Abstract
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 TiO2-based enrichment (230). Moreover, the phosphopeptides were extracted with low sequence bias and showed no evidence for the characteristic preference of TiO2 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.
Highlights
Such as immobilized metal affinity chromatography (IMAC) or titanium dioxide (TiO2), widely used in phosphoproteomics, lack site selectivity for phosphorylation at serine, threonine or tyrosine[8,9] and exhibit a sequence bias in favor of peptides rich in aspartic (D) and glutamic (E) acid
The molecularly imprinted polymers (MIPs) selectively retained phosphorylation at serine (pS) and pY Fmoc derivates with a preference for the complementary amino acid side chains, whereas the nonimprinted polymer displayed weak retention of all analytes (Supplementary Fig. 4)
The presented human embryonic kidney (HEK) 293T cell experiments showed that the strong cation exchange (SCX)/pS-MIP enrichment approach resulted in a larger number of phosphopeptides (924) compared with the TiO2 approach (711) while using 10-fold less peptide sample
Summary
Such as immobilized metal affinity chromatography (IMAC) or titanium dioxide (TiO2), widely used in phosphoproteomics, lack site selectivity for phosphorylation at serine (pS), threonine (pT) or tyrosine (pY)[8,9] and exhibit a sequence bias in favor of peptides rich in aspartic (D) and glutamic (E) acid These methods require access to a large amount, typically in the milligram range, of complex protein digest[5,10,11]. A phosphotyrosine imprinted polymer (pY-MIP) was used to selectively enrich tyrosine-phosphorylated peptides spiked at low levels into proteolytic digests with only minor cross-reaction with pS peptides This approach has yet to be used for biological samples or extended beyond pY recognition. The versatility of the MIP receptors was demonstrated for four different biological samples (trypsinized HEK 293T and SH-SY5Y cell lines, mouse brain and human cerebrospinal fluid (CSF)), paying special attention to the minimum required sample amounts, analytical throughput and amino acid sequence bias
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