Surreal illustration of HILIC and HPRP chromatography columns leading to a mass spectrometer, representing advanced proteomic analysis.

Unlock Deeper Insights: How Tandem Liquid Chromatography is Revolutionizing Proteomics

Mira Elwood in Science & Nature November 2025 • 4 min read.

"A Simplified Guide to HILIC-HPRP for Enhanced Protein Analysis and PTM Discovery"

In the rapidly evolving world of biological research, proteomics – the large-scale study of proteins – stands as a cornerstone. Understanding the intricate details of proteins, their functions, and modifications is crucial for advancements in medicine, biotechnology, and various other fields. Shotgun proteomics, a popular approach, involves breaking down proteins into smaller peptides and analyzing them using liquid chromatography coupled with tandem mass spectrometry (LC-MSMS). While powerful, this technique faces challenges due to the complexity of proteomes and the vast dynamic range of protein abundances.

One significant hurdle lies in the diverse array of post-translational modifications (PTMs). These modifications, which alter a protein's properties and function, are often present in low quantities, making them difficult to detect. Effective separation and enrichment techniques are essential to isolate and identify these modified peptides. Traditional methods often fall short in capturing the full spectrum of PTMs, hindering our ability to comprehensively understand cellular processes and disease mechanisms.

To overcome these limitations, scientists are exploring multidimensional liquid chromatography (MDLC) strategies, employing different separation techniques to reduce sample complexity before mass spectrometry analysis. Among these, Hydrophilic Interaction Liquid Chromatography (HILIC) has emerged as a promising alternative, offering unique advantages in separating polar analytes and modified peptides. By combining HILIC with other separation methods like High-pH Reverse-Phase (HPRP), researchers aim to achieve greater proteome coverage and gain deeper insights into the world of protein modifications.

What is Tandem HILIC-HPRP and Why is it a Game-Changer for Protein Analysis?

Surreal illustration of HILIC and HPRP chromatography columns leading to a mass spectrometer, representing advanced proteomic analysis.

Tandem HILIC-HPRP is a sophisticated separation strategy that combines two distinct liquid chromatography techniques to maximize peptide separation and improve protein identification. It leverages the complementary nature of HILIC and HPRP, each of which separates peptides based on different properties. This approach allows researchers to capture a wider range of peptides and PTMs, leading to more comprehensive proteomic analyses.

In essence, this tandem approach works by first separating peptides using one technique (either HILIC or HPRP) and then subjecting the unretained species to the other technique. This ensures that peptides that may not be well-resolved by one method are captured and analyzed by the other. The fractions from both separation modes are then combined for downstream analysis by LC-MSMS.

Increased Proteome Coverage: By combining HILIC and HPRP, the tandem strategy significantly expands the range of peptides identified, providing a more complete picture of the proteome.
Enhanced PTM Discovery: The ability to capture and separate modified peptides improves the detection and identification of PTMs, which are crucial for understanding protein function and regulation.
Improved Separation of Complex Samples: The multidimensional approach reduces sample complexity, allowing for better resolution and identification of individual peptides.
Versatile Application: Tandem HILIC-HPRP can be applied to various proteomic studies, including biomarker discovery, drug target identification, and understanding disease mechanisms.

The key to the success of tandem HILIC-HPRP lies in the orthogonality of the two separation techniques. HILIC separates peptides based on their hydrophilicity, while HPRP separates them based on their hydrophobicity. This difference in selectivity allows for the capture of a broader range of peptides, including those that are difficult to resolve using a single separation method.

The Future of Proteomics: A Deeper Dive with Tandem HILIC-HPRP

Tandem HILIC-HPRP represents a significant advancement in proteomics, offering researchers a powerful tool to explore the complexities of the proteome and discover novel PTMs. As technology advances and analytical techniques improve, we can expect even greater insights into the world of proteins and their modifications, leading to breakthroughs in our understanding of health and disease.

