Methods for LTQ Orbitrap A Guided Tour with Examples Dr. Michaela Scigelova

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

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

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 MS/MS in Orbitrap For de novo sequencing, PTM discovery

Mass Accuracy and Resolution Settings Parallel Mode Resolution Mass Accuracy Survey MS (OT) 60,000 3 ppm MS/MS (LTQ) Unit ~200 ppm 0.9 s 0.1 s 0.35 s 0.2 s Serial Mode Resolution Mass Accuracy Survey MS (OT) 15,000 3 ppm MS/MS (OT) 7,500

AGC settings LTQ Full MS 3.00e+04 IT 50ms SIM 1.00e+04 IT 100ms MSn 1.00e+04 IT 100ms

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

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%

OT_LTQ_Big6 MS in Orbi + 6 MS/MS in ion trap

Parallel Mode – Method Setup Step-by-step guide Method BIG 6 Tune settings Max fill time 100 ms Number of microscans 1

Parallel Method – starting the method

Parallel Method – Survey MS

Parallel Method – Add another 6 scan events

Parallel Method – BIG 6 Save yourself the typing: use copy and paste!

Parallel Method – DDA Scan event 2 Can be used for directing MS/MS to a preferred region – e.g. 800-1300 in glycopeptide analysis

Parallel Method – DDA Scan event 2

Parallel Method – DDA Scan event 2 Ignore Exclusion list size – use max number available. The list will contain also all the isotopes of excluded ions so it fills up quite quickly. Exclusion duration - should be about ½ peak width – this ensures another MS/MS spectrum is taken hopefully around the peak apex. Exclusion mass width - is set very narrow. Isotopes will be also excluded.

Parallel Method – DDA Scan event 2 ‘most intense if no parent masses found’ will be used for NL MS3 method for phosphopeptides (peak corresponding to the detected neutral loss of phosphate is considered as a parent mass for this purpose) ‘exclude parent mass from MSn selection’ – used mainly for drugs. When parent fragmentation is not efficient, the leftover parent ion is still the biggest in the spectrum – when ticking this button the system will ignore such parent ion leftover and go for the next biggest ion in the MSn spectrum. This is the key to PARALLEL mode

Parallel Method – DDA Scan event 2 The NCE settings can vary for different MSn levels: good for phosphopeptides where MS2 energy should be set lower (i.e. 20) to just knock off the labile modification, and MS3 energy can go back to normal 30 to fragment the dephosphorylated peptide with maximum efficiency.

Parallel Method – DDA Scan event 2 Import lists of target parent ions (parent list) or contaminants (reject list)

Parallel Method – DDA Scan event 2 Reject ‘1+’ and ‘unassigned’ for max number of PROTEIN ID

Parallel Method – DDA Scan event 2 Leave unticked if going for max sequence COVERAGE

Parallel Method – DDA Scan event 2 Even better COVERAGE: if you inject sample 3 times and set the following:

Parallel Method – DDA Scan event 2

Parallel Method – DDA Scan event 3 Default settings: WRONG!!!!!

Parallel Method – DDA Scan event 3

Parallel Method – DDA Scan event 4

Parallel Method – DDA Scan event 5

Parallel Method – DDA Scan event 6

Parallel Method – DDA Scan event 7

When you have built your method.. Check it!

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

OT_3OT MS in Orbi + 3 MS/MS fragmented in LTQ and measured in OT

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 Can resolve 4+ ions Resolution Mass Accuracy Survey MS (OT) 15,000 3 ppm MS/MS (OT) 7,500 0.35 s 0.2 s

Serial Method – Full scan MS

Serial Method – MS + 3 MS/MS Save yourself the typing: use copy and paste!

Serial Method – Major change in ‘Current Segment’ There is NO box ticked!

Serial Method – DDA Scan event 2

Serial Method – DDA Scan event 3

Serial Method – DDA Scan event 4

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

Want ppb Mass Accuracy? Use Lock mass in the method Olsen et al., MPC 2005, 4, 2010-2021

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

NL_MS3_AccMass MS in Orbi + MS/MS in Orbi + MS3 in LTQ

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

NL MS3 Method – starting the method

NL MS3 Method – Survey MS

NL MS3 Method – MS2

NL MS3 Method – MS2 NOTE: phosphopeptide analysis Accurate NL determination avoids false triggers of MS3  more efficient analysis

NL MS3 Method – MS2 NOTE: phosphopeptide analysis ‘most intense if no parent masses found’ will be used for NL MS3 method for phosphopeptides (peak corresponding to the detected neutral loss of phosphate is considered a parent mass for this purpose)

NL MS3 Method – MS2 NOTE: phosphopeptide analysis The NCE settings can vary for different MSn levels: good for phosphopeptides where MS2 energy should be set lower (i.e. 20) to just knock off the labile modification, and MS3 energy can go back to normal 30 to fragment the dephosphorylated peptide with maximum efficiency.

NL MS3 Method – MS2 NOTE: phosphopeptide analysis The system recalculates NL for all relevant charge states

NL MS3 Method – MS2 NOTE: phosphopeptide analysis

NL MS3 Method – MS3

NL MS3 with accurate mass support – MS3

NL_MSA MS in Orbi + MSA in LTQ

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

Phosphopeptide analysis with MSA APPDNLPSPGGpSR 5 fmol CID MS² CID MS³ [M+2H-98]2+ MSA Weitere Gegenüberstellung: wie vorherhegehende Folie, nur auch mit pulsed-q fragmentierung. Bei phosphopeptid mit starken NL kein Vorteil sichtbar (keine zB. Immoniumionen oder iTRAQ) 1.) 060522OT_14PP_CAD-ITnl-ms3x5_par14PP_MPSon.1588.1588.2.dta 2.) 060522OT_14PP_CAD-ITnl-ms3x5_par14PP_MPSon.1589.1589.2.dta 3.) 060420FTc2_CCBSAPP_mstgMS2_09.523.523.2.dta The problem for SEQUEST – does not label NL-fragments in MSA spectrum. Therefore our customers who want to use SEQUEST do not like MSA method and still use NL MS3 method. This is not a problem for Mascot – does NL-ion labeling really well. Data courtesy of Karl Mechtler

MSA Method – starting the method

MSA Method – Survey MS

MSA – data dependent MS2

Dynamic exclusion

Neutral Loss – measured in LTQ

Current Segment

MSn Settings

Monoisotopic precursor selection

Neutral Loss – no need to spell it our for different charge states

Multistage Activation - ON

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

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.