The world leader in serving science Methods for LTQ Orbitrap A Guided Tour with Examples Dr. Michaela Scigelova.

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.