Online Inquiry

Hydrophilic Chromatography Protocol for Phosphoproteome Enrichment

Phosphorylation, the reversible addition of phosphate groups to proteins, plays a crucial role in regulating various cellular processes, including signal transduction, cell cycle regulation, and metabolism. Therefore, studying the phosphoproteome, the entire complement of phosphorylated proteins within a cell or tissue, is essential for understanding cellular signaling networks and disease mechanisms. One widely used approach for phosphoproteome analysis is phosphopeptide enrichment, which selectively isolates phosphorylated peptides from complex biological samples for subsequent mass spectrometry analysis. Among the various enrichment strategies, hydrophilic chromatography has emerged as an effective method due to its compatibility with diverse sample types and its ability to enrich phosphopeptides with high specificity and efficiency.

Why Choose Hydrophilic Chromatography?

Hydrophilic chromatography, also known as hydrophilic interaction liquid chromatography (HILIC), exploits the polar interactions between hydrophilic stationary phases and hydrophilic analytes such as phosphopeptides. In this method, samples are loaded onto a hydrophilic chromatography column under conditions of high organic solvent content (e.g., acetonitrile) and low pH to facilitate peptide retention. Phosphopeptides, which typically contain multiple charged phosphate groups, exhibit strong affinity for the hydrophilic stationary phase and are retained on the column, while non-phosphorylated peptides are eluted more rapidly. By subsequently adjusting the chromatographic conditions, such as reducing the organic solvent concentration or increasing the pH, phosphopeptides can be selectively eluted from the column for downstream analysis.

Experimental Materials

Cell Culture Materials:

Dulbecco's Modified Eagle's Medium (DMEM) supplemented with 10% fetal bovine serum.

Serum-free Dulbecco's Modified Eagle's Medium (DMEM).

Solution of trypsin (0.25%) and ethylenediamine tetraacetic acid (EDTA) (1 mM).

100 × 20 mm treated tissue culture dishes.

Dulbecco's phosphate buffered saline without calcium or magnesium.

Teflon disposable cell scrapers.

Microson Ultrasonic Cell Disruptor (Misonix) with probe tip or equivalent.

Cell lysis buffer: 8 M Urea/20 mM Tris–HCl pH 8.0.

Protein Extraction and Digestion Materials:

400 mM ammonium bicarbonate in water.

Reduction: 45 mM dithiothreitol (DTT) in water.

Alkylation: 100 mM iodoacetamide in water.

Digestion: Immobilized trypsin agarose (Pierce).

Stop: 100% trifluoroacetic acid.

Vacuum extraction manifold (Waters).

Desalt: 1 g Sep-Pak C18 6 cc/1 g cartridge (Waters).

Wash buffer: 0.1% TFA in water.

Elution buffer: 0.1% TFA containing 60% acetonitrile in water.

Speedvac concentrator (Savant).

HILIC column: 4.6 × 250 mm TSKgel Amide-80 5 μm particle column (TOSOH Biosciences).

Chromatography Materials:

Solvent A: 98% water with 0.1% TFA.

Solvent B: 98% acetonitrile with 0.1% TFA.

HPLC grade acetonitrile.

HPLC grade water.

Trifluoroacetic acid (TFA), sequanal grade.

IMAC resin: PHOS-Select Iron Affinity Gel.

0.22 μm Nylon Spin-X centrifuge tube filter.

Wash solution 1: 250 mM acetic acid with 30% acetonitrile.

Wash solution 2: water.

Elution buffer: 400 mM ammonium hydroxide.

Speedvac concentrator.

Sample resuspension: 0.1% formic acid/0.02% TFA in water.

LC-MS/MS Materials:

Trapping column: C18 PepMap100 300 μm × 5 mm 5-μm enrichment cartridge (LC Packings).

Analytical column: C18 PepMap100 75 μm × 15 mm 5-μm analytical column (LC Packings).

Load solution: 0.1% formic acid/0.05% TFA.

Solvent A: 0.1% formic acid in water.

Solvent B: 0.1% formic acid in acetonitrile.

Peak lists from individual LC-MS/MS runs are merged into one file using MasCat software (Agilent).

Data Analysis Materials:

Database search software: Mascot version 2.2.

Database: IPI Human version 3.37 containing 69,164 entries; forward and reverse.


