Phosphotyrosine Biased Enrichment of Tryptic Peptides from Cancer Cells by Combining pY-MIP and TiO₂ Affinity Resins

Abstract

Protein phosphorylation at distinct tyrosine residues (pY) is essential for fast, specific, and accurate signal transduction in cells. Enrichment of pY-containing peptides derived from phosphoproteins is commonly facilitated by use of immobilized anti-pY antibodies prior to phosphoproteomics analysis by mass spectrometry. We here report on an alternative approach for pY-peptide enrichment using inexpensive pY-imprinted polymer (pY-MIP). We assessed by mass spectrometry the performance of pY-MIP for enrichment and sequencing of phosphopeptides obtained by tryptic digestion of protein extracts from HeLa cells. The combination of pY-MIP- and TiO-based phosphopeptide enrichment provided more than 90% selectivity for phosphopeptides. Mass spectrometry signal intensities were enhanced for most pY-phosphopeptides (approximately 70%) when using the pY-MIP-TiO combination as compared to TiO alone. pY constituted up to 8% of the pY-MIP-TiO-enriched phosphopeptide fractions. The pY-MIP-TiO and the TiO protocols yielded comparable numbers of distinct phosphopeptides, 1693 and 1842, respectively, from microgram levels of peptide samples. Detailed analysis of physicochemical properties of pY-MIP-TiO-enriched phosphopeptides demonstrated that this protocol retrieved phosphopeptides that tend to be smaller (<24 residues), less acidic, and almost exclusively monophosphorylated, as compared to TiO alone. These unique properties render the pY-MIP-based phosphopeptide enrichment technique an attractive alternative for applications in phosphoproteomics research.

Phosphopeptide enrichment for phosphoproteomic analysis - A tutorial and review of novel materials

“…As mentioned above, pTyr sites make up a smaller fraction of the p-proteome, and are 937 often under-represented due to sampling bias. Commercially available anti-tyrosine 938 antibodies have a high affinity for pTyr and can be used for selective enrichment of pTyr 939 peptides; but poor reproducibility, low sensitivity, and limited availability/variability of 940 antibody/bulk starting materials limit enrichment capacity -particularly for complex peptide mixtures [100], and high costs limit their wider application [101]. To solve the affinity specificity and quantification accuracy issue caused by sequence bias, pTyr antibody cocktails (combined different pTyr antibodies together) have been proposed [102].…”

“…The combination of phosphotyrosine-imprinted polymer with TiO2 (pY-MIP-TiO2) shed light on the study of pThr and pTyr, based on the finding that pY-MIP-TiO2 protocol caused comparable identification numbers with TiO2 alone, with enhanced ion signal intensities for pThr and pTyr, but not pSer [100]. Moreover, the incorporation of alternative β-elimination followed by the Michael approach is required for the precise assignment about the location of multisite-phosphorylated (especially higher than triply-phosphorylated peptides) Ser/Thr-rich regions after the MS detection of p-peptides using TiO2 treatment [108], for which the presence of high number of phosphorylated residues on the same peptide decrease the IEP and thus challenge MS sequencing and detection.…”

Recent Advances in Protein Kinase Activity Analysis Based on Nanomaterials

“…Therefore, these metal oxide nanoparticles can be utilized for phosphopeptide enrichment [20,21,22]. For instance, TiO 2 combined with specific modified imprinted polymer was designed and provided more than 90% selectivity for phosphopeptides [23]. A ZnO nanorods array-based field-effect transistor biosensor was developed for phosphate detection [24].…”

Zirconium(IV)-IMAC for phosphopeptide enrichment in phosphoproteomics

“…IMAC uses metal cations, and MOAC metal oxides, that interact with the oxygen atoms of phosphate groups in peptides [17,22,23]. More recently, molecularly imprinted polymers (MIPs) have emerged as potential tools for selective phosphopeptide enrichment [24].…”