Phosphoproteomics is a specific type of proteomics that characterizes proteins with the reversible post-translational modification of phosphorylation. Peptide phosphorylation has a vital role in cellular processes such as cell cycle regulation, signal transduction, and protein targeting.
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Though important to cellular processes, phosphopeptides are found in lower abundance than non-phosphorylated peptides. This means that techniques for detection and quantification of non-phosphorylated peptides have had to be adapted for phosphoproteomics.
Basic Phosphoproteomic Analysis
The basic methodology for large scale analysis of phosphopeptides first involves SILAC encoding of cultured cells. SILAC, stable isotope labeling by/with amino acids in cell culture, provides differential labeling of cells ready for mass spectrometry (MS). The technique is based on the addition of a ‘heavy’ and ‘light’ form of amino acids to proteins.
Two cell populations are grown differing only in whether the ‘heavy’ or ‘light’ form of amino acid is incorporated. The mass spectrometer differentiates pairs of chemically identical peptides because of the mass difference. Before entering the mass spectrometer, cells are stimulated, lysed and enzymatically digested.
The separation of peptides occurs through ion-exchange chromatography. A final step of phosphopeptide enrichment then takes place before analysis through mass spectrometry to counteract the low abundance of phosphopeptides.
Different phosphopeptide enrichment techniques are used in phosphoproteomics. In cases where one particular phosphorylated amino acid is being searched for, immunoprecipitation can be used for phosphopeptide enrichment. This involves the employment of antibodies raised against phosphorylated amino acids that bind and isolate the peptide.
For large-scale phosphopeptide enrichment, immobilized metal affinity chromatography (IMAC) is commonly used where phosphopeptides are separated according to their affinity for metal ions immobilized on the solid resin.
Metal oxide affinity chromatography (MOAC) is also employed being composed of a metal oxide or hydroxide matrix, removing the need for resin anchoring. Titanium dioxide is a frequent choice for MOAC because of its known affinity to organic compounds and ability to retain phosphopeptides during high-performance liquid chromatography.
Challenges to Phosphoproteomics
The field of phosphoproteomics is currently limited because of:
- The low abundance of proteins in comparison to the vast range of cells.
- The impact of fractional stoichiometry found in phosphorylation.
- The potential impairment of digestion efficiency during sample preparation.
- The loss of phosphopeptides during sample preparation.
- The impairment of phosphopeptide ionization efficiency.
- The impairment of peptide sequence identification from poor quality MS/MS spectra, caused by the behavior of the labile phosphate group.
- Problems evaluating the localization of phosphorylation sites.
Phosphopeptide site identification and quantification is more difficult than the measurement of non-phosphorylated proteins. Phosphorylation is a dynamic process prone to errors, meaning that some protein phosphorylation events occur without major functional relevance. When studying large amounts of proteins, random phosphorylation events will be reported because of the high sensitivity of modern mass spectrometers.
When random phosphorylation occurs on highly abundant proteins, there is greater difficulty in identifying phosphorylation sites on low abundant proteins. A potential solution to this problem is the formation of temporal profiles for protein phosphorylation upon specific treatment. This would create a strategy for interpreting large amounts of phosphoproteomic data.
Advances in Phosphoproteomics
Advances in MS instrumentation, phosphopeptide enrichment, peptide chromatography and computational proteomics have enhanced the field of phosphoproteomics. Nanoflow liquid chromatography-tandem mass spectrometry (LC-MS/MS) in particular is a contemporary method of identifying and quantifying protein phosphorylation.
Though SILAC is the most common method of labeling for LC-MS/MS phosphopeptide quantification, alternative methods such as label-free quantification (LFQ) and isobaric tandem mass tags (e.g. iTRAQ, TMT) are increasing in popularity. LFQ works through the comparison of peptide MS signal intensities between MS runs meaning that stable isotope labeling is not required.
Isobaric tandem mass tags have an advantage over LFQ which requires the measurement of individual samples; both iTRAQ and TMT can simultaneously measure up to eleven samples in an approach called multiplexing. Choice of quantification techniques is dependent on utilization, with SILAC and LFQ providing greater accuracy in a mixed-species comparison with fixed phosphopeptide ratios whilst algorithms for determining phosphorylation sites are improved with multiplexing.
- Olsen, J.V. et al. 2006. Global, In Vivo, and Site-Specific Phosphorylation Dynamics in Signaling Networks, Cell, 127, pp. 632-648. https://www.cell.com/cell/fulltext/S0092-8674(06)01274-8
- Fila, J. & Honys, D. 2012. Enrichment techniques employed in phosphoproteomics, Amino Acids, 43, pp. 1025-1047. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3418503/
- Solari, F.A. et al. 2015. Why phosphoproteomics is still a challenge, Molecular BioSystems, 11, pp. 1487-1493. http://pubs.rsc.org/en/Content/ArticleHtml/2015/MB/c5mb00024f
- Hogrebe, A. et al. 2018. Benchmarking common quantification strategies for large-scale phosphoproteomics, Nature Communications, 9, e1045. https://www.nature.com/articles/s41467-018-03309-6