Protein phosphorylation is one of the most ubiquitous post-translational modifications. Many low-abundance endogenous phosphoproteins and phosphopeptides in the body fluids or tissues are biomarkers with higher clinical sensitivity and specificity, which could provide valuable information for the detection of many diseases and elucidation of pathology. It is still a great challenge to detect the phosphoproteins and phosphopeptides from complex biological samples directly due to reversibility of protein phosphorylation and the extremely low concentrations of phosphoproteins. Nanostructured materials have attracted particular attentions in enrichment, separation, and purification of phosphoproteins/phosphopeptides due to their larger surface area, numerous affinity sites and unique structures. The research subject has become one of research hotspots in phosphoproteomics presently. Various multifunctional nanostructured materials such as core-shell particles with mesoporous affinity shell and magnetic core, hybrids of multiple components and composite affinity materials have been synthesized for selective enrichment and fast purification of phosphoproteins/phosphopeptides. In this review, we are focusing on recent advancements of nanostructured materials for phosphoprotein/phosphopeptide enrichment and purification prior to MS analysis. Definition, structure characteristic, unique physicochemical properties and potential in bio-separation of the nanostructured materials are first introduced. Subsequently, the related research background on phosphoproteomic studies in proteomics using mass spectrometric strategies in combination with phosphospecific enrichment techniques is briefly presented. After that, two types of affinity enrichment mechanisms to phosphoproteins/phosphopeptides are compactly discussed. Next, mesoporous, hybrid, and composite nanostructured materials for enrichment of phosphoproteins/phosphopeptides as well as application of multifunctional nanostructured materials in phosphoproteomics are summarized in detail. Finally, some unsolved problems and a brief perspective and outlook on phosphopeptide enrichment are proposed.

Contents
1 Introduction
2 Protein phosphorylation and enrichment detection of phosphoproteins/phosphopeptides
2.1 Protein phosphorylation
2.2 Enrichment detection of phosphoproteins/phosphopeptides
3 Mechanism of affinity enrichment of phosphoproteins/phosphopeptides
4 Mesoporous nanostructured materials for enrichment of phosphoproteins/phosphopeptides
4.1 Mesoporous SiO₂ nanostructured materials with immobilized affinity material
4.2 Mesoporous nanostructured materials doped with affinity material
4.3 Mesoporous metal oxide nanostructured materials
5 Composite nanostructured materials for enrichment of phosphoproteins/phosphopeptides
5.1 Magnetic core-shell nanostructured materials
5.2 Mesoporous magnetic nanostructured materials
5.3 Hybrid composite nanostructured materials
6 Application of multifunctional nanostructured materials in phosphoproteomics
6.1 Multifunctional nanostructured materials for fast proteolysis and phosphopeptide enrichment
6.2 Multifunctional nanostructured materials for enrichment and identification of phosphopeptides
6.3 Multifunctional nanostructured materials for enrichment of various biomolecules
6.4 Nanostructured materials modified target plate for on-plate enrichment and analysis of phosphopeptides
7 Conclusions and outlook