamic Exit™
Algorithm●
During MS/MS acquisition, the spectral quality is monitored and then terminated once a user defined quality is achieved
●
On intense precursors, fewer laser shots are used, conserving the sample spot for data acquisition on lower level precursors
●
On lower level precursors, more laser shots are used to obtain higher quality spectra
Only 250 laser shots acquired
12 ASMS 2009 © 2009 Applied Biosystems Inc. and MDS Inc. Joint Owners ABOVE & BEYOND
EasyAccessTM
Job Set Up Wizard A User-Friendly One Page Data Acquisition Tool
13 ASMS 2009 © 2009 Applied Biosystems Inc. and MDS Inc. Joint Owners ABOVE & BEYOND
Depth of Coverage for Single Spot Analysis DynamicExit™
Algorithm on the AB SCIEX TOF/TOF™
5800 System
●
Sample Preparation:–
Dilution series of a trypsin digested 20 protein mixture (25ng-1.25 ng)
●
4800 MALDI TOF/TOF™
Analyzer Acquisition–
MALDI stage was moving in discrete motion mode in all acquisition modes (move after each sub-spectrum).
–
MS data was acquired at 200 Hz with 1000 laser shots/spectrum.–
MS/MS data was acquired at 200 Hz in 1kV MS/MS mode with 4000 laser shots/spectrum. Interpretation Method: Top 200 precursors
●
TOF/TOF™
5800 System Acquisition–
MALDI stage was moving in continuous motion mode. –
MS data was acquired at 400 Hz with 1000 shots/spectrum. Sub-
spectra acquired every 250 shots
–
MS/MS data was acquired at 1000 Hz in 1kV mode with 4000 shots/spectrum. Sub-spectra acquired every 250 shots with Dynamic Exit ™
Algorithm either turned on or off. –
Interpretation Method: Top 200 precursors.●
Database searching:–
The protein identification was performed on ProteinPilotTM
version 3.0 with ParagonTM
and Pro GroupTM
algorithm .
14 ASMS 2009 © 2009 Applied Biosystems Inc. and MDS Inc. Joint Owners ABOVE & BEYOND
Intelligent MS/MS Acquisition DynamicExit™
Algorithm
MSMS of the 159th
precursors
Peptide ID: VTLTSEEEAHLKK (m/z=1483.780)
5800 system
5800 system with DynamicExit™
Algorithm
15 ASMS 2009 © 2009 Applied Biosystems Inc. and MDS Inc. Joint Owners ABOVE & BEYOND
Faster Protein Identifications Faster Laser + DynamicExit™
Algorithm
●
Variable rate 1000Hz laser provides >5x increase in MS/MS acquisition speed–
Improved throughput–
Ideal solution for labs with many samples to process
●
DynamicExit™
Algorithm further increases acquisition speed (~2x) and minimizes sample spot consumption–
More protein identifications–
Higher sequence coverage
16 ASMS 2009 © 2009 Applied Biosystems Inc. and MDS Inc. Joint Owners ABOVE & BEYOND
Extending the Limits of Protein Discovery Protein ID from Complex Samples using LC MALDI
●
Sample Preparation:–
A 500 ng trypsin digest of an E.coli
lysate
was subjected to LC MALDI reversed-
phase fractionation using the Tempo™
LC MALDI Spotting System.
●
4800 MALDI TOF/TOF™
Analyzer Acquisition –
MALDI stage was moving in discrete motion mode in all modes (move after each sub-spectrum).
–
MS data was acquired at 200 Hz with 1000 laser shots/spec. Sub-spectra acquired every 50 shots
–
MS/MS data was acquired at 200 Hz in 1kV MS/MS mode with 4000 laser shots/spectrum. Sub-spectra acquired every 25 shots with usual stop criteria
–
Interpretation Method: Top 25 precursors
●
TOF/TOF™
5800 System Acquisition –
MALDI stage was moving in continuous motion mode. –
MS data was acquired at 400 Hz with 1000 shots/spectrum. Sub-spectra acquired every 250 shots
–
MS/MS data was acquired at 1000 Hz in 1kV mode with 4000 shots/spectrum. Sub-spectra acquired every 250 shots with Dynamic Exit ™
Algorithm either turned on or off.
