Hydroxylation Analysis – LC-MS/MS Profiling of Protein Hydroxylation

Hydroxylation transfers hydroxyl (-OH) groups to specific amino acid residues and associated with collagen-related disorders, cancer, and hypoxia pathways. Creative proteomics provide high-precision hydroxylation research services focused on the identification and quantification of amino acid hydroxylation modifications.

Comprehensive detection: Identify and quantify hydroxylated proteins and site-specific amino acid modifications.
Advanced technology: LC-Ms/Ms + immunoprecipitation enrichment for precision and depth.
Functional insights: Integrated bioinformatics for motif, pathway, and network interpretation.
Research impact: Clarify hydroxylation critical roles in protein stability, enzymatic activity, and disease pathogenesis.

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What Is Hydroxylation?

Hydroxylation is a post-translational modification of proteins in which an enzyme catalyses the introduction of a hydroxyl (-OH) group onto specific amino acid residues. Hypoxia-inducible factor (HIF) represents the primary oxygen-sensitive regulatory modification involving proline hydroxylation. This modification is primarily catalyzed by 2-oxoglutarate-dependent dioxygenases, including prolyl hydroxylases (PHDs), lysyl hydroxylases, and factor inhibiting HIF (FIH). These enzymes require oxygen (O₂), iron (Fe²⁺), α-ketoglutarate (also known as 2-oxoglutarate; 2-OG), and ascorbic acid (vitamin C). PHDs utilize one oxygen atom from O₂ during the oxidative decarboxylation of 2-OG to produce succinate and CO₂, while another directly incorporates into the oxidized amino acid residues of HIF-α. Furthermore, enzymatic hydroxylation reactions have been observed in asparagine, aspartic acid, arginine, histidine, and lysine residues. Hydroxylation plays central roles in oxygen sensing, transcriptional regulation, and protein stability. It controls the hypoxic response through HIF degradation, modulates protein–protein interactions, and influences phase separation and organelle assembly. Collectively, hydroxylation represents a versatile modification linking environmental signals to structural and functional outcomes in diverse proteins^[1].

Hydroxylation Service at Creative Proteomics

Hydroxylation is an important post-translational modification of proteins, and its dysregulation is associated with collagen-related disorders, cancer, and hypoxia pathways. Systematic investigation of hydroxylated proteins is essential for understanding their biological and pathological functions. Our hydroxylation analysis service provides precise identification and quantification of hydroxylated peptides using high-resolution LC–MS/MS and immunoprecipitation enrichment. This service enables researchers to explore enzyme-specific hydroxylation events and uncover their biological significance across different models.

Hydroxylation Research Platforms

Thermo Fisher Easy-nLC 1000 and Thermo Fisher LTQ Obitrap ETD
(Figure from Thermo Scientific)

Workflow of Hydroxylation Analysis

Advantages Our Hydroxylation Service

Professional detection and analysis capability: Supported by an experienced research team and well-established, rigorously validated methodologies.
Broad Sample Compatibility: Proficient in handling protein samples derived from diverse sources, including cells, animal and plant tissues, and microorganisms.
High specificity and accuracy: Advanced quantitative proteomics techniques combined with robust PTM enrichment strategies ensure precise and reliable measurements.
High stability and reproducible: Consistently delivers robust and reproducible results with excellent inter- and intra-assay consistency, enabling reliable downstream data analysis.
Advanced Analytical Platforms: Use of high-sensitivity mass spectrometry and state-of-the-art tools, ensuring precise, repeatable results.
End-to-End Support: Offer full-spectrum services from design to data interpretation, with continuous technical support throughout the research process.

Applications of Hydroxylation

Protein interactions and regulations

JMJD family enzymes catalyze lysine hydroxylation on histones and non-histone proteins, linking hydroxylation to transcriptional regulation, RNA processing, and cancer-related signaling pathways.^[2].

Therapeutic Targeting

Pharmacological inhibition of prolyl hydroxylases stabilizes HIF and enhances erythropoiesis, demonstrating hydroxylation as a therapeutic target for anemia in chronic kidney disease^[3].

Physiological state of animals

Hydroxyproline modulation supports collagen synthesis, antioxidant defense, and signal regulation in response to nutritional and hypoxic conditions^[4].

Demo Result of Hydroxylation Service

PCA plot

Heat map

GO analysis

Heatmap

The plot is used to show the enrichment of hydroxylated protein in different pathways.

KEGG analysis

The protein-protein interaction network demonstrated the interactions between all hydroxylation modification differential proteins.

PPI

FAQs of Hydroxylation Service

Which sample types are accepted by the service?

We accept a variety of sample types including cells, tissues, plant tissue and more. If you have other special sample types, please consult in advance.

Whether the sample needs pretreatment?

We recommend that customers provide fresh or frozen samples, which should avoid repeated freezing and thawing, and we will be responsible for subsequent protein extraction and pretreatment. If you have already extracted the protein, please communicate with us in advance about the sample handling method.

What are the common targets for hydroxylation detection?

We can detect hydroxylation modifications of various amino acid residues such as proline (Pro), lysine (Lys), asparagine (Asn), and histidine (His), and it is particularly suitable for studies on HIF pathway proteins, collagen family proteins, and metabolic-related enzymes.

