HILIC-Based Peptide Fractionation Service

MtoZ Biolabs provides high-resolution HILIC-based peptide fractionation service to achieve efficient separation and quantitative analysis of complex peptide samples, offering reliable sample pretreatment solutions for proteomics, post-translational modification studies, and biomarker identification. This service utilizes hydrophilic interaction Llquid chromatography (HILIC) to perform highly selective fractionation of complex peptide samples, effectively improving mass spectrometry sensitivity and protein identification coverage. It is widely applicable to multi-omics research in academic and biopharmaceutical fields.

 

The Principle of HILIC Fractionation

HILIC is a separation method based on polar interactions. Its stationary phase is usually composed of polar materials such as silica or amide-bonded phases, while the mobile phase contains a high proportion of organic solvent such as acetonitrile and a small proportion of aqueous solvent.

 

• When samples enter the chromatographic system, polar compounds form a hydration layer on the stationary phase surface.

• Peptides with higher polarity interact more strongly with this hydration layer and elute later.

• By gradually changing the aqueous phase ratio, precise separation is achieved according to polarity differences.

 



Jensen, PH. et al. Methods Mol Biol. 2013.

Figure 1. The Separation Principle of HILIC

HILIC separation offers excellent reproducibility and compatibility, making it particularly suitable for analyzing peptides, polar metabolites, and modified peptides such as phosphopeptides. It is an essential component of proteomics fractionation strategies.

 

HILIC-Based Peptide Fractionation Service at MtoZ Biolabs

The HILIC-based peptide fractionation service provided by MtoZ Biolabs is specifically designed for complex protein digests or modified peptide samples. It significantly enhances the depth and quantitative accuracy of subsequent LC-MS/MS analysis.

 

Service Features:

•  Applicable to the whole proteome, peptide mixtures, and post-translationally modified peptides (PTMs).

•  Supports high-throughput fractionation on both nano-scale and conventional HPLC systems.

•  Fully compatible with TMT, iTRAQ, and label-free quantification strategies.

 

Service Objective:

Through an optimized HILIC fractionation approach, this service reduces sample complexity and expands the dynamic detection range of mass spectrometry, providing clients with reproducible and traceable sample preparation data support.

 

Workflow of HILIC-Based Peptide Fractionation Service

1. Sample Preparation

Protein extraction, enzymatic digestion, desalting, and drying to ensure sample purity and solubility.

 

2. HILIC Fractionation

Separation of peptide fractions using a polar stationary phase column such as amino or amide under gradient elution conditions.

 

3. Fraction Collection

Peptide fractions are collected based on time or peak area and then dried for storage.

 

4. Mass Spectrometry and Data Analysis (Optional)

LC-MS/MS analysis can be performed to evaluate fractionation efficiency and protein identification depth.

 



Sun, Z. et al. Anal Bioanal Chem. 2019.

Figure 2. Schematic Illustration of HILIC-HPRP and HPRP-HILIC Workflows for Peptide Fractionation

All procedures are conducted under temperature-controlled and low-adsorption conditions to maximize sample stability and signal consistency.

 

Why Choose MtoZ Biolabs

✅ Professional analytical platform equipped with high-resolution HILIC systems and Orbitrap mass spectrometers

✅ Standardized workflows with precise gradient control and reproducible fractionation performance

✅ Excellent compatibility with multiple labeling and modification analysis techniques

✅ Experienced technical team specializing in peptide fractionation and proteomics sample preparation

✅ Advanced HILIC–RP Integration for High-Resolution Peptide Fractionation

 

Applications of HILIC-Based Peptide Fractionation Service

1️⃣ Proteomics Research: Improves detection coverage of low-abundance proteins.

2️⃣ Post-Translational Modification Studies: Enhances enrichment and separation of phosphorylated, acetylated, and glycosylated peptides.

3️⃣ Biomarker Discovery: Increases detection sensitivity and dynamic range for clinical samples.

4️⃣ Quantitative Protein Analysis: Improves peptide uniformity and quantitative accuracy in TMT and iTRAQ experiments.

5️⃣ Metabolomics and Microbiome Research: Enables fractionation of polar molecules and metabolic peptides.

 

Deliverables

1. Fractionated peptide samples with detailed numbering records (typically 6–20 fractions)

2. Experimental report including chromatographic parameters and profiles

3. Optional LC-MS/MS identification and data analysis results

4. Summary files and statistical reports in PDF or Excel format

 

All reports are quality-checked to ensure accuracy and traceability of data.

 

FAQs

Q1: What is the service's general workflow?

 

Q2: What data formats are provided?

MtoZ Biolabs provides comprehensive deliverables in multiple standard formats to facilitate downstream data processing and visualization. The typical deliverables include:

• Raw chromatographic data files from HILIC and LC-MS platforms

• Processed data tables (CSV or Excel format) listing fraction numbers, retention times, and peak intensities

• Comprehensive summary reports in PDF format including experimental conditions, gradient settings, and QC metrics

• Chromatograms and peak distribution figures in high-resolution image formats (TIFF or PNG)

 

Additional data formats or customized report templates can be supplied upon request to meet specific research or publication requirements.

 

Q3: How should I prepare my samples?

To ensure optimal performance of the HILIC-Based Peptide Fractionation Service, please follow the guidelines below:

1. Sample Type: Peptide mixtures derived from protein digestion, modified peptide samples, or lyophilized peptide powders are accepted.

2. Sample Purity: Samples should be free from salts, detergents (SDS, Triton), or organic solvents that may interfere with chromatographic retention or MS ionization.

3. Quantity Requirement: Provide at least 200 µg of total peptide or equivalent dry weight material. Additional quantity may be required for replicate analyses.

4. Storage and Shipping: Store samples at −80°C. Ship on dry ice in sealed, leak-proof containers to maintain sample integrity.

5. Documentation: Include detailed information such as sample source, digestion method, buffer composition, and desired number of fractions.

 

For more information, please refer to Sample Submission Guidelines for Proteomics and Sample Submission Guidelines for Metabolomics.

 

If you are unsure about your sample preparation, our technical team can provide personalized pre-analysis consultation and a sample submission guide to help you optimize preparation and shipping conditions.

 

Start Your Project with MtoZ Biolabs

MtoZ Biolabs is committed to providing efficient, stable, and reproducible HILIC-based peptide fractionation service for proteomics and peptide research. Through professional fractionation strategies and standardized quality control systems, we help researchers maximize sample information content, enhance mass spectrometry detection depth, and improve data quality. Contact us to obtain customized experimental solutions and quotations to make your peptide separation and quantification research more efficient and precise.