Cell Culture and Stimulation:

a. Maintain HeLa cells in Dulbecco's Modified Eagle's Medium (DMEM) supplemented with 10% fetal bovine serum (FBS) in a humidified incubator at 37°C with 5% CO2.

b. Passage cells when they reach approximately 80-90% confluency using trypsin/EDTA solution.

c. For phosphoproteomic studies, grow HeLa cells in 100 × 20 mm tissue culture dishes until they reach near confluency.

d. Before harvesting, starve the cells by incubating them in serum-free DMEM for 16-24 hours to synchronize signaling pathways.

e. Stimulate the cells with 100 nM Calyculin A in serum-free DMEM for 30 minutes to induce phosphorylation events.

Cell Lysis and Protein Digestion:

a. Wash stimulated cells twice with ice-cold Dulbecco's phosphate buffered saline (DPBS) to remove residual media and debris.

b. Lyse cells by adding 1 mL of cold lysis buffer (8 M urea/20 mM Tris–HCl, pH 8.0) per 100 mm dish. Scrape the cells and transfer them into a 15-mL conical tube.

c. Disrupt the cellular lysate by sonication using a microprobe tip set at 20 W output for three brief 15-second pulses on ice.

d. Centrifuge the lysate at 10,000 × g for 15 minutes at 4°C to clarify the supernatant.

e. Reduce disulfide bonds in proteins by adding 1:9 volume of 45 mM DTT in water and incubate at 37°C for 30 minutes.

f. Alkylate cysteine residues by adding 1:9 volume of 100 mM iodoacetamide in water and incubate at room temperature in the dark for 30 minutes.

g. Dilute the lysate with 4 mL of 100 mM ammonium bicarbonate in water.

h. Add 250 μL of immobilized trypsin agarose beads to the sample and digest overnight at room temperature on a rocking platform.

i. Acidify and quench the digestion reaction by adding 40 μL of trifluoroacetic acid (TFA) to a final concentration of 1%.

Peptide Desalting and Fractionation:

a. Desalt the digested peptides by applying the sample to a prewetted and equilibrated 1 g Waters Sep-Pak C18 cartridge using a vacuum manifold apparatus.

b. Wash the cartridge with 20 mL of 0.1% TFA and elute the peptides with 4 mL of 60% acetonitrile containing 0.1% TFA.

c. Concentrate the eluent to dryness using a Speedvac concentrator.

Preparative HILIC Chromatography:

a. Perform preparative HILIC chromatography using a TSKgel Amide-80 column with a specific gradient of water and acetonitrile containing 0.1% TFA.

b. Dissolve the desalted peptides in 80% solvent B (acetonitrile) and spin in a microcentrifuge to remove particulates.

c. Collect fractions at specified intervals throughout the gradient and pool fractions of interest based on experimental design.

IMAC Enrichment of Phosphopeptides:

a. Equilibrate PHOS-Select Iron Affinity Gel by incubating it in 80% HILIC solvent B on a rocking platform for 15 minutes at room temperature. Centrifuge briefly and remove liquid. Resuspend the gel in 80% solvent B and add it to each fraction.

b. Vortex the fractions with the gel at room temperature for 30 minutes. Transfer the fractions to individual Spin-X Centrifuge Tube Filters and centrifuge to remove unbound material.

c. Discard the unbound material and wash the gel with 250 mM acetic acid containing 30% acetonitrile. Vortex for 5 minutes, centrifuge, and discard the wash.

d. Repeat the wash step using water.

e. Transfer the filter inserts to clean tubes and elute the bound phosphopeptides with 400 mM ammonium hydroxide by vortexing for 10 minutes.

f. Collect the eluates and concentrate them to dryness to obtain fractionated and highly enriched phosphopeptide pools.

LC-MS/MS Analysis:

a. Inject the concentrated phosphopeptide samples into an LC-MS/MS system equipped with a nano-flow HPLC column.

b. Perform tandem mass spectrometry (MS/MS) analysis to identify and quantify phosphopeptides.

c. Use Mascot software to search against a protein database with specific parameters including H. sapiens restriction, fully tryptic peptides with no more than one missed cleavage, fixed modification of carboxamidomethylated cysteine, and variable modifications of phosphorylation on serine/threonine/tyrosine residues.

d. Filter search results to achieve a false-positive rate of approximately 1.0%.

e. Manually validate peptide sequence and phosphosite assignments using Mascot ion scores, verifying features such as neutral loss of H3PO4 from the precursor, series of y or b ions, prominent y ion cleavage N-terminal to a proline, and absence of unexplained high mass fragment ions.

* For Research Use Only. Not for use in diagnostic procedures.
Our customer service representatives are available 24 hours a day, 7 days a week. Inquiry

Online Inquiry

Please submit a detailed description of your project. We will provide you with a customized project plan to meet your research requests. You can also send emails directly to for inquiries.

* Email
* Service & Products of Interest
Services Required and Project Description
* Verification Code
Verification Code