–
Interpretation Method: Top 25 precursors.●
Database searching: –
All data was processed with ProteinPilot™
Software v.3.0
17 ASMS 2009 © 2009 Applied Biosystems Inc. and MDS Inc. Joint Owners ABOVE & BEYOND
Improved Protein Identification Faster Laser Speed and Improved Hardware
●
The 5x faster laser speed on the TOF/TOF™
5800 System provides the same number of MS/MS acquisitions at a >7x shorter acquisition time
●
5800 system hardware improvements provide higher quality MS/MS to almost double the peptides found, providing more sequence coverage and therefore more confidence in the proteins identified
* No DynamicExit
Algorithm Used
18 ASMS 2009 © 2009 Applied Biosystems Inc. and MDS Inc. Joint Owners ABOVE & BEYOND
Improved Protein Identification
Dynamic Exit™
Algorithm
●
Dynamic Exit Algorithm provides an additional 2x in speed of acquisition, while continuing to provide additional peptides
●
Efficiency of sample utilization will enable additional precursors to be analyzed per spot and further increased depth of coverage if additional analysis time is used.
19 ASMS 2009 © 2009 Applied Biosystems Inc. and MDS Inc. Joint Owners ABOVE & BEYOND
Improved Protein Identification Faster Laser Speed and Improved Hardware
●
Hardware improvements, a 5x faster laser speed and the intelligent DynamicExit™
Algorithm on the TOF/TOF™
5800 System provide:–
50% more proteins–
100% more peptides–
In ~1/15th
the acquisition time●
Improved depth of coverage in less time using LC MALDI workflow is key for biomarker discovery experiments
20 ASMS 2009 © 2009 Applied Biosystems Inc. and MDS Inc. Joint Owners ABOVE & BEYOND
Assessing the Complementarity of MALDI and ESI for Protein Identification in Complex Mixtures
●
Sample Preparation:–
E.coli
total cell lysate, digested (trypsin) reduced (dithiothreitol) and alkylated
(iodoacetamide), sample loadings of 20, 100 and 500 ngs, three replicates.
●
LC MALDI Acquisition:–
Peptides were separated on a Monolithic CapRod
column (100 µm ID x 15cm, Merck, Germany) with a 90 minute of ACN gradient at 2 µL/min using the Tempo™
LC MALDI Spotting System. –
720 spots collected per LC run with online mixing of α-cyano-4-hydroxycinnamic acid matrix
–
MS/MS spectra were acquired on the 25 most intense precursors per spot
●
LC ESI Acquisition: –
Peptides were separated on a Magic AQ C18 column (75 µm x 10cm, self packed) using a 90 min gradient at 300 nL/min
–
The eluant
was directed into an Orbitrap (ThermoFisher). –
Data acquisition parameters were optimized according to the vendor’s recommendations, acquiring 7 MS/MS per cycle.
●
Database searching: –
All data was searched with ProteinPilot™
Software 3.0 against a database of all E.coli
proteins in UniProt
as of Jan. 27, 2009 (4379) plus 77 common contaminant proteins using the Paragon™
Algorithm in Thorough mode.–
False discovery rate (FDR) analysis was done using the embedded PSPEP tool. –
*.mgf
file was generated using ReAdw4Mascot2.exe, a NIST derivative of the ISB tool, ReAdW.
21 ASMS 2009 © 2009 Applied Biosystems Inc. and MDS Inc. Joint Owners ABOVE & BEYOND
Comparing Sample Loading at the Protein Level ESI vs MALDI
●
Similar protein ID rates achieved at 20 ●
More proteins found by LC MALDI at 100 and 500 ng–
Acquisition time was 2.5x longer though
22 ASMS 2009 © 2009 Applied Biosystems Inc. and MDS Inc. Joint Owners ABOVE & BEYOND
Comparing Sample Loading at the Peptide Level ESI vs MALDI
●
Similar peptide ID rates achieved at 20●
More peptides found by LC MALDI at 100 and 500 ng
–
Acquisition time was 2.5x longer though
23 ASMS 2009 © 2009 Applied Biosystems Inc. and MDS Inc. Joint Owners ABOVE & BEYOND
Protein / Peptide Intersection 500 ng Loading Level
●
ESI with MALDI acquisitions appear complementary and suggest greater depth of coverage from combination than a signal technique alone.
