Methods and Libraries

These methods have been carefully optimized for the relevant workflows by our application specialists.

They are compatible with otofControl 6.2.6 and timsControl 4.0 plus Service Releases.

Please download the data acquisition software package Compass 2023 here.

If you need a default method for an older software version, please contact our support team (esitof.support@bruker.com).

Proteomics TIMS on

• DDA PASEF-low_sample_amount_1.9sec_cycletime.m
  Low sample amount with increased sensitivity due to longer TIMS ramp time
• dia-PASEF_low-sample-amount_0.64-1.45-Mobility_0.96sec-cycletime.m
  This dia-PASEF method can be used as a QC method for the qualification of the LC-MS system or for any complex proteomics sample of low concentration (single cell up to 5 ng):
• DDA PASEF-short_gradient_0.5sec_cycletime.m
  Short LC separation times (30 min or less)
• DDA PASEF-standard_1.1sec_cycletime.m
  Default PASEF method for most shotgun applications
• DDA PASEF-PepMap_1.1sec.m
• DDA PASEF-TMT_2.2sec_cycletime.m
  Method for TMT / iTRAQ up to 8-plex using TIMS- and collision energy-stepping
• dia-PASEF - long gradient.m
  dia-PASEF method with 1.8 sec.cycle time for longer LC separation times
• dia-PASEF - short gradient.m
  dia-PASEF method with 0.9 sec.cycle time for short LC times (30 min or less)

Proteomics TIMS off

• Protein identification - Instant Expertise - AUTO MSMS.m
  Standard proteomics method utilizing variable MS / MS speed(3 sec.fixed cycle time)
• PepMap - Instant Expertise - Auto MSMS.m
• Intact antibodies - MS.m
• Intact proteins - MS.m

4D-Lipidomics 

(TIMS on, PASEF, lipids)

• 4D-Lipidomics_neg.m
• 4D-Lipidomics_pos.m
  Covered mass range: 100 - 1350 m/z
  Covered mobility range (1/K0): 0.55 - 1.9 V*s/cm2. 

Application Type:
These methods cover a typical mass range for lipidomics applications and are available in positive and negative ion mode for the timsTOF, timsTOF Pro and timsTOF fleX systems. The timsTOF Pro and fleX systems offer the PASEF acquisition mode, while the timsTOF systems use the so-called AutoMS/MS mode to acquire MS2 spectra. TIMS off methods for lipid profiling are not available.

Important Notes:
The source gas settings have been optimized for an HPLC solvent flow of about 400 µL/min. Please adjust the gas settings if you change the flow rate. Higher LC flow rates require an improved desolvation by increased Nebulizer gas [bar], Dry gas [L/min] and Dry Temp [°C]. Instead of switching polarity inside a method, please simply load the method of the respective other polarity.

4D-Metabolomics

(TIMS on, PASEF, small molecules)

• 4D-Metabolomics_neg.m
• 4D-Metabolomics_pos.m
  Covered mass range: 20 - 1000 m/z
  Covered mobility range (1/K0): 0.45 - 1.45 V*s/cm2 

Application Type:
These methods cover a rather low molecular weight range for metabolomics applications and are available in positive and negative ion mode for the timsTOF, timsTOF Pro and timsTOF fleX systems. The focus is on a mass range below 650 m/z. The timsTOF Pro and fleX systems offer the PASEF option, while the timsTOF systems use the so-called AutoMS/MS acquisition mode to acquire MS2 spectra. Both MS/MS modi use a stepping of the collision cell RF value and the collision energy in order to improve the MS/MS quality.

Important Notes:
The source gas settings have been optimized for an HPLC solvent flow of about 400 µL/min. Please adjust the gas settings if you change the flow rate. Higher LC flow rates require an improved desolvation by increased Nebulizer gas [bar], Dry gas [L/min] and Dry Temp [°C]. Instead of switching polarity inside a method, please simply load the method of the respective other polarity.

MALDI Methods for timsTOF fleX

MALDI

• MSMS lipids tims off pos.m
• small molecules tims off pos DD.m
  
  

MALDI Imaging TIMS off

• lipids tims off imaging 20 um pos.m
• lipids tims off imaging 20 um pos_MALDI-2.m
• peptides tims off imaging 50 um pos.m
• small molecules tims off imaging 20 um pos.m
  
  

MALDI Imaging TIMS on

• lipids tims on imaging 20 um pos.m
• small molecules tims on imaging 20 um pos.m

Proteomics TIMS on

• DDA PASEF-low_sample_amount_1.9sec_cycletime.m
  Low sample amount with increased sensitivity due to longer TIMS ramp time
• DDA PASEF-short_gradient_0.5sec_cycletime.m
  Short LC separation times (30 min or less)
• DDA PASEF-standard_1.1sec_cycletime.m
  Default PASEF method for most shotgun applications
• DDA PASEF-PepMap_1.1sec.m
• DDA PASEF-TMT_2.2sec_cycletime.m                      
  Method for TMT/iTRAQ up to 8-plex using TIMS- and collision energy-stepping
• dia-PASEF - long gradient.m                                                   
  dia-PASEF method with 1.8 sec. cycle time for longer LC separation times
• dia-PASEF - short gradient.m
  dia-PASEF method with 0.9 sec. cycle time for short LC times (30 min or less)

