Ana Pereira Navaza, Jorge Ruiz Encinar and Alfredo Sanz-Medel of the University of Oviedo in Spain explain why elemental mass spectrometry is a high flier in the world of quantitative phosphoproteomics

Proteins carry out most of the biological functions in a cell. So a thorough understanding of cell functions and biochemical mechanisms in cells requires information about the proteins present, how and why they interact, their functions and when exactly they carry them out. Also, dynamic control of a protein's conformation, and hence its function, is achieved mostly through chemical changes after it has been translated from messenger RNA - so-called post-translational modifications. Of these, phosphorylation is implicated in regulating protein activity and signalling pathways in cells and has received enormous attention recently, due mainly to its connection with cancer.

However much phosphoprotein analysis is needed though, it is far from being straightforward. Firstly, phosphoproteins function at very low levels within cells. Secondly, a single protein can be phosphorylated and dephosphorylated by different kinases and phosphatases, respectively, on one or more different residues, and at different times. These variations may lead to very important biological effects, which should be detectable only if quantitative information (quantitative phosphoproteomics) is possible.

Phosphopeptides in complex samples are destroyed and ionised during ICP-MS and can be quantified using the 31P signal obtained

Classical approaches to analysing protein phosphorylation have consisted of labelling the proteins with radioactive ³²P or Western Blot analysis, which uses gel electrophoresis to separate proteins of different length or structure. However, only advanced molecular mass spectrometry (MS)-based methodologies developed during the past decade have really boosted quantitative phosphorylation analysis.

Quantitative data in phosphoproteomics are often obtained as relative phosphoprotein levels between two cell states. Relative strategies are very useful to evaluate phosphorylation changes with experiment, but they fail to provide absolute phosphopeptide abundance levels. In fact, absolute quantification of phosphoproteins at given phosphorylation sites has barely been addressed using molecular MS. Moreover, the few methods reported for this purpose so far require the previous chemical synthesis of each individual phosphopeptide, preferably as an isotopically-labelled form. Therefore, application of these methods is restricted to proteins with well-known phosphorylation behaviour and is limited by the availability of the labelled phosphopeptides.

Absolute phosphoprotein concentration determination by molecular MS techniques is constrained by the fact that they provide matrix- and species-dependent signals. Conversely, elemental MS (for example inductively coupled plasma MS, ICP-MS) gives an analytical response that can be made directly proportional to the absolute amount of the element present (P in this case). The ³¹P signal obtained will be independent of the amino acid sequence of the phosphopeptide and of the matrix in which it is analysed.

As a highly accurate and precise method, ICP-MS will allow very small changes in protein phosphorylation levels to be followed, aiding, for example, cell signalling studies. The exceptional features of the ICP-MS technique also open the door to species-independent calibration, providing a generic approach for absolute quantification of biomolecules containing ICP-MS heteroatoms (elements different from C, H, N and O). In heteroatom-tagged proteomics, absolute quantitative results can be traced directly to a simple certified P standard and so will allow sound data comparisons among different laboratories. 

Yet plasma MS comes with a price: the loss of molecular information. Complex biological mixtures call for high resolution separation and molecular MS of the individual components is still mandatory to find the amino acid sequences of the phosphopeptides quantified by ICP-MS.

Complementary techniques, both molecular and elemental MS are required to translate an amount of phosphorus into an absolute amount of phosphopeptide and, of course, to determine the phosphorylation sites in the peptide - the first step to quantitative phosphoproteomics.

Read Sanz-Medel  et al's critical review 'Quantitative protein phosphorylation analysis: the role of ICP-MS' in issue 10 of Journal of Analytical Atomic Spectrometry.