●
But how similar are replicates from the same instrument?
234 466 78(30.1%) (59.9%) (10.0%)
5800
700 proteins
Orbitrap
544 proteins
3302 1408 851(59.4%) (25.3%)
(15.3%)
5800
4710 peptides
Orbitrap
2259 peptides
24 ASMS 2009 © 2009 Applied Biosystems Inc. and MDS Inc. Joint Owners ABOVE & BEYOND
●
Average Unused ProtScores
for all proteins were aligned from each instrument (500 ng replicates).
●
Points above the diagonal mean those proteins have more peptides and therefore get better Protein scores with 5800 system.
●
The ‘clumps’
at the lowest limits are proteins not found at all by one instrument or the other.
Comparison of Protein Identification Quality Protein Pilot Data of Orbitrap and 5800
25 ASMS 2009 © 2009 Applied Biosystems Inc. and MDS Inc. Joint Owners ABOVE & BEYOND
Replicates vs
combination ●
Orbitrap
(red) and 5800 system (blue) data demonstrate similar relative gains from increasing replicates,
●
Multiple replicate acquisitions on the Orbitrap
did not equal the peptide or protein yield from a single LC MALDI 5800 system acquisition
●
Two 5800 system runs are ~equal to the combination of one 5800 and one Orbitrap runs.
●
Since one ESI and one MALDI runs can be done more quickly than two MALDI runs, combined use is valuable.
●
New LTQ OT Velos
data shows increase in peptide and protein ID over the Orbi
XL. But complimentarity
is the same.
26 ASMS 2009 © 2009 Applied Biosystems Inc. and MDS Inc. Joint Owners ABOVE & BEYOND
–
1 kHz OptiBeam
on-axis laser
and continuous motion sample plate for faster data acquisition
–
User-tunable laser acquisition rate for optimal results from all types of molecules
–
Best-in-class QuantTIS™
precursor ion selection for superior MS/MS
of small molecules and proteins (for identification)
–
Heated laser mirror and automated self-
cleaning of source eliminates the contamination
that forces time-consuming cleaning between samples in other systems
MALDI Mass Spectrometry Imaging (MSI) Fastest, most sensitive and most flexible platform for MSI
27 ASMS 2009 © 2009 Applied Biosystems Inc. and MDS Inc. Joint Owners ABOVE & BEYOND
Benefits of a High Repetition Rate Laser for Imaging●
Data acquisition times for imaging experiments at different laser frequencies:–
100 shots fired per pixel.–
100µm x 100µm pixel resolution.
1.8cm
0.8cm
2.3cm
0.8cm
Rat braincoronal section
Rat brainsagittal section
Tissue Surface Area Acquisition times (minutes)Analyzed 200Hz 400Hz 1000Hz
1.5 cm2
189min 128min 94min
2.0 cm2
244min 167min 118min
28 ASMS 2009 © 2009 Applied Biosystems Inc. and MDS Inc. Joint Owners ABOVE & BEYOND
MS Imaging of Rat Brain
(distribution of ion m/z
= 734.6 Da)
Optical image of coronal section Extracted ion image (EII) of ion with m/z = 734.6
(EII) of ion m/z = 734.6 overlaidonto optical image
734.6
100µm x 100µm pixel resolution
Mass spectrumat defined position
29 ASMS 2009 © 2009 Applied Biosystems Inc. and MDS Inc. Joint Owners ABOVE & BEYOND
MS Imaging of Mouse Heart
(mass range 400-1000 Da)
Averaged mass spectrum over region of interest (entire tissue)
Peptides/lipid species observed in Reflector Mode
MS/MS-based imaging experiment performed on species with precursor mass m/z = 782.6 Da.
