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![Page 1: The world leader in serving science Methods for LTQ Orbitrap A Guided Tour with Examples Dr. Michaela Scigelova.](https://reader036.fdocuments.us/reader036/viewer/2022081512/5697bfeb1a28abf838cb7c19/html5/thumbnails/1.jpg)
The world leader in serving science
Methods for LTQ OrbitrapA Guided Tour with Examples
Dr. Michaela Scigelova
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Outline
Data acquisition strategies Method setup and examples
• OT_LTQ_Big6• MS in Orbi + 6 MS/MS in ion trap
• OT_3OT• MS in Orbi + 3 MS/MS in Orbi
• NL_MS3_AccMass• Phosphopeptides with accurate NL • MS in Orbi, MS2 in Orbi, and MS3 in ion trap
• OT_MSA• Phosphopeptides• ‘composite’ MS2 and MS3 spectrum = MSA• MS in Orbi, MSA in LTQ
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What is the objective of the analysis?
One or more of these:• Maximise the protein ID• Boost the sequence coverage• Phosphorylation• De novo sequencing• Quantitation
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Data Acquisition Strategies
Parallel mode• Survey MS in Orbitrap• MS/MS in LTQ
For highest protein ID and coverage
assuming very complex mixtures
Serial mode• Survey MS in Orbitrap • MS/MS in Orbitrap
For de novo sequencing, PTM discovery
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Mass Accuracy and Resolution Settings
Resolution Mass Accuracy
Survey MS (OT) 15,000 3 ppm
MS/MS (OT) 7,500 3 ppm
Resolution Mass Accuracy
Survey MS (OT) 60,000 3 ppm
MS/MS (LTQ) Unit ~200 ppm
Parallel Mode
Serial Mode
0.9 s
0.1 s
0.35 s
0.2 s
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AGC settings LTQ
Full MS 3.00e+04 IT 50ms SIM 1.00e+04 IT 100ms MSn 1.00e+04 IT 100ms
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AGC settings orbitrap
Full MS 5.00e+05 - 1.00e+06 IT 500ms SIM 1.00e+05 - 2.00e+05 IT 500ms MSn 1.00e+05 - 2.0e+05 IT 500ms
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Settings for HCD, Lock mass
Act.Q 0.13-0.14 Normalized Collision Energy 60-70
specify the FT lock mass abundance (%) Diagnostics/Set Device Default value 10%
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OT_LTQ_Big6
MS in Orbi + 6 MS/MS in ion trap
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Parallel Mode – Method Setup
Step-by-step guide Method BIG 6 Tune settings
• Max fill time 100 ms• Number of microscans 1
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Parallel Method – starting the method
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Parallel Method – Survey MS
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Parallel Method – Add another 6 scan events
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Parallel Method – BIG 6
Save yourself the typing: use copy and paste!
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Parallel Method – DDA Scan event 2
Can be used for directing MS/MS to a preferred region – e.g. 800-1300 in glycopeptide analysis
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Parallel Method – DDA Scan event 2
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Parallel Method – DDA Scan event 2
Ignore
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Parallel Method – DDA Scan event 2
This is the key to PARALLEL mode
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Parallel Method – DDA Scan event 2
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Parallel Method – DDA Scan event 2
Import lists of target parent ions (parent list) or contaminants (reject list)
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Parallel Method – DDA Scan event 2
Reject ‘1+’ and ‘unassigned’ for max number of PROTEIN ID
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Parallel Method – DDA Scan event 2
Leave unticked if going for max sequence COVERAGE
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Parallel Method – DDA Scan event 2
Even better COVERAGE: if you inject sample 3 times and set the following:
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Parallel Method – DDA Scan event 2
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Parallel Method – DDA Scan event 3
Default settings:
WRONG!!!!
!
WRONG!!!!
!
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Parallel Method – DDA Scan event 3
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Parallel Method – DDA Scan event 4
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Parallel Method – DDA Scan event 5
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Parallel Method – DDA Scan event 6
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Parallel Method – DDA Scan event 7
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When you have built your method.. Check it!
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Who Is Working this Way?
