The Mass Spectrometry Unit at Stellenbosch University in the Western Cape performs quantitative and qualitative analysis of organic molecules using Mass spectrometry. Services include GC-MS analysis, LC-MS analysis, accurate mass determinations and Proteomic analysis.
The Mass Spectrometry Unit, part of the Central Analytical Facilities of Stellenbosch University, serves as a resource offering contemporary mass spectrometric techniques and the "state-of-the-art" instrumentation needed for the diversity of research interests on- and off-campus.
During electrospray ionisation (ESI), multiply charged ions of macromolecules are produced. This enables the accurate determination of molecular masses of molecules such as proteins, oligonucleotides and oligosaccharides. This technique is used for the analysis of polar non-volatile molecules. The ionisation is softer than electron impact (EI) and very few if any fragment ions are observed.
During electron impact ionisation (EI), ions are generated by colliding electrons with the sample molecule in gas phase, resulting in the loss of an electron from the sample molecule generating a molecular ion M+. Due to the excess in the internal energy, the molecular ion is often fragmented and may thus not show up in the spectrum. Fragmentation that occurs is highly reproducible and the spectra may often be used to search library databases for the tentative identification of the compound. This technique is used for the analysis of volatile, more non-polar compounds.
The Proteomics facility is equipped with two state of the art mass spectrometers a LTQ Orbitrap Velos and a Fusion which were both the first of their kind in South Africa. These sophisticated mass spectrometers provide South African researchers the unique opportunity to analyse thousands of proteins in one run. It also allows SA researchers the chance to compete with international proteomic researchers albeit at a local price without compromise in quality.
Qualitative proteomics asks the “what is there” questions and, in the case of label free proteomics, requires multiple injections for statistical purposes.
Quantitative proteomics requires a reference sample for comparison and therefore, the reference is included in the cost/comparison. Therefore the cost for 1 comparison equals the reference as well as the sample to be compared. As only one reference is required per comparative group subsequent comparisons do not include the reference sample i.e. four samples for comparison equals samples and one reference. Statistical analyses are performed on the data sets to obtain regulation data. This requires at least three injections per sample. The cost is for 3 injections of unfractionated samples and needs to be multiplied for the number of fractions required.
The CAF unit is able to perform labelled quantitation using iTRAQ (isobaric Tag for Relative and Absolute Quantitation; AB Sciex) and TMT (Tandem Mass Tag; Thermo Scientific). This method is based in the covalent attachment of an isobaric mass tag to all the samples, depending on the choice of tag 4-10 samples can be quantified. The technique is based on the detection of reporter ions after fragmentation and therefore all the samples for analysis is combined prior to LCMS/MS. The standard cost options are unfractionated (single LC-MS/MS injection) or fractionated in either 5 or 10 fractions. The cost does not include the cost of the labels but do include the labelling of the samples. Separations is the agent for both iTRAQ and TMT and the reagent cost can be obtained from them.
Qualitative proteomics asks the “what is there” questions and is usually performed using single injections. No quantitative data can be obtained as this analysis option is not designed for downstream statistics required for quantitation.
Complete Proteome Extracts
The complexity of total proteome demands long gradients and preferably the use of an advanced mass spectrometer such as the Thermo Scientific fusion. Samples can either be analysed unfractionated or as multiple fractions and the data combined after database interrogation. Typically 2-3 hour gradients are sufficient depending on proteome complexity.
Purified or Partially Purified Samples
The much reduced complexity of purified samples allow the use of shorter gradient times and does not require the use advanced mass spectrometers. For purified samples such as a single protein band from an SDS-PAGE gel, a 35 min gradient on the Velos mass spectrometer is sufficient.
MS Unit: Services
- Mass spectrometry (MS) with electrospray ionization (ESI) or atmospheric pressure chemical ionization (APCI)
- Accurate mass determination
- LC-MS and LC-MS/MS for both qualitative and quantitative work
- LC-MS and HPLC method development and consulting services
- A do-it-yourself service for HPLC with UV or RI detection for internal users
- GC-MS and GC method development and consulting services
- Proteomics (peptide sequencing and protein identification via capillary LC-MS/MS & Database search)
- Amino acid analysis
- Ochratoxin A, natamycin, and sorbic acid analysis in wine
- Melamine analysis
- Plasticizers and phthalate analysis in various matrices including wine and spirits
- Biogenic amine analysis in wine including tryptamine, tyramine and histamine
- Various medicinal plant analysis including Hoodia, Harpagoside and Mesembrine
- SPME GC-MS analysis of volatile compounds