Relevant phosphoproteomic and mass spectrometry: approaches useful in clinical research

Abstract

Background “It's not what we do, it's the way that we do it”. Never has this maxim been truer in proteomics than now. Mass Spectrometry‐based proteomics/phosphoproteomics tools are critical to understand the structure and dynamics (spatial and temporal) of signalling that engages and migrates through the entire proteome. Approaches such as affinity purification followed by Mass Spectrometry (MS) have been used to elucidate relevant biological questions disease vs. health. Thousands of proteins interact via physical and chemical association. Moreover, certain proteins can covalently modify other proteins post‐translationally. These post‐translational modifications (PTMs) ultimately give rise to the emergent functions of cells in sequence, space and time. Findings Understanding the functions of phosphorylated proteins thus requires one to study proteomes as linked‐systems rather than collections of individual protein molecules. Indeed, the interacting proteome or protein‐network knowledge has recently received much attention, as network‐systems (signalling pathways) are effective snapshots in time, of the proteome as a whole. MS approaches are clearly essential, in spite of the difficulties of some low abundance proteins for future clinical advances. Conclusion Clinical proteomics‐MS has come a long way in the past decade in terms of technology/platform development, protein chemistry, and together with bioinformatics and other OMICS tools to identify molecular signatures of diseases based on protein pathways and signalling cascades. Hence, there is great promise for disease diagnosis, prognosis, and prediction of therapeutic outcome on an individualized basis. However, and as a general rule, without correct study design, strategy and implementation of robust analytical methodologies, the efforts, efficiency and expectations to make biomarkers (especially phosphorylated kinases) a useful reality in the near future, can easily be hampered.

Enrichment of Phosphorylated Peptides and Proteins by Selective Precipitation Methods

“…Mass spectrometric methods such as MALDI-TOF MS or ESI-MS have become the most powerful tools for the identification of proteins in phosphoproteomics [77,78]. However, owing to the huge dynamic range of biological samples a prior reduction of sample complexity is often essential before mass spectrometric analysis.…”

A proteomic approach to obesity and type 2 diabetes

“…This type of complementary data may or may not be easily interpreted but many clinical research studies show that analysing both types of samples offers the possibility to explain linked biological processes. Additionally, the selection of a given proteomic platform must be made according to the goal of the experiment . For example, high‐throughput–proteomic strategies may be useful for discovery of biomarkers specifically related to various stages of obesity and DM2, and they may help to improve current treatments and innovative therapies.…”

“…On the other hand, once the target proteins are selected by the discovery phase of the research, analysis of those proteins in the complex mixture of biological samples can be conducted by selected reaction monitoring (SRM) or multiple reaction monitoring (MRM) assays. The sample‐preparation of body fluids is a tedious but critical step in obtaining reliable results . For example, urine contains low levels of proteins compared to blood and a large amount of sample and/or a concentration procedure may be required for analyses.…”

Clinical proteomics and OMICS clues useful in translational medicine research

“…For the future, greatly improved involvement and collaboration from all interested parties – including experts in discovery and assay development, in health policy, in clinical trial units, in the diagnostics industry and in laboratories responsible for providing clinical testing – will almost certainly lead to earlier identification and implementation of promising new biomarkers [68-71]. …”