John Yates (Scribbs Inst.) Ruedi Aebersold (ETH Basel) David Goodlet (ISB Washington)
Yates et al., AC 2006, 78, 493-450
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OT_3OT
MS in Orbi + 3 MS/MS fragmented in LTQ and measured in OT
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Serial Method Setup
Full scan in Orbi + 3 MS/MS in Orbi Tune settings
• Max fill time 100 ms• Number of microscans 2 to improve S/N in MSn
Resolution Mass Accuracy
Survey MS (OT) 15,000 3 ppm
MS/MS (OT) 7,500 3 ppm
Can resolve 4+ ions
0.35 s
0.2 s
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Serial Method – Full scan MS
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Serial Method – MS + 3 MS/MS
Save yourself the typing: use copy and paste!
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Serial Method – Major change in ‘Current Segment’
There is NO box ticked!
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Serial Method – DDA Scan event 2
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Serial Method – DDA Scan event 3
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Serial Method – DDA Scan event 4
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Who Is Doing It This Way?
Matthias Mann (MPI Martinsreid)• Lock mass ‘trick’• Divide the mass range is sections
Roman Zubarev (Uppsala)
Olsen et al., MPC 2005, 4, 2010-2021
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Want ppb Mass Accuracy?
Use Lock mass in the method
Olsen et al., MPC 2005, 4, 2010-2021
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Lock Mass - Caveats
Lock mass should be present in your analysis all the time • Can be a background ion• Or spike in a lock mass solution via a Y-connector (P-773,17 nL
swept volume) with fused silica tubing attached to a syringe pump Use multiple lock masses For calibrating MS/MS spectra the lock mass closest to your
parent mass of interest is taken (good for peptides) Once you fill in the lock mass dialog box, you can export it, save
it, and import it to other methods
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NL_MS3_AccMass
MS in Orbi + MS/MS in Orbi + MS3 in LTQ
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Strategy for Phosphopeptides
Enrichment!!! Use prominent neutral loss of phosphate in MS2 to trigger MS3 Accurately defined neutral loss
• Use Orbi for MS and MS2• Cuts down on false positive MS3 triggers
MS3 is highly informative for peptide sequence and PTM position Data review is fast and easy – search MS3 scans only Thorough search (MS2 + MS3) provides high confidence ID
Olsen and Mann, PNAS 2004, 101, 13417-22
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NL MS3 Method – starting the method
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NL MS3 Method – Survey MS
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NL MS3 Method – MS2
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NOTE: phosphopeptide analysis
NL MS3 Method – MS2
Accurate NL determination avoids false triggers of MS3 more efficient analysis
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NOTE: phosphopeptide analysis
NL MS3 Method – MS2
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NOTE: phosphopeptide analysis
NL MS3 Method – MS2
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NOTE: phosphopeptide analysis
The system recalculates NL for all relevant charge states
NL MS3 Method – MS2
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NOTE: phosphopeptide analysis
NL MS3 Method – MS2
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NL MS3 Method – MS3
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NL MS3 with accurate mass support – MS3
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NL_MSA
MS in Orbi + MSA in LTQ
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Multistage Activation - MSA
To improve the signal, it is advantageous to lump together the fragments from both MS2 and MS3 scans and search them as one ‘strong’ scan
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CID MS² CID MS³ [M+2H-98]2+
Phosphopeptide analysis with MSA
APPDNLPSPGGpSR 5 fmol
MSA
Data courtesy of Karl Mechtler
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MSA Method – starting the method
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MSA Method – Survey MS
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MSA – data dependent MS2
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Dynamic exclusion
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Neutral Loss – measured in LTQ
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Current Segment
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MSn Settings
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Monoisotopic precursor selection
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Neutral Loss – no need to spell it our for different charge states
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Multistage Activation - ON
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MSA - Conclusions
MSA enables to activate ions from MS2 that show neutral loss from parent, while still keeping the rest of the MS2 fragments in the trap. The MS3 fragments from activated NL candidates are ‘added’ to the rest of the MS2 fragments
Data can be processed by SEQUEST/BioWorks or Mascot
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Who is Doing it this Way
Donald Hunt (Uni Virginia) Karl Mechtler (IMP, Vienna) Andy West, Peter Francis (GSK, Stevenage)
Schoeder et al, AC2004, 76, 3590-3598.