Selective Enrichment of Phosphopeptides on
MALDI Plates
The
intracellular phosphorylation of serine,
threonine, or tyrosine residues of proteins provides a mechanism for
the cell
to switch on or off many diverse processes, such as kinase cascade
activation
and gene transcription. Hence, a profile of
protein
phosphorylation is vital for understanding cell regulation.
Currently, MS
is the most powerful technique for studying protein post-translational
modifications, but profiling of phosphorylation typically requires
enrichment
of phosphopeptides after proteolytic digestion because of the low
stoichiometry
of protein phosphorylation. One
strategy to minimize sample consumption and
simplify enrichment is capture of the desired species directly
on the matrix assisted laser desorption/ionization (MALDI) plate. We
recently developed a method for growing polymer brushes on MALDI plates
and modifying them with titania clusters (Figure a below) that
selectively bind phosphopeptides prior to rinsing, elution, and
MALDI-MS (Figure b below). 
(a)
Schematic
drawing
of
the formation of the polymer-oxoTi
hybrid structure, and the binding and elution of phosphopeptides in
this
material. (b) Procedures for on-plate phosphopeptide enrichment using
Au-P-oxoTi.
Enrichment of phosphoprotein digests lead to mass spectra dominated with signals from phosphopeptides. In a recent paper, J. Proteome Res. 9, 3005–3015 (2010), we used these modified MALDI plates to identify phosphoproteins that are associated with p65, an ubiquitous protein that is partially responsible for regulating inflammation as well as the life cycle of lymphoid cells during the immune response. Overall, these techniques facilitated the identification of five p65-associated proteins, two of which were not previously reported to interact with p65.
Enrichment of phosphoprotein digests lead to mass spectra dominated with signals from phosphopeptides. In a recent paper, J. Proteome Res. 9, 3005–3015 (2010), we used these modified MALDI plates to identify phosphoproteins that are associated with p65, an ubiquitous protein that is partially responsible for regulating inflammation as well as the life cycle of lymphoid cells during the immune response. Overall, these techniques facilitated the identification of five p65-associated proteins, two of which were not previously reported to interact with p65.