In-Depth Phosphoproteome Analyses Using PolyMAC

Protein phosphorylation plays critical roles in the regulation of many cellular functions. Analysis of phosphoproteomes by mass spectrometry depends on an efficient method to enrich phosphopeptides from complex mixtures. The current prevailing enrichment methods lack required efficiency and reproducibility due to complex sample conditions. Here, we utilize a novel soluble nanopolymer-based phosphopeptide enrichment approach termed PolyMAC (Polymer-based Metal ion Affinity Capture) for in-depth phosphoproteome analysis. The central piece of PolyMAC reagents lies in a water soluble and highly accessible polymer support. PolyMAC reagents are synthesized from polyamidoamine (PAMAM) dendrimers functionalized with metal ions to specifically capture O-phosphorylated peptides through bidentate chelation chemistry. An extensive comparison was made between PolyMAC reagents and the TiO₂ method, in terms of the specificity, reproducibility and sensitivity. We also compared different types of PolyMAC reagents functionalized with several metal ions. The enriched phosphopeptides were analyzed by nanoflow liquid chromatography tandem mass spectrometry (nLC-MS/MS). The MS spectra were acquired on an Eksigent ultra 2D LC system that is coupled to hybrid linear ion trap orbitrap mass strometer (LTQ Orbitrap velos, Thermo Fisher). Data were searched using the SEQUEST^TM algorithm within the Proteome Discoverer software. Using only 50 ug of total cell lysate, PolyMAC enrichment has led to the identification of over 1000 phosphopeptides with over 95% selectivity in a single run. Overall, the PolyMAC method demonstrated excellent selectivity, outstanding recovery yield and high reproducibility, thus rendering it one of the most effective phosphopeptide isolation techniques to date. The PolyMAC enrichment approach has been applied to examine the differences in signaling pathways in breast cancer cell lines modulated by a tumor suppressor, spleen tyrosine kinase (Syk). MDA-MB-231 human breast cancer cell line was transfected with a tetracycline-inducible Syk vector. Proteins from cells with or without Syk induction were processed and phosphotyrosine peptides were enriched using the combination of pTyr peptide immunoprecipitation and PolyMAC to obtain the phosphorylation profiles. We identified 794 sites of tyrosine phosphorylation in malignant breast cancer cells, 514 of which are dependent on the expression of Syk. Proteins with changes in pTyr phosphorylation were manually validated and a number of them confirmed through immunoprecipitation-Western blot experiments. They were mapped in a variety of major signaling networks including cell migration and apoptosis, therefore offering numerous leads to future breast cancer studies.