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Protocol for Quantitative Phospho-proteomics Based on Soluble Nanopolymers

Why Choose Soluble Nanopolymers?

Phosphoproteomic studies demand robust and efficient methods for phosphopeptide enrichment to uncover signaling cascades accurately. Soluble nanopolymers, exemplified by polyamidoamine (PAMAM) dendrimers, emerge as a promising avenue due to their unique physicochemical properties and versatile functionalities.

Versatility and Selectivity

Soluble nanopolymers offer unparalleled versatility and selectivity in targeting phosphorylated peptides. Through functionalization, these dendrimers can be tailored to selectively bind phosphate groups, facilitating precise enrichment of phosphopeptides from complex biological samples.

Enhanced Reproducibility and Sensitivity

By leveraging homogeneous solution-phase reactions, soluble nanopolymers ensure uniform binding kinetics and distribution of phosphopeptides. This enhances reproducibility and sensitivity in phosphoproteomic analyses, crucial for deciphering subtle phosphorylation dynamics in biological systems.

Material for Quantitative Phospho-proteomics Based on Soluble Nanopolymers

The success of any phosphoproteomic study hinges on the meticulous selection and preparation of materials to ensure accurate and reproducible results. Here, we provide a detailed elucidation of the materials utilized in the quantitative phospho-proteomics based on soluble nanopolymers developed by Creative Proteomics.

Cell Culture and Lysis

The choice of cell culture medium profoundly influences cellular physiology and subsequently, the phosphoproteome. RPMI medium 1640, a widely used basal medium, provides essential nutrients and growth factors for diverse cell types. Supplemented with 10% fetal bovine serum (FBS), it ensures optimal cell growth and maintenance of cellular functions. Additionally, the inclusion of 2 μM L-glutamine, 100 μg/mL streptomycin sulfate, and 100 units/mL penicillin G minimizes microbial contamination and promotes cell viability throughout the culture period.

Upon achieving desired confluency, cells undergo lysis to release their intracellular contents, including proteins and phosphopeptides. The lysis buffer composition, comprising 1% Triton, 150 mM NaCl, and 50 mM Tris-HCl (pH 7.8), is meticulously formulated to solubilize proteins while preserving their phosphorylation status. Triton, a non-ionic detergent, disrupts cellular membranes, facilitating the release of cytoplasmic and nuclear proteins. The inclusion of NaCl maintains physiological ionic strength, while Tris-HCl buffers the lysate at a near-neutral pH, minimizing protein denaturation and enzymatic degradation.

Phosphopeptide Enrichment

Creative Proteomics employs two innovative strategies for phosphopeptide enrichment, each leveraging soluble nanopolymers to enhance specificity and reproducibility.

1. Derivatization with PAMAM Dendrimers: The first method involves the use of polyamidoamine (PAMAM) dendrimers, specifically generation 5 dendrimers, functionalized to selectively target phosphate groups on peptides. The reaction solution comprises 50 mM 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC), 100 mM imidazole, 200 mM 2-(N-morpholino) ethanesulfonic acid (MES, pH 6.0), and 9 mg of PAMAM dendrimer. This homogeneous solution-phase reaction facilitates the coupling of phosphopeptides to amine groups on the dendrimer surface, enabling efficient phosphopeptide isolation and subsequent mass spectrometry analysis.

2. Chelation with Zirconia or Titania Functionalized Nanopolymers: In the second method, soluble nanopolymers, such as PAMAM dendrimers generation 4, are functionalized with zirconia or titania to form stable bidentate complexes with phosphorylated peptides. The reaction solution comprises zirconium oxychloride solution (100 mM), which provides zirconia functional groups for phosphate chelation. Subsequent bioconjugation between the coupling groups on the dendrimer and solid-phase beads facilitates phosphopeptide isolation. The phosphopeptide-bound nanopolymer is then eluted under mild basic conditions for downstream mass spectrometry analysis.

Procedure of Quantitative Phospho-proteomics Based on Soluble Nanopolymers

Method 1: Phosphoramidate Chemistry on Soluble Nanopolymer

Method 1 involves enriching phosphopeptides through a chemical process that forms covalent bonds between phosphate groups and amines on soluble nanopolymers. Initially, phosphoramidate bonds are catalyzed by EDC and imidazole. Subsequently, the mixture is filtered through a specialized membrane-based spin column, isolating phosphopeptides bound to the polymer based on size. Following this, phosphopeptides are detached under acidic conditions for subsequent mass spectrometry analysis.

Jurkat Cell Culture and Lysis

Jurkat cells undergo culture in RPMI medium 1640 and are stimulated with sodium pervanadate before lysis in a specific buffer solution. After centrifugation, soluble proteins are collected from the supernatants, with insoluble fractions discarded. Optionally, phosphotyrosine-containing proteins can be further isolated if necessary, followed by concentration and drying of the protein samples.

Protein Digestion and Isotopic Tagging

Proteins undergo denaturation, reduction, alkylation, and digestion with trypsin. The resulting peptides are desalted and subjected to methyl esterification using methanolic HCl, followed by drying for subsequent enrichment.

Phosphopeptide Enrichment

Dried peptides are resuspended in a reaction solution containing EDC, imidazole, MES, and PAMAM dendrimer generation 5 for phosphoramidate bond formation. The reaction mixture is then filtered through a membrane-based spin column, washing away unbound peptides. Bound phosphopeptides are cleaved off the dendrimers with TFA, collected, and dried for further analysis.

Mass Spectrometry-Based Phosphopeptide Analysis

Dried phosphopeptides are resolubilized and loaded onto a reverse-phase C18 column for LC-MS analysis. Peptides are eluted and analyzed in data-dependent mode, with MS data searched against a protein database using the SEQUEST algorithm. Static and variable modifications are considered during database search, with additional manual validation for result filtering.

Method 2: Immobilized Metal Oxide Affinity Isolation on Soluble Nanopolymers

Method 2 utilizes the strong affinity of zirconium oxide to phosphate groups for phosphopeptide enrichment. PAMAM dendrimer G4 is functionalized with zirconia-phosphonate groups and hydrazide for efficient binding in homogeneous solutions. The bound phosphopeptides are isolated by specific handle-based binding, followed by elution and mass spectrometry analysis.

Synthesis of Zirconia-Functionalized Dendrimer

PAMAM dendrimer generation 4 is functionalized with phosphonic acid and zirconium oxychloride, followed by purification steps. The functionalized dendrimer undergoes further treatment to remove unreacted reagents and purification via dialysis.

Synthesis of Aldehyde Beads

Controlled pore glass beads are coupled with Fmoc-serine-OH using DIPCI and HOBT. After washing and treatment with acetylation, deprotection, and oxidation, aldehyde-functionalized beads are obtained.

Sample Preparation and Phosphopeptide Enrichment

Peptide samples are prepared, and phosphopeptides are enriched using synthesized zirconia-functionalized dendrimer and aldehyde beads. Phosphopeptides are eluted, dried, and resolubilized for mass spectrometry analysis.

Mass Spectrometry Analysis of Phosphopeptides

Mass spectrometry analysis follows a similar protocol as described in Method 1 for the identification and validation of phosphopeptides.


  1. de Graauw, Marjo. Phospho-Proteomics. Humana Press, 2009.
* For Research Use Only. Not for use in diagnostic procedures.
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