Orbitrap Fusion Lumos Tribrid MS

The Thermo Scientific Orbitrap Fusion Lumos Tribrid mass spectrometer allows systems biology researchers to test new limits of quantitation and protein characterization. It incorporates the brightest ion source, a segmented quadrupole mass filter with improved selectivity and ion transmission, Advanced Vacuum Technology for improved ion transmission to the Orbitrap mass analyzer. Combined, these hardware improvements enable the new tribrid instrument to excel in the most challenging applications, including analysis of low level PTMs, multiplexed relative quantitation using isobaric tags, intact protein characterization, as well as MSn analysis of small molecules..

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1/8/2016 Fusion (MP025) Installed

    Scan Rate
  • Orbitrap MSn up to 20 Hz
  • Ion trap MSn up to 20 Hz
  • 15,000-500,000 (FWHM) at m/z 200
    Mass accuracy
  • < 3 ppm RMS using external calibration
  • < 1 ppm RMS using internal calibration
  • CID/HCD Fragmentation
  • High Capacity Transfer Tube (HCTT) and Electrodynamic Ion Funnel (EIDF) for for increased ion flux and lower limits of detection
  • Segmented quadrupole mass filter allows for 0.4 u to 1200 u precursor isolation
  • Ultra high field Orbitrap mass analyzer
  • Spectral multiplexing for enhanced duty cycle
  • Synchronous Precursor Selection Up to 15 precursors per MS2 scan, for MS3 analysis only

Parallelization on the Orbitrap Fusion
To improve the spectral acquisition rate the acquisition process on the Fusion Series is extensively parallelized. The figure below shows a schematic of parallel execution and event pipelining: Precursors identified in the Orbitrap full scan (top row) are fragmented and accumulated in the IRM (middle row) concurrent with LT acquisition of the previous MS/MS (bottom row).

Senko MW et al. Novel parallelized quadrupole/linear ion trap/Orbitrap tribrid mass spectrometer improving proteome coverage and peptide identification rates. Anal Chem 85(24), 11710 - 4 (2013)

How is AGC performed on Orbitrap Fusion Series Instruments?
Ion population control is an important concept for all trapping instruments. To ensure proper functioning of the Orbitrap and Linear Ion trap analyzers, the ion populations are regulated at a given target level, in a process referred to as automatic gain control (AGC). The mechanism for the regulation is a fast ion trap full scan referred to as the "prescan". This scan is "hidden", and is not recorded in the raw file. The prescan makes a measurement of the ion flux, and from this information, the injection time for subsequent scans can be set to deliver the target number of ions.
For data dependent experiments, the prescan uses the same settings as the MS master scan. The prescan is executed immediately before the master scan, and the total ion current (TIC) from the prescan is used to set the injection time for the master scan. The identities (m/z and charge) of the precursors for the dependent scans are extracted from the previous master scan, but the injection times for these dependent scans are calculated from the intensities in the prescan.
For targeted experiments, the prescan analyzes the mass range spanning the smallest target up to the largest target, and the injection times for each target are calculated from intensities in the prescan. In the context of LC-MS, analyte intensity varies considerably as each compound elutes from the column, making the length of time between prescan and subsequent scans an important factor to consider. When the list of targeted scans is large, the time between execution of the prescan and execution of the later targeted scans could become significant, negatively affecting the accuracy of the ion population regulation. For this reason, the Loop Control mechanism was added to the targeted experiment methods. For example, when Loop Control is set to Time, the prescan will be executed at a period less than or equal to the specified time period, ensuring a certain proximity between prescan and targeted scans, which in turn can improve the accuracy of the calculated injection times, and hence the quality of the acquired data.

Resolving Power and Transient Length
Here is a full table of the available resolution settings. Note: higher resolution does not always result in better mass accuracy. The higher the resolution increases your ability to distinguish between m/z ions. As you can see in this table, there is always a trade of higher resolution with time. 15k is the lowest resolution setting, with the fastest scan speed of ~15 Hz. On the Fusion, the highest resolution setting is 450K that is <1 Hz.
To fully take advantage of the parallel fill and detect capabilities of the Fusion, we need to balance the max fill times with the transient length. For resolving power 30,000, detection time is about 64 ms. There is always a small amount of inter-scan delay. Up to 54 ms can be used to fill the C-trap with ions- and without increasing cycle time.
In SW version 2.1 50k resolution OT scans for TMT 10plex experiments shows a ~10% improvement over 60k resolution. We successfully used 30k resolution as well.

