We offer a range of mass spectrometry services within our mass spectrometry facility (MSF). Read more information about these services below. 

For further information on any of these workflows, please contact the facility manager at msf-uqccr@uq.edu.au

Protein identification

The mass spectrometry facility currently houses the TripleTOF® 5600 System and Eksigent NanoLC Ultra system that is used routinely for the identification of proteins and peptides. The TripleTOF® 5600 System is a hybrid quadrupole time-of-flight (TOF) LC-MS/MS system that uniquely integrates comprehensive qualitative exploration, rapid profiling and high resolution quantitation workflows on a single platform. This system is used primarily for protein identification studies and SWATH acquisition for comprehensive MS/MS quantitation in proteomics. This system is currently set up with the Eksigent NanoLC Ultra technologies to achieve high sensitivity and low flow rates for working with limited amounts of biological material.

Protein complexes are digested with an enzyme (Trypsin) and injected onto a Nano LC system that is coupled to the mass spectrometer. Ions are continually monitored in the mass spectrometer until a peptide elutes which results in the fragmentation of this peptide (MS/MS).  

Once completed, the data is processed using either ProteinPilot ™ Software or Mascot Software against user-defined databases to identify the proteins within the sample.    
 

SWATH analysis

SWATH analysis (commonly referred to as data independent analysis (DIA)) is a new technique that combines the traditional mass spectrometry workflows of data dependent acquisition and MRM or targeted workflows. The advantage of this technique is it provides a complete MS/MS profile of a biological sample that can be stored and re-interrogated at a later time.

There are two steps to this workflow:

  1. a complete list of all of the proteins identified are generated for the samples of interest (referred to as an ion library) using the data dependent acquisition (DDA) techniques.  
  2. the data independent acquisition (DIA) is achieved by stepping across a set precursor mass range (25Da windows or less) and the MS/MS information is captured for each window. The data is processed by searching the MS/MS profile generated against the respective ion library for the biological samples of interest.

At the MSF, we have the TripleTOF® 5600 System and the NanoLC Ultra system that has been set up to run these workflows routinely. The samples are processed with ProteinPilot ™ software to generate the ion library and then imported into the PeakView™ software containing the SWATH acquisition software to extract the ions from the DIA resulting data. Once this data has been processed and reviewed, student t-tests and PCA analysis is completed using MarkerView ™ software.   

 

Quantitation of peptides, small molecules

Targeted or Multiple Reaction Monitoring (MRM or Selective reaction monitoring (SRM)) analysis of biological samples can be used to verify and validate discovery findings such as proteins identified by protein ID and SWATH analysis, western blotting or ELISA workflows.  

This technique using a tandem mass spectrometer of which an ion of a specific mass (compound/ peptide of interest) is selected in the first stage of the instrument, fragmented and then a daughter ion is selected and monitored in the second stage of the mass spectrometer. Resulting data is processed using MultiQuant software for the QTRAP® 5500 system and the Insight and Post run for the LCMS 8050 system.  

Our MSF houses two tandem systems: QTRAP® 5500 System and the LCMS-8050 System.   

The QTRAP® 5500 system is a hybrid triple quadrupole, linear ion trap (LIT) mass spectrometer designed for biomarker, lipidomic and metabolite identification, quantitation and validation. It is currently used for targeted proteomic, lipidomics and metabolomic workflows. It houses the most sensitive ion trap (patented Linear Accelerator Trap) along with ultra-fast scan speeds and full MS3 capabilities. This system has been set up to run with either the Eksigent MicroLC 200 system or the Shimadzu Nexera X2 UHPLC system depending on the application and amount of biological material available for use.  

The LCMS-8050 is a triple quadrupole mass spectrometer that provides high sensitivity quantitation at high speed whilst maintaining quantitative accuracy. It is capable of simultaneously obtaining both qualitative and quantitative information in a single analysis. It is designed for lipidomics, metabolomic and proteomic workflows. The Shimadzu Nexera X2 UHPLC is currently set up on this system allowing sub 2 micron columns to be used thus resulting in narrower peak widths and shorter chromatographic run times.

Post translational modifications

Post translational modification (PTM) analysis can be conducted within the MSF. Depending on the modification of interest (phosphorylation, glycosylation etc), samples are prepared to enhance these features to enable the instrumentation to capture and report on these modifications. Our facility has the TripleTOF® 5600 system and the MALDI TOFTOF 5800 System, capable of reporting these modifications for the biological samples of interest.

Intact protein analysis

Intact protein analysis using mass spectrometry can be achieved using the LC MS or MALDI TOF MS systems. The MSF currently houses the TripleTOF® 5600 system or the MALDI TOFTOF 5800 system that can be used to determine the mass of a protein of interest. Samples are prepared in a number of ways depending on the sample and what instrument the work is being conducted on. 

Non-covalent binding experiments and/or antibody drug conjugate (ADC) studies can also be conducted within the MSF. These experiments are run on the TripleTOF® 5600 System as it provides high resolution data required to distinguish between the varied forms of drug and antibody. Resulting data is analysed using BioPharmaView™ software which provides an accurate deconvolution of the protein, provide the drug to antibody ratio (DAR) for ADC workflows. 

 

Lipidomics

Discovery and targeted lipidomics applications can be conducted within the MSF. Discovery based lipidomics can be completed using the TripleTOF® 5600 System as it has the capability of screening multiple samples using the SWATH technology (MSMSALL) and provides an accurate mass lipid profile. The sample is introduced by infusion or by using the TriVersa NanoMate system, and the MS/MS profile is obtained by stepping the across of mass range of interest by 1Da. The resulting data is processed using the LipidView™ software which contains a comprehensive reference library of over 25,000 lipid species characterised by head groups, fatty acid based and long chain based fragments.

Targeted lipidomics workflows are best completed using triple quadrupole instruments such as the QTRAP® 5500 System or the LCMS-8050 System. Precursor Ion or neutral loss experiments can be set up to monitor the lipid classes as they are detected within the systems. Samples are processed using vendor specific software.  

 

Contact us for bookings and enquiries: 

General Email: ccd-uqccr@uq.edu.au
Phone: +61 7 3346 5500

Postal Address: 71/918 Royal Brisbane Hospital, HERSTON QLD 4029