Mass spectrometry: proteomics

Mass spectrometry (MS) has emerged as one of the most valuable tools in modern biology.

In proteomics, it facilitates the measurement of protein mass and protein identity, as well as quantification with extraordinary sensitivity, speed and accuracy.

MS can also be used to analyse other molecules such as lipids, glycans and oligonucleotides for identification and often quantification. It's a core technology for "omics" research.

Contact us

Contact Dr Amanda Nouwens for more information on sample preparation, experimental design, prices, and seminar dates.

Analyses and applications

Analyses

Protein identification from complex mixtures and purified proteins using bottom-up or top-down methods.
Quantitative workflows (SWATH, multiple reaction monitoring, labelled techniques).
Analysis of post-translational modifications (eg glycopeptides, dimethylarginine, succinylation, phosphorylation, disulphide-bond mapping, etc).
Intact mass analyses to determine exact protein size.
Native MS for protein complexes.
De novo sequencing of novel proteins.

Applications and projects

Identification of proteins (eg those not encoded by main ORFs; confirmation of recombinant protein purification; identification of proteins from co-IP experiments).
Relative quantification of proteins from different growth conditions using SWATH (wildtype versus mutant yeast, normal versus Alzheimer brain, normal versus diseased patient saliva samples).
Identification of biomarkers in sera and saliva for human diseases.
Mapping post-translational modifications, including type, amino acid location/s and analysis of when proteins are modified (eg glycosylation status in yeast mutants, dimethyarginine status on hnRNP proteins, disulphide bond linkages in recombinant proteins).
Confirming intact protein size so as to identify PTMs or truncations on proteins.

Our facility has enabled a large number of School researchers and collaborators to answer their biological questions.

Equipment and location

Instruments

ABSciex ZenoTof 7600

The Zenotof 7600 mass spectrometer is the newest quadrupole-hybrid time-of-flight instrument released by ABSciex. It's a sensitive state-of-the-art instrument with high resolution, mass accuracy and fast acquisition rates. The new design has a Zeno trap, meaning 90% of frament ions are collected, resulting in far superior MS/MS spectra. The system also has Electron Activated Disscoiation, an alternate method to fragmenting compounds, allowing for structural characterisation of lipids and natural products, as well as retaining labile protein modifications on peptides (eg phosphorylation and glycosylation). Teh system is suitable for both discovery project (ie what proteins are in my sample?), as well as relative and abolute quantitation of proteins (SWATH, iTRAQ, SILAC, etc). A Waters M-class UPLC system capable fo both micro and nanofllow LC separations is available. For robustness, the LCMS system is typically set up in microflow operation, but can be run for specific projects with nanoflow for ultimate sensitivity.

ABSciex TripleTof 5600

The TripleTof 5600 mass spectrometer is a quadrupole-hybrid time-of-flight instrument. It's a sensitive state-of-the-art instrument with high resolution, mass accuracy and fast acquisition rates (~50 MS/MS per second), suitable for discovery projects (ie what proteins are in my sample?), relative quantitation of proteins (SWATH, iTRAQ, SILAC etc), intact mass analysis (<10 ppm error) and (some) native MS. A Shimadzu Prominence 1D nano-LC system is used to provide upstream separation of samples with reversed phase chromatography.

ABSciex QTRAP 5500

The QTRAP 5500 mass spectrometer is a quadrupole-TRAP instrument most suitable for quantification of samples and structural analyses (eg MS^3, neutral loss and precursor ion scans). It has at least 10-fold sensitivity over previous models. An Agilent 1100/1200 capLC and a Shimadzu UHPLC system are available for upstream separation of samples.

Orbitrap Elite with ETD

The Orbitrap Elite with ETD is a state-of-the-art hybrid trap mass spectrometer. Suitable for protein identification from both bottom-up and top-down methods, as well as quantification of molecules (labelled samples), intact mass analyses, and characterisation of post-translational modifications. Accurate to < 1ppm and with resolution >240,000. A Dionex nano-UPLC system is used to provide upstream separation of samples with reversed-phase chromatography.

Sciex X500B QTOF

The Sciex X500B QTOF system is a high resolution, accurate mass instrument suitable for proteomics, metabolomics, biopharmaceuticals and small molecules. An Exion UPLC system and an Agilent capillary HPLC system are available for use with the mass spectrometer to allow separation of complex mixtures. The LCMS system is suitable for both identification and quantification of compounds, with all relevant downstream data analysis software also available for researchers to use.

Associated equipment and software

Shimadzu Prominence nano-LC system.
Agilent 1100/1200 capLC system.
Shimadzu Prominence UHPLC system.
Various MS software programs (ProteinPilot, MRMPilot, MultiQuant, LipidView).
Speed-vacuum concentrators, freeze-driers, water-bath sonicators, offline HPLC fractionators and incubators.

Location

Molecular Biosciences Building (Building 76), Room 441A

Sample requirements and training

Sample requirements

Sample preparation is critical: samples need to be detergent- and salt-free.

In general, for protein identification and quantification, samples need to be enzymatically digested (eg trypsin) followed by SPE (solid phase extraction) clean-up.

The amount required depends on sample complexity with sensitivity on the order of femtomol to attomol range. As an easy guide, a Coomassie stained gel slice should provide excellent results for gel slices, or 1-5ug of a complex mix of digested proteins in solution.

To provide greater proteome sequence coverage, we offer an offline HPLC system for upstream fractionation of complex samples prior to LC-MS/MS analysis.

For intact mass analysis, proteins need to be in a detergent-free buffer or solvent.

The amount required depends on size:

5 kDa: ~25 pmol
20 kDa: ~100 pmol
60 kDa: ~500 pmol.

Training

We offer twice-yearly introductory seminars on MS that cover:

an introduction to MS
sample preparation for proteomics
database searching.

Charges and considerations

Fees

We charge an access fee to cover consumable and maintenance costs.

Charges

Internal users

External users

Individual analyses: $390 per sample

If you submit more than 20 samples within two months, prices will convert to tier charges.

Tier charges

Tier 1 group user	<400 samples or <300 hours	$8,736 for 1 year or $4,368 for 6 months (at half the usage rate)
Tier 2 group user	401–800 samples or 300–600 hours	$15,288 for 1 year or $7,644 for 6 months (at half the usage rate)
Tier 3 group user	801–1600 samples or 600–1200 hours	$16,800 for 1 year or $8,400 for 6 months (at half the usage rate)

Note:

For usage above 1600 samples (or 1200 hours annually), prices are by negotiation.
As some samples require long acquisition times, tiers are based on sample number and hours.

Considerations

UQ users have priority of access.

You'll need to undertake the relevant building and other inductions before using equipment.