Proteomics TIMS off

• Protein identification - Instant Expertise - AUTO MSMS.m
  Standard proteomics method utilizing variable MS/MS speed (3 sec. fixed cycle time)
• PepMap - Instant Expertise - Auto MSMS.m
• Intact antibodies - MS.m
• Intact proteins - MS.m

4D-Lipidomics 

• 4D-Lipidomics_neg.m
• 4D-Lipidomics_pos.m

Covered mass range 100-1350 m/z in MS mode, mobility range 0.55-1.9 V*s/cm2. 

Application Type: Please adjust proportionally the source parameters (“Nebulizer”, “Dry gas”, “Dry Temp”) if flows below or higher than 0.4 mL/min are applied.

4D-Metabolomics

• 4D-Metabolomics_neg.m
• 4D-Metabolomics_pos.m

Covered mass range ~150-800 m/z in MS mode, mobility range: 0.45-1.45 V*s/cm2. 

Application Type: MS/MS stepping, including default values of collision cell RF, transfer time, and collision energy, is applied to improve the MS/MS quality. Please adjust proportionally the source parameters (“Nebulizer”, “Dry gas”, “Dry Temp”) if flows below or higher than 0.4 mL/min are applied.

• 4D-Metabolomics_Stepping_neg.m
• 4D-Metabolomics_Stepping_pos.m

Covered mass range ~75-750 m/z in MS mode, mobility range 0.1-1.5 V*s/cm2. 

Application Type: TIMS stepping, including default mobility ranges and values of collision cell RF, transfer time, and collision energy, is applied to improve the MS/MS quality and widen the mass/mobility ranges analysed in a single analysis. Please adjust proportionally the source parameters (“Nebulizer”, “Dry gas”, “Dry Temp”) if flows below or higher than 0.4 mL/min are applied.

To apply TIMS Stepping: the following default profiles (a given subset of instrument parameters) are available within each method.  When using these profiles, several values present in the Tune Tab in timsControl are specified and not subject to customization.

• TIMS Stepping PASEF neg. Metabolomics 75-750 m/z
• TIMS Stepping PASEF pos. Metabolomics 75-750 m/z

Covered mass range 75-750 m/z in MS mode. 

Application Type: Can be used in PASEF-based methods and has been optimized for the comprehensive untargeted metabolome analysis. 

Additional Notes: TIMS stepping can be applied in MS, PASEF, and bbCID modes. Data acquired using PASEF methods and profiles can be processed in MetaboScape. Data acquired with bbCID methods and profiles can be evaluated using the TASQ software.

3D-Metabolomics

• 3D-Metabolomics_neg.m
• 3D-Metabolomics_pos.m

Covered mass range 20-1300 m/z in MS Mode. 

MS and MS/MS stepping of collision cell RF, transfer time, and collision energy are applied to improve the MS/MS quality. Please adjust proportionally the source parameters (“Nebulizer”, “Dry gas”, “Dry Temp”) if flows below or higher than 0.4 mL/min are applied.

MALDI Methods for timsTOF fleX

MALDI

• MSMS lipids tims off pos.m
• small molecules tims off pos DD.m
  
  

MALDI Imaging TIMS off

• lipids tims off imaging 20 um pos.m
• lipids tims off imaging 20 um pos_MALDI-2.m
• peptides tims off imaging 50 um pos.m
• small molecules tims off imaging 20 um pos.m
  
  

MALDI Imaging TIMS on

• lipids tims on imaging 20 um pos.m
• small molecules tims on imaging 20 um pos.m

Some of these methods are restricted to a particular hardware configuration. Use the current instrument control software to find out about your hardware configuration. 

Not sure? Feel free to ask our support hotline (esitof.support@bruker.com)

In otofControl 6.2.1, open the “About” dialog, click “more”. Look up the line “Instrument Controller”. 
Options 0x001AB023 -> Hardware Configuration A
Options 0x005AB023 -> Hardware Configuration B

In timsControl 2.0, open the “About” dialog.
TIMS cartridge # 1848291 -> Hardware Configuration A
TIMS cartridge # 1875139 -> Hardware Configuration B

Proteomics TIMS on

• DDA PASEF-low_sample_amount_1.9sec_cycletime.m
• DDA PASEF-short_gradient_0.5sec_cycletime.m
• DDA PASEF-standard_1.1sec_cycletime.m
• DDA PASEF-TMT_2.2sec_cycletime.m
• dia-PASEF - long gradient.m
• dia-PASEF - short gradient.m

Proteomics TIMS off

• Protein identification - Instant Expertise - AUTO MSMS.m

4D-Lipidomics

• 4D-Lipidomics_neg.m
• 4D-Lipidomics_pos.m

4D-Metabolomics

• 4D-Metabolomics_neg.m
• 4D-Metabolomics_pos.