30 ASMS 2009 © 2009 Applied Biosystems Inc. and MDS Inc. Joint Owners ABOVE & BEYOND
38.9586.09
146.97
184.06
723.40
599.45
198.07162.95
1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphatidylcholine
characteristic phosphatidylcholinefragment
MS/MS Imaging of Mouse Heart
(precursor m/z
= 782.5 Da)
Averaged mass spectrum over region of interest (entire tissue)
31 ASMS 2009 © 2009 Applied Biosystems Inc. and MDS Inc. Joint Owners ABOVE & BEYOND
14175
4968
9991
Mass spectrumat defined position
MS Imaging of Rat Brain (mass range 2,000-20,000 Da)
Optical image of sagittal section Extracted ion image (EII) of ion with m/z = 4968
Extracted ion image (EII) of ion with m/z = 14175
Extracted ion image (EII) of ion with m/z = 9991
100µm x100µm pixel resolution
32 ASMS 2009 © 2009 Applied Biosystems Inc. and MDS Inc. Joint Owners ABOVE & BEYOND
The AB SCIEX TOF/TOFTM
5800 System ASMS 2009: Presentations and Posters
●
Luncheon Workshop–
Mon 12:00 –
1:00 pm Location: Marriott Hotel Salon B & F–
Introducing the AB SCIEX TOF/TOF™
5800 System: Complimentarity
of ESI and MALDI TOFTOF technologies–
Session is Full. Registered invitees only.
●
Additional Workshop–
Sun 3:15 –
4:15 pm Location: Lowes Hotel Hall C and D
●
Posters:–
New MALDI TOF/TOF system data acquisition strategy that minimizes MS/MS acquisition time while maintaining high quality data and increasing depth of coverage poster number is #142, Monday session.
–
Proteome analysis of superior temporal gyrus
in schizophrenics and non-human primates treated with anti-psychotics demonstrate disease and drug specific changes. poster number is #600, Monday session.
–
High speed MALDI TOF/TOF analysis –
its advantages for identification of digested proteins and screening for phosphorylated
peptides in LC-MSMS–
poster number is #400, Wednesday session.
33 ASMS 2009 © 2009 Applied Biosystems Inc. and MDS Inc. Joint Owners ABOVE & BEYOND
Acknowledgment/ Trademarks / Licensing ●
Acknowledgment–
Jenny Van Eyk
and Rebekah
Gubdry
Johns Hopkins University for running the OrbiTrap
samples–
Aaron Booy, MDS Analytical technologies.–
Sean Seymour, Kathy Lowis-Torpy, Shixin
Sun and Christie Hunter Applied biosystems
●
Trademarks / Licensing–
For Research Use Only. Not for use in diagnostic procedures.–
AB (Design) and Applied Biosystems
are registered trademarks of Applied Biosystems
or its subsidiaries in the U.S. and/or certain other countries.
OptiBean, Paragon, ProGroup, ProteinPilot
and MALDI TOF/TOF are trademarks of Applied Biosystems/MDS Analytical Technologies, a joint venture between Applied Biosystems
and MDS Inc. All other trademarks are the sole property of their respective owners.
–
©
2009 Applied Biosystems
and MDS Inc. Joint Owners. All rights reserved. Information subject to change without notice.
34 ASMS 2009 © 2009 Applied Biosystems Inc. and MDS Inc. Joint Owners ABOVE & BEYOND
Trademarks / Licensing
●
For Research Use Only. Not for use in diagnostic procedures.●
AB (Design) and Applied Biosystems are registered trademarks of Applied Biosystems or its subsidiaries in the U.S. and/or certain other countries. OptiBean, Paragon, ProGroup, ProteinPilot and MALDI TOF/TOF are trademarks of Applied Biosystems/MDS Analytical Technologies, a joint venture between Applied Biosystems and MDS
Inc. All other trademarks are the sole property of their respective owners.
●
©
2009 Applied Biosystems and MDS Inc. Joint Owners. All rights reserved. Information subject to change without notice.