Simplification of complex mixtures is of great interest, especially in the aspect of mass spectrometric (MS) identification and analysis of biological components. Due to some limitions in the dynamic range of MS detections, effective protein/peptide sample separation strategies are usually required prior to MS measurement to improve the MS detection capabilities for highly complex biological samples like proteomes. Our group has successfully used self-assembled amphiphilic polymers to selectively simplify peptide mixtures via a bi-phasic extraction format and sensitively detect different fractions using matrix assisted laser desorption ionization mass spectrometry (MALDI-MS). Amphiphilic random copolymers with different functional groups (carboxylate, sulfonate, and phosphonate) have been synthesized and allowed to form reverse micelles in toluene. Mixtures of peptides and protein digests were prepared in aqueous buffers, and peptides of interest were extracted in a biphasic system after mixing the aqueous-phase peptide mixture with the toluene phase containing the reverse micelles. The peptides extracted into the organic phase were dried, reconstituted in THF, mixed with matrix, and then analyzed using a Bruker Autoflex III MALDI-TOF mass spectrometer. Using pH dependent capacities of standard peptides as an indicator, we have understood that pKa of functional group on the micellar state is consistent with its counterpart on the small-molecule state. The decreased capacity at high pHs is both polymer- and peptide- dependent. By optimizing the polymer structure, the decreased capacity can be recovered. Using polymer with low pKa value, complex peptide mixtures are able to be efficiently simplified at acidic pH. Furthermore, extraction and bracketing of acidic peptides within narrow pI range improves the detection sensitivity. Overall, our studies explore the pKa of functional groups in the context of supramolecular assembly, which provides fundamental understanding of these materials and also broadly impacts the area of separation. Taking the advantage of low pKa polymers, specific isolation and detection of acidic peptides is realized, which would potentially benefit the proteomics study.

Reverse micelles with different functional group pKas and their application for the detection of acidic peptides

255th National Meeting (2018)

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