The identification of proteins and their biological function has become an important part of biological research and the drug discovery process.
However, the identification of the proteins present in a sample is not sufficient to fully characterise a system as the proteome is dynamic and protein expression levels changes in response to an organism's environment or disease state.
The latest research, published in the journal Analytical Chemistry , uses an elemental MS technique known as ICPMS (inductively coupled plasma MS) to determine the quantity of proteins in a sample - even those whose structure and composition are unknown.
The analysis of proteins is usually conducted by chopping them into small peptide fragments using protease enzymes before separating those using techniques such as high performance liquid chromatography (HPLC) or electrophoresis.
The peptide fragments are then analysed using mass spectrometry (MS) techniques that 'weigh' individual peptides. In addition, by using collision cells in the mass spectrometer these peptides can be further degraded to give information about the specific peptide sequences.
"It should be stressed that elemental MS and molecular MS are really complimentary techniques for proteomics," write the authors.
"Elemental MS has a unique quantitative ability and unmatched sensitivity for elemental detection, whereas molecular MS can probe the structure, dynamics and functional properties of proteins."
The major drawback of molecular MS techniques is that quantification is somewhat ambiguous due to the diverse ionisation efficiencies of the different compounds.
To overcome this problem various external peptide standards have been developed as rulers against which the size of the fragments can be measured against.
In addition, stable isotope labelling of proteins and peptides using iTRAQ reagents can be used to enable relative quantification and many successful applications of the technique have been reported.
The new research shows that elemental MS on an ICPMS instrument can be used to quantitatively analyse proteins based on their sulphur content.
While a typical proteomics experiment uses a soft ionisation source, such as ESI (electropray ionisation) or MALDI (matrix assisted laser desorption ionisation) that leave the peptides mostly intact, the ICPMS system operates at around 7000K breaking all the chemical bonds in the sample as it is turned into a plasma.
This means that all the ions are pumped into the MS with the response of any element being completely independent of the molecular environment it was in before being plasmarised.
The researchers had to overcome various obstacles to enable the study including ensuring that the ICPMS remained stable when linked to the HPLC and did this by using a postcolumn isotope dilution analysis method.
To carry out the research a Thermo Fisher Scientific X7 ICPMS was coupled with a Waters HPLC system with a UV/Vis (ultra violet / visible) detector that was used to monitor the various fractions.
The system enabled the quantification of the bovine serum albumin (BSA), superoxide dismutase (SOD) and metalothionein-II model proteins with detection limits as low as 8pmol.
The system could also be used to measure the amount of copper (Cu) and zinc (Zn) in the proteins, enabling the ratios of S/Cu and S/Zn to be calculated. Variation in the metal content of a biological system has previously been linked with the onset of various diseases such as Alzheimer's and Parkinson's diseases.
The author's write that: "the method has abilities not only to quantify proteins via sulphur, but also to characterise protein stoichiometric composition, which will be helpful for protein identification."
