ABOVE & BEYOND your expectations - Thermo Fisherdocs.appliedbiosystems.com/cms/groups/portal/documents/general... · ABOVE & BEYOND your expectations Introducing the AB SCIEX 5800

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


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


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



5800 System Technology and Hardware Improvements

Heated

laser mirror1kHz laser

New heated source New Detector

New Reflectron Assembly

High resolution QuanTIS

On Axis Ionization


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


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 -


1668 1670 1672 1674 1676 1678Mass (m/z)

1460.8

0102030405060708090

100

Final -

Shots 2000 -


1672.24(R20294,S196)

1668 1670 1672 1674 1676 1678Mass (m/z)

4166.9

0102030405060708090

100

Final -

Shots 2000 -


1668 1670 1672 1674 1676 1678Mass (m/z)

1771.6

0102030405060708090

100

Final -

Shots 7500 -


1672.17(R18461,S58)

250 amol

150 amol

50 amol

200 amol

100 amol

25 amol


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)


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


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


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


464.25510(R2430,S142)

465.25266(R2362,S39)469.15463(R24

MSMS resolution >7500


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


5800 System Higher Resolution with sub-ppm

mass accuracy

<1 ppm

mass accuracy


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


EasyAccessTM

Job Set Up Wizard A User-Friendly One Page Data Acquisition Tool


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 .


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


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


Extending the Limits of Protein Discovery Protein ID from Complex Samples using LC MALDI

●


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


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


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.


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


Assessing the Complementarity of MALDI and ESI for Protein Identification in Complex Mixtures

●


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.


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


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


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


●

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


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.


–

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


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


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


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.


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)


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



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.


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.


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.

Documents

ABOVE & BEYOND your expectations - Thermo Fisherdocs.appliedbiosystems.com/cms/groups/portal/documents/general... · ABOVE & BEYOND your expectations Introducing the AB SCIEX 5800