Low-bias phosphopeptide enrichment from scarce samples using plastic antibodies

Phosphospecific enrichment techniques and mass spectrometry (MS) are essential tools for comprehending the cellular phosphoproteome. Here , we report a fast and simple approach for low sequence-bias phosphoserine (pS) peptide capture and enrichment that is compatible with low biological or clinical sample input. The approach exploits molecularly imprinted polymers (MIPs , plastic antibodies) featuring tight neutral binding sites for pS or pY that are capable of cross-reacting with phosphopeptides of protein proteolytic digests. The versatility of the resulting method was demonstrated with small samples of whole-cell lysate from human embryonic kidney (HEK) 293T cells , human neuroblastoma SH-SY5Y cells , mouse brain or human cerebrospinal fluid (CSF). Following pre-fractionation of trypsinized proteins by strong cation exchange (SCX) chromatography , pS-MIP enrichment led to the identification of 924 phosphopeptides in the HEK 293T whole-cell lysate , exceeding the number identified by TiO 2 -based enrichment (230). Moreover , the phosphopeptides were extracted with low sequence bias and showed no evidence for the characteristic preference of TiO 2 for acidic amino acids (aspartic and glutamic acid). Applying the method to human CSF led to the discovery of 47 phosphopeptides belonging to 24 proteins and revealed three previously unknown phosphorylation sites.