2015R-molecular biosystems-Why Phosphoproteomics remains a challenge

Protein activity is mainly modulated by dynamic reversible (and
irreversible) post-translational modifications (PTMs).1

Phosphorylation is a reversible PTM involved in numerous regulatory
mechanisms in eukaryotic cells, such as cell division, apoptosis, response to extracellular signals and growth factor stimulation,2 and is regulated by the interplay of protein kinases and phosphatases.3,4

The fundamental importance of protein phosphorylation and the continuous development in the field of protein mass spectrometry render phosphoproteomics a valuable tool in current life science, allowing the study of regulatory changes
over time and/or between conditions. However, despite its routine use in many laboratories around the world several limitations still have to be faced.5,6 Among those are
(i) the analysis of low abundant proteins in the light of the vast dynamic range of cells,
(ii) the low stoichiometry of phosphorylation,
(iii) potentially impaired digestion efficiency,7
(iv) phosphopeptide losses during sample preparation and chromatography,8–11
(v) impaired ionization efficiency of phosphopeptides,
(vi) the peculiar behavior特异动态 of the labile phosphate group 不稳定磷酸基团upon collision induced dissociation, often resulting in poor quality MS/MS spectra that can impair both identification of the peptide sequence and the
(vii) correct localization of the phosphorylation site(s),12–14 and
(viii) the general limitations of peptide-centric proteomics.15

Taken together, these issues can complicate the identification and quantification of phosphopeptides (and particularly phosphorylation sites) compared to their non-phosphorylated counterparts, and can produce wrong, misleading and inaccurate
identification and quantification results.

Here, we discuss current obstacles that can emerge in phosphoproteomic analysis and demonstrate why beside all the advances achieved in recent years, phosphoproteomics is still a challenge.