Recent advances in phosphoproteomics and application to neurological diseases

Abstract

Phosphorylation has an incredible impact on the biological behavior of proteins, altering everything from intrinsic activity to cellular localization and complex formation. It is no surprise then that this post-translational modification has been the subject of intense study and that, with the advent of faster, more accurate instrumentation, the number of large-scale mass spectrometry-based phosphoproteomic studies has swelled over the past decade. Recent developments in sample preparation, phosphorylation enrichment, quantification, and data analysis strategies permit both targeted and ultra-deep phosphoproteome profiling, but challenges remain in pinpointing biologically relevant phosphorylation events. We describe here technological advances that have facilitated phosphoproteomic analysis of cells, tissues, and biofluids and note applications to neuropathologies in which the phosphorylation machinery may be dysregulated, much as it is in cancer.

Figure 1

Typical bottom-up phosphoproteomics workflow. Proteins are first extracted from cell lines, tissues, or biofluids and digested into peptides by site-specific proteases such as GluC, trypsin, or LysC. Phosphopeptides are then enriched and subjected to tandem mass spectrometry before bioinformatics analyses, including quantitation, signal transduction pathway mapping, or kinase motif extraction.

Figure 2

Common strategies for phosphopeptide enrichment.

Figure 3

Popular approaches for quantitative LC-MS.