About this Article -

This article was crafted using a human-AI hybrid and collaborative approach. AI assisted our team with initial drafting, research insights, identifying key questions, and image generation. Our human editors guided topic selection, defined the angle, structured the content, ensured factual accuracy and relevance, refined the tone, and conducted thorough editing to deliver helpful, high-quality information.See our About page for more information.

This article is based on research published under:

DOI-LINK: 10.1007/s00216-018-1462-3, Alternate LINK

Title: Improving Deep Proteome And Ptmome Coverage Using Tandem Hilic-Hprp Peptide Fractionation Strategy

Subject: Biochemistry

Journal: Analytical and Bioanalytical Chemistry

Publisher: Springer Science and Business Media LLC

Authors: Zeyu Sun, Feiyang Ji, Zhengyi Jiang, Lanjuan Li

Published: 2018-11-20

Everything You Need To Know

What is Tandem Hydrophilic Interaction Liquid Chromatography (HILIC) and High-pH Reverse-Phase (HPRP), and how does it work?

Tandem Hydrophilic Interaction Liquid Chromatography (HILIC) and High-pH Reverse-Phase (HPRP) is a dual separation strategy in liquid chromatography that maximizes peptide separation and protein identification. It combines the principles of HILIC, which separates peptides based on their hydrophilicity, with HPRP, which separates them based on their hydrophobicity. This combination allows for a more comprehensive capture and analysis of a wider range of peptides and post-translational modifications (PTMs) than using either technique alone. This approach works by separating peptides using one technique, and then subjecting the unretained species to the other technique.

What does 'orthogonality' mean in the context of Tandem Hydrophilic Interaction Liquid Chromatography (HILIC) and High-pH Reverse-Phase (HPRP), and why is it important?

Hydrophilic Interaction Liquid Chromatography (HILIC) separates peptides based on their hydrophilicity, while High-pH Reverse-Phase (HPRP) separates them based on their hydrophobicity. This difference in selectivity is crucial because it allows for the capture of a broader range of peptides, including those that are difficult to resolve using a single separation method. The 'orthogonality' refers to how different the separation mechanisms are between HILIC and HPRP, ensuring a more comprehensive coverage of the proteome.

How does Tandem Hydrophilic Interaction Liquid Chromatography (HILIC) and High-pH Reverse-Phase (HPRP) improve proteomics analysis compared to using a single separation method?

Tandem Hydrophilic Interaction Liquid Chromatography (HILIC) and High-pH Reverse-Phase (HPRP) improves proteomics by increasing proteome coverage, enhancing post-translational modification (PTM) discovery, and improving the separation of complex samples. This leads to better resolution and identification of individual peptides. The tandem approach provides a more complete picture of the proteome and PTM landscape, which is crucial for understanding protein function and regulation.

What are post-translational modifications (PTMs), and how does Tandem Hydrophilic Interaction Liquid Chromatography (HILIC) and High-pH Reverse-Phase (HPRP) aid in their discovery?

Post-translational modifications (PTMs) are chemical alterations to a protein after its translation. They play a crucial role in regulating protein activity, localization, and interactions. However, they often occur in low quantities and can be challenging to detect. Tandem Hydrophilic Interaction Liquid Chromatography (HILIC) and High-pH Reverse-Phase (HPRP) enhances PTM discovery by providing better separation and resolution of modified peptides, making it easier to identify and characterize these important modifications using mass spectrometry.

What are some potential applications of Tandem Hydrophilic Interaction Liquid Chromatography (HILIC) and High-pH Reverse-Phase (HPRP) in biological and medical research?

Tandem Hydrophilic Interaction Liquid Chromatography (HILIC) and High-pH Reverse-Phase (HPRP) can be applied to various proteomic studies, including biomarker discovery, drug target identification, and understanding disease mechanisms. It is particularly useful in situations where a comprehensive analysis of the proteome and its modifications is required. By providing a more complete and detailed view of the protein landscape, this technique can help researchers identify potential therapeutic targets and understand the molecular basis of disease.