Metabolomics - TIMS off

• Metabolomics TIMS off_neg.m
• Metabolomics TIMS off_pos.m 

DI TIMS check

• DI check neg.m
• DI check pos.m

Proteomics TIMS on

• DDA PASEF-low_sample_amount_1.9sec_cycletime.m
• DDA PASEF-short_gradient_0.5sec_cycletime.m
• DDA PASEF-standard_1.1sec_cycletime.m
• DDA PASEF-TMT_2.2sec_cycletime.m
• dia-PASEF - long gradient.m
• dia-PASEF - short gradient.m

Proteomics TIMS off

• Protein identification - Instant Expertise - AUTO MSMS.m

4D-Lipidomics

• 4D-Lipidomics_neg.m
• 4D-Lipidomics_pos.m

4D-Metabolomics

• 4D-Metabolomics_neg.m
• 4D-Metabolomics_pos.

Metabolomics - TIMS off

• Metabolomics TIMS off_neg.m
• Metabolomics TIMS off_pos.m 

DI TIMS check

• DI check neg.m
• DI check pos.m

These methods are included in the ESI downloads for timsTOF Pro and timsTOF fleX. Download the file suitable for your Hardware Configuration and then select the appropriate instrument when installing the methods.

4D-Lipidomics

• 4D-Lipidomics_neg.m
• 4D-Lipidomics_pos.m 

4D-Metabolomics

• 4D-Metabolomics_neg.m
• 4D-Metabolomics_pos.m

Metabolomics - TIMS off

• Metabolomics TIMS off_neg.m
• Metabolomics TIMS off_pos.m  

DI TIMS check

• DI check neg.m
• DI check pos.m

MALDI

• MSMS lipids tims off pos.m
• small molecules tims off pos DD.m
   

MALDI Imaging TIMS off

• lipids tims off imaging 20 um pos.m
• lipids tims off imaging 20 um pos_MALDI-2.m
• peptides tims off imaging 50 um pos.m
• small molecules tims off imaging 20 um pos.m
   

MALDI Imaging TIMS on

• lipids tims on imaging 20 um pos.m
• small molecules tims on imaging 20 um pos.m

These methods have been carefully optimized for the relevant workflows by our application specialists.

Please download the data acquisition software package Compass 2025 here.

If you need a default method for an older software version, please contact our support team (esitof.support@bruker.com).

These methods have been carefully optimized for the relevant workflows by our application specialists.

Please download the data acquisition software package Compass 2023b here.

If you need a default method for an older software version, please contact our support team (esitof.support@bruker.com).

Now coming with CCS analyte lists for 137 compounds

The Bruker Sumner MetaboBASE® Plant Libraries 1.0 were generated in a collaboration between Bruker and Drs. Dennis Fine, Daniel Wherritt*, and Lloyd W. Sumner* from the Plant Biology Division of the Noble Foundation, Ardmore, OK, USA. The libraries contain MS and MS/MS spectra that were acquired using a Bruker impact QToF MS, and enable identification of metabolites through spectral library searches and matching.

NOTE: Please keep in mind that a library search result indicates which spectrum from the utilized library matches best to the acquired MS/MS spectrum. This does not necessarily mean that this compound was unambiguously identified. Therefore, the tentative identifications from spectral library queries should be validated, e.g. by comparison of retention times, CCS and MS/MS spectra to a reference standard.

*Current affiliations: 
Daniel Wherritt, Department of Chemistry, University of Texas at San Antonio, San Antonio, TX, USA
Lloyd W. Sumner, Department of Biochemistry, University of Missouri, Columbia, MO, USA

NOTE: The Bruker Sumner MetaboBASE® Plant Libraries 1.0 are for research use only.
NOTE: Please keep in mind that a library search result indicates which spectrum from the utilized library matches best to the acquired MS/MS spectrum. This does not necessarily mean that this compound was unambiguously identified. Therefore, the tentative identifications from spectral library queries should be validated, e.g. by comparison of retention times and MS/MS spectra to a reference standard.

This library contains MS/MS spectra collected from mixtures of drug compounds from the Reframe drug library (https://reframedb.org/). The library contains information on the mean collision cross section (CCS) values from triplicate acquisition, and COV <2%. Mass Spectral and ion mobility data was collected on a timsTOF Pro 2 in positive ESI mode at 20eV and 40eV CE. Feature extraction and annotations were processed in Metaboscape 2025b. These annotations have been filtered for an MSMS score >200. The spectral library was created by Victoria Deleray videleray@ucsd.edu in the Dorrestein Lab at the University of California, San Diego.

NOTE: The GNPS-Reframe Drug CCS Library is for research use only.
NOTE: Please keep in mind that a library search result indicates which spectrum from the utilized library matches best to the acquired MS/MS spectrum. This does not necessarily mean that this compound was unambiguously identified. Therefore, the tentative identifications from spectral library queries should be validated, e.g. by comparison of retention times and MS/MS spectra to a reference standard.