In-depth Analyses of Kinase-dependent Tyrosine Phosphoproteomes Based on Metal Ion-functionalized Soluble Nanopolymers

Abstract

The ability to obtain in-depth understanding of signaling networks in cells is a key objective of systems biology research. Such ability depends largely on unbiased and reproducible analysis of phosphoproteomes. We present here a novel proteomics tool, polymer-based metal ion affinity capture (PolyMAC), for the highly efficient isolation of phosphopeptides to facilitate comprehensive phosphoproteome analyses. This approach uses polyamidoamine dendrimers multifunctionalized with titanium ions and aldehyde groups to allow the chelation and subsequent isolation of phosphopeptides in a homogeneous environment. Compared with current strategies based on solid phase micro-and nanoparticles, PolyMAC demonstrated outstanding reproducibility, exceptional selectivity, fast chelation times, and high phosphopeptide recovery from complex mixtures. Using the PolyMAC method combined with antibody enrichment, we identified 794 unique sites of tyrosine phosphorylation in malignant breast cancer cells, 514 of which are dependent on the expression of Syk, a protein-tyrosine kinase with unusual properties of a tumor suppressor. The superior sensitivity of PolyMAC allowed us to identify novel components in a variety of major signaling networks, including cell migration and apoptosis. PolyMAC offers a powerful and widely applicable tool for phosphoproteomics and molecular signaling. Molecular & Cellular Proteomics 9:2162-2172, 2010.Reversible phosphorylation of proteins is a major mechanism for the regulation of multiple cellular processes (1, 2). Mass spectrometry-based phosphoproteomics provides a method for the global analysis of protein phosphorylation and molecular signaling in cells (3,4). Despite the great progress that has been made over the past few years, the isolation of phosphopeptides and their analysis by mass spectrometry are still a considerable challenge because of the typically low stoichiometry of protein phosphorylation and the resulting low abundance of phosphopeptides. An early step in any phosphoproteome analysis is the isolation of phosphopeptides, preferably with high efficiency, selectivity, sensitivity, and reproducibility. Currently, there are three major strategies for the isolation of phosphopeptides: antibody-based affinity capture, chemical derivatization of phosphoamino acids, and metal ion-based affinity capture. Antibody-based methods are used mainly for the selective isolation of phosphotyrosinecontaining proteins or peptides (5-8). Chemical derivatization methods begin with the ␤-elimination of phosphates from phosphoserine and phosphothreonine (9) or the formation of phosphoramidates by reactions with amines (10) to selectively immobilize phosphopeptides. Metal ion-based affinity capture techniques use immobilized metal affinity chromatography (IMAC) with Fe(III) (11,12) or Ga(III) (13) and, for the past a few years, more successful metal oxide approaches (i.e. TiO 2 (14, 15) and ZrO 2 (16, 17)) for the selective binding of phosphorylated peptides. Almost all of the current isolation methods are b...

Sensitive kinase assay linked with phosphoproteomics for identifying direct kinase substrates

“…To enhance our ability to identify true substrates, we compared endogenous phosphotyrosine-containing peptides derived from phosphoproteins from DG75 cells that were treated with or without the Syk inhibitor, piceatannol. Isolation of phosphopeptides by a tandem purification scheme using antiphosphotyrosine antibody and PolyMAC (20) and analysis by mass spectrometry resulted in the identification of over 400 phosphotyrosinecontaining peptides. A larger number were identified in samples from untreated as compared to inhibitor treated cells.…”

“…Note that the kinase reaction is performed at the peptide stage which efficiently eliminates any problem related to endogenous kinase contaminations (all endogenous kinases were digested before the kinase reaction). Phosphopeptides generated by in vitro phosphorylation with the kinase of interest are enriched using highly efficient polymer-based metal ion affinity capture (PolyMAC) (20), followed by mass spectrometric analyses to identify their sequences and sites of phosphorylation. This procedure generates a list of peptide substrates that can be compared to reveal the substrate specificity of the enzyme.…”

“…The resulting phosphopeptides were enriched using PolyMAC, a highly efficient, titanium functionalized dendrimer support (20), and then analyzed by mass spectrometry. We identified 142 tyrosine phosphorylated peptides (out of a total of 156 peptide identifications; Dataset S1) in a sample that originated from 3 mg of DG75 whole cell extract, whereas virtually no phosphopeptides were detected in the sample without the addition of Syk.…”

Perspectives of Comprehensive Phosphoproteome Analysis Using Shotgun Strategy

“…9 Recently, nanosize polymer based metal ion affinity capture (PolyMAC) was developed, and high reproducibility, selectivity, and recovery for phosphopeptide enrichment were obtained. 21,22 Phosphotyrosine dependent networks play a key role in transmitting signals, which is important for elucidating the regulatory mechanisms of each biological effect. For tyrosine phosphorylation analysis, antibody based enrichment is still the mainstream method due to its low abundance and the high specificity of antibody for phosphotyrosine.…”

“…23,24 Furthermore, the combination of antibody based enrichment with other phosphopeptide enrichment methods also shows a promising strategy for high sensitive tyrosine phosphorylation detection. 22 …”