On-target titanium dioxide-based enrichment for characterization of phosphorylations in the Adenovirus pIIIa protein

• On-target phosphopeptide enrichment and separation are performed.
• Phosphorylations in the Adenovirus type 2 protein pIIIa are investigated.
• Seven phosphorylations are detected, out of which three are novel.
• The first evidence of multi-phosphorylated peptides in pIIIa is presented.
• The used method is able to characterize phosphorylations in real biological samples.

A recently developed titanium dioxide (TiO2) based on-target method for phosphopeptide enrichment and matrix assisted laser desorption-ionization mass spectrometry (MALDI MS) analysis was used to investigate phosphorylations in the Adenovirus type 2 structural protein pIIIa. Lysates of purified virus particles were separated on 1-D SDS-PAGE and the band for the pIIIa protein was excised for tryptic digestion into peptides that were enriched with the on-target method. The enrichment provided by the method clearly improved the detectability of phosphorylated peptides and the results show for the first time evidence for multi-phosphorylated peptides in pIIIa. Moreover, three novel phosphorylations were identified in the protein sequence, even though the precise positions could not be determined. These results illustrate the potential of the method for the characterization of novel phosphoproteomes in biological samples of medical relevance.