Protein phosphorylation is a highly abundant and important post-translational modification in eukaryotes, affecting over 2/3 of proteins in a given cellular proteome. The modification is implicit in processes including: protein degradation, enzyme regulation, protein-protein interactions, protein kinase and receptor kinase signalling, cell cycle control, and more. These important roles, coupled with the powerful capabilities of modern LC-MS technologies, has made protein phosphorylation an incredibly prolific area of investigation with over 278,000 research articles cited in PubMed as of September 2017.
Past approaches towards phosphorylation analysis included labelling with P32 followed by extensive enzyme digestion and detection by electrophoresis or chromatography methods. The techniques involved in identifying phosphorylation events and pinpointing modified sites were both tedious and time-consuming. With the advent of soft ionization techniques including electrospray ionization coupled and sensitive mass spectrometry detection platforms came a paradigm shift in the range of phosphoprotein analyses and the scope of investigations that could be explored.
Soft ionization through ESI or MALDI methods permits the analysis of intact peptides, small proteins, or protein digestions, without the threat of complete fragmentation and loss of mass information. Using this approach, mass shifts due to phosphorylation can easily be mapped to the base peptide or protein. Soft fragmentation techniques, such as metastable decay in MALDI-TOF analysis, can subsequently identify the exact residue modified in a given reaction. Although MALDI is a method of choice when analyzing samples with excipients or complex matrix components, ESI has excelled due largely to the fact that it is a solution based ionization technique and thus directly compatible with LC separations. This coupling has made pre-fractionation of samples routine and resolution of complex proteomic experiments possible.
Beyond identification of the sites of modification and the abundance, the sensitivity to detect changes in proteins at sub stoichiometric levels in cells is a real advantage of LC-MS. The ability to trace the temporal patterns of phosphorylation, for instance as a cell moves through the cell cycle, is another very powerful advantage.
There is a wide range of different phosphoprotein enrichment methods in circulation, including those using phospho-specific antibodies and binding groups such as titanium dioxide (TiO2). There is an equal number of methods for quantitative detection across wide dynamic ranges and between multiple samples. Challenges include unknown alterations in ionization efficiency between phosphorylated and non-phosphorylated peptides as well as ion suppression in complex backgrounds thereby limiting high-resolution analyses and interpretations.
Modern LC-MS instruments are making sense of complex data sets and are contributing to quantitative power through the use of isobaric tagging and other approaches. SILAC, or stable isotope labelling with amino acids in cell culture, is a powerful method to measure protein abundance between cell cultures grown under different experimental conditions. TMT, or tandem mass tag, labelling solutions serve as an alternative to SILAC, allowing up to 10 protein targets can be labelled at once. Upon fragmentation, the isotope tag reporter ions are used to compare relative abundance with control tags. A third quantitative approach involves label-free analysis, measuring signal abundance integrated over the LC time scale – with the advantages of unlimited sample numbers and fragmentation modes. At the backend, state-of-the-art proteomics data analysis software is striving to apply biological meaning to the large amounts of data accrued.
Manufacturers like Thermo Fisher have created complete workflow solutions to suit given applications, with sample processing kits, sensitive and powerful instrumentation, and integrated software platforms. As an example, the Thermo Fisher Orbitrap Lumor Tribrid MS platform is designed to meet the latest challenges of low level post-translational modification analyses, multiplexed quantitative isobaric tagging, and top-down analysis of intact proteins. The instrument includes a segmented quadrupole mass filter with improved sensitivity and ion transmission, Advanced Vacuum Technology for improved ion transmission to the Orbitrap mass analyzer, and the HD ETD for high capacity fragmentation. The Advanced Peak Determination (APD) algorithm improves data-dependent experimentation. The UVPD is a new fragmentation technique using a 213 nm UV laser and 1M provides ultra-high-resolution detection at 1,000,000 FWHM for improved structural detailing and quantitative analyses.
Total workflow solutions are the new paradigm for phosphorylation and other post-translational modification analyses. The power of modern MS instrumentation has expanded the depth and range of data that can be extracted from a given experiment. Moreover, the latest proteome discovery approaches have necessitated complete solutions to interpret the increasing flow of information.