Learn about other Q&A.

Case Study

Article

Publications

Here are some publications in Proteomics research from our clients:

Pair bonding and disruption impact lung transcriptome in monogamous Peromyscus californicus. 2023. https://doi.org/10.1186/s12864-023-09873-6
Impact of alanyl-tRNA synthetase editing deficiency in yeast. 2021. https://doi.org/10.1093/nar/gkab766
Protein interactors of Spindle Pole Body (SPB) components and septal proteins in fungus Neurospora crassa: A mass spectrometry-based dataset. 2024. https://doi.org/10.1016/j.dib.2023.109980
In Vitro Identification of Phosphorylation Sites on TcPolβ by Protein Kinases TcCK1, TcCK2, TcAUK1, and TcPKC1 and Effect of Phorbol Ester on Activation by TcPKC of TcPolβ in Trypanosoma cruzi Epimastigotes. 2024. https://doi.org/10.3390/microorganisms12050907
Trypanosoma cruzi DNA Polymerase β Is Phosphorylated In Vivo and In Vitro by Protein Kinase C (PKC) and Casein Kinase 2 (CK2). 2022. https://doi.org/10.3390/cells11223693

More Publications

Hydroxylation Service Sample Requirements

Sample	Sample Quantity
Tissue	animal tissue	200-500mg
Tissue	fresh plant	200-500mg
Cell	suspension cell	> 3x10⁸
	adherent cell	> 3x10⁸
	microorganism	> 50 mg or 3 × 10⁸ cells

References

Zurlo, G., Guo, J., Takada, M., Wei, W., & Zhang, Q. (2016). New Insights into Protein Hydroxylation and Its Important Role in Human Diseases. Biochimica et biophysica acta, 1866(2), 208–220. https://doi.org/10.1016/j.bbcan.2016.09.004
Oh, S., & Janknecht, R. (2024). Versatile JMJD proteins: juggling histones and much more. Trends in biochemical sciences, 49(9), 804–818. https://doi.org/10.1016/j.tibs.2024.06.009
Ren, S., Yao, X., Li, Y., Zhang, Y., Tong, C., & Feng, Y. (2023). Efficacy and safety of hypoxia-inducible factor-prolyl hydroxylase inhibitor treatment for anemia in chronic kidney disease: an umbrella review of meta-analyses. Frontiers in pharmacology, 14, 1296702. https://doi.org/10.3389/fphar.2023.1296702
Hu, S., He, W., & Wu, G. (2022). Hydroxyproline in animal metabolism, nutrition, and cell signaling. Amino acids, 54(4), 513–528. https://doi.org/10.1007/s00726-021-03056-x.

Proteomics Sample Submission Guidelines

Ensure your samples are prepared and submitted correctly by downloading our comprehensive Proteomics Sample Submission Guidelines. This document provides detailed instructions and essential information to facilitate a smooth submission process. Click the link below to access the PDF and ensure your submission meets all necessary criteria.

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* For Research Use Only. Not for use in diagnostic procedures.

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From Our Clients

"I recently used their proteomics service for a project analyzing protein interactions in yeast models. The team was very responsive and helped clarify the methodology they employed, which made me feel confident in the results. The data quality was solid, with clear identification of several key proteins involved in our study. Their thorough analysis enabled me to pinpoint specific interactions that I hadn't considered before, which significantly improved the direction of my research. I appreciate their professionalism and support throughout the process."

Sarah Thompson, University of California, Berkeley

"Our lab collaborated with them on a project studying cancer biomarkers. The proteomics analysis provided was detailed and focused, specifically highlighting the differential expression of proteins between healthy and tumor samples. Their clear explanations of the data helped my team understand the biological implications. I also appreciated their willingness to revise the reports based on our feedback, ensuring that we had everything we needed for our publication. This collaborative spirit was invaluable."

Emily Rodriguez, Stanford University

"Our lab worked with them on a project studying the effects of diet on gut microbiota using proteomics. They used a label-free quantification method to analyze proteins in fecal samples before and after dietary intervention. The results showed significant changes in protein expression linked to microbial activity. This was pivotal for our hypothesis about diet-microbiota interactions. The clarity of their data presentation made it easy for our team to integrate these findings into our ongoing research."

Dr. Lisa Wong, University of Toronto

"My experience with Creative Proteomics during the mass spectrometry analysis was excellent. We sent in human saliva and mouse brain tissue samples, which they expertly analyzed using both LC-MS and GC-MS techniques. The results were invaluable, revealing key metabolites in the saliva and identifying biomarkers linked to brain function in the brain tissue."

Dr. Emily Carter, Senior Research Scientist

"The overall service from Creative Proteomics was outstanding. They made the entire process seamless and efficient, allowing us to focus on our research. We worked with leaf and root samples from various Arabidopsis genotypes for targeted metabolomics analysis. Their thorough profiling of primary and secondary metabolites gave us important insights into how the plants respond metabolically to environmental stress."

Dr. Laura Henderson, Plant Physiologist

"We had a pleasant collaboration with Creative Proteomics on mass spectrometry analysis of lipids. They conducted a detailed analysis of lipid species, providing us with important insights into lipid metabolism and its relationship with metabolic syndrome disease states."

Dr. Sarah Mitchell, Research Scientist

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