Res. at m/z 200 Transient length [ms] Approx. scan speed [Hz] "Free" fill time [ms]
15,000 32 na 22
30,000 64 15 54
50,000 96 na 86
60,000 128 7.5 118
120,000 256 4 246
240,000 512 2 502
450,000 1024 <1 1014

The table below lists some of the instrument method settings we tested on our Lumos.
We use a nanoAcquity UPLC with home made columns (35cm x 75μm, Reprosil Pur C18AQ 120Å 5μm) and trap column (3cm x 100μm, Reprosil Purc C18AQ 120Å 5μm), with 5-30% B (ACN, 0.1%FA) in 90min gradient. The sample is a Hela cell whole cell tryptic digest (ThermoFisher), 100 ng on column injection. We typically get ~17000 unique peptides with Comet search and peptide prophet p>0.9,
The results below are based on the number of unique peptide ID's with Comet search and peptide prophet p>0.9.

Best Result (100%)Tested Values (x%)
Software Version Fusion 2.0
OTMS2 comparisons
MS1 resolution 60k MS1 resolution 60k (100%), 120k (99.6%), 240k (90%)
MS1 AGC target 7e5 MS1 AGC target 5e5 (98.2%), 6e5 (98.8%), 7e5 (100%)
5-30 %B in 150 min 5-30 %B in 90 min (84.3%), in 120min (92.2%), in 150 min (100%)
5-30 %B in 90 min curve 6 5-30 %B in 90 min curve 6 (100%), curve 7 (89.2%), curve 8 (77.1%)
Isolation Width 1.6 Isolation Width 0.4 (90.1%), 0.7 (95.6%), 1.2 (95.1%), 1.6 (100%)
HCD NCE 25 HCD NCE 25 (100%), 29 (99.5%), 30 (98.9%), 31 (98.1%), 32 (99.8%), 33 (99.7%), 35 (99.7%)
OTMS2 Resolution 30k OTMS2 Resolution 15k (98.1%), 30k (100%)
OTMS2 AGC 5e3 (IT 100ms) OTMS2 AGC 5e3 IT100ms (100%), 1e4 (97.9%), 2e4 (95.9%), 3e4 (93.4%), 4e4 (93.2%), 5e4 (92.4%), 2e4 IT300ms (87.3%)
OTMS2: Dynamic Excl 60 sec Dynamic Excl 15 sec (97.0%), 60 sec (100%)
OTMS2 OTMS2 (100%), ITMS2 (91%)
OTMS2 (DE15s) 18933 unique and 32227 total peptide ID's
ITMS2 (DE15s) 17227 unique and 31621 total peptide ID's
OTMS2 cycle time 2 sec OTMS2 cycle time 1 s (90.7%), 2 s (100%), 3 s (99.0%)
Fusion vs Lumos vs QEplus (OTMS2) Fusion 15900(89.8%), Lumos 17700 (100%), QE 15760 (89.0%)
Fusion vs Lumos (ITMS2) Fusion 18100 (100%), Lumos 18000 (99.4%)
ITMS2 comparisons
ITMS2 AGC target 2e3 ITMS2 AGC target 2e3 (100%), 3e3 (99.1%), 4e3 (98.5%), 5e3 (97.1%), 2e4 (91.0%), 3e4 (85.5%), 4e4 (85.3%)
HCD HCD (100%) CID (96.8%)
Dynamic Exclusion DE15 sec unique peptide ID's: ITMS2 DE 15 s (100%), 30 s (97.7%), 60 s (94.6%)
PSM's: ITMS2 DE 15 s (100%), 30 s (76.27%), 60 s (67.1%)
ITMS2 cycle time ITMS2 cycle time 1 s (99.9%), 2 s (100%), 3 s (99.9%)
Fusion vs Lumos vs QEplus (OTMS2) Fusion (SW1.2) 15900 (89.8%), Lumos (SW2.0 beta) 17700 (100%), QE (SW2.5) 15760 (89.0%)
Fusion vs Lumos (ITMS2) Fusion (SW1.2) 18100 (100%), Lumos (SW2.0 beta) 18000 (99.4%)
5-30 %B in 180 min unique ID's 5-30% B in 60 mins (77.8%), 90 mins (88.2%), 120 mins (93.7%), 180 mins 100%)
5-30 %B in 180 min PSM's 5-30% B in 60 mins (45.3%), 90 mins (62.9%), 120 mins (76.5%), 180 mins (100%)
Software Version Fusion 2.1
OTMS2 AGC 5e3 OTMS2 AGC 5e3 (100%), 1e4 (96.5%), 5e4 (79.4%)
OTMS2 signThreshold 3e4 OTMS2 signThreshold 3e4 (100%), 2e4 ( 88.4%)

Precursor m/z and charge state distribution
The chart below shows the charge state and precursor m/z distribution for all the peptides identified with a peptide prophet cutoff 0.9 The average of four LCMS runs with 90 min gradients, two replicates using a HeLa tryptic digest (Pierce, 100 ng on column) and two replicates of a tryptic digest from a Ramos cell whole cell lysate 100ng on column. The combined total number of peptide identifications p>= 0.9 is 38574; the total number of unique peptide identifications p>= 0.9 is 19218.

m/z and charge state distribution