Middle-Down MS-Based PTM Analysis Service

Post-translational modifications (PTMs) of proteins play crucial roles in cellular regulation, physiological processes, and disease progression. Modifications such as phosphorylation, acetylation, methylation, and ubiquitination intricately regulate protein structure and function through precise spatial-temporal dynamics, forming complex regulatory networks. However, due to the diverse nature and complexity of PTMs, traditional proteomic approaches, like bottom-up methods, have significant limitations in accurately quantifying co-occurring PTMs and identifying combinatorial modification patterns. Middle-down proteomics has emerged as a powerful strategy that bridges the analytical gap between bottom-up and top-down approaches.

 



Kim, DN. et al. Bioengineering (Basel). 2024.

Figure 1. Various Types of Post-Translational Modifications and Their Effects

 

MtoZ Biolabs leverages advanced middle-down proteomics technology integrated with high-resolution mass spectrometry (MS) to offer a specialized Middle-Down MS-Based PTM Analysis Service. This service helps clients precisely quantify complex PTMs and their combinatorial patterns, uncovering deeper biological insights into protein modifications.

 

Technical Principles

Middle-down proteomics is an innovative analytical strategy situated between bottom-up and top-down proteomics. Protein samples are first enzymatically digested into peptides of intermediate length (approximately 10-50 amino acids), preserving valuable combinational PTM information while providing high sensitivity and accuracy during MS analysis. Initially, proteins undergo targeted enzymatic digestion, followed by chromatographic separation to simplify the sample complexity, enhancing the effectiveness of subsequent MS analyses.

 



Figure 2. Workflow of PTM Analysis Based on Middle-Down Proteomics

 

MtoZ Biolabs employs high-sensitivity Orbitrap mass spectrometry platforms combined with advanced fragmentation techniques such as Electron Transfer Dissociation (ETD) and Electron Capture Dissociation (ECD). These technologies facilitate high-precision identification and quantification of PTM sites. Additionally, Parallel Reaction Monitoring (PRM) methods significantly enhance analytical depth and quantitation accuracy of targeted PTMs.

 

Analysis Workflow

The standard workflow for Middle-Down MS-Based PTM Analysis service comprises:

1. Sample Preparation

Proteins are extracted and enzymatically digested using selective proteases (e.g., Glu-C, Asp-N) to produce peptides of suitable length, preserving co-occurring PTM site information.

 

2. Chromatographic Separation

Peptide mixtures undergo preliminary online liquid chromatography separation to reduce sample complexity, improving subsequent MS analysis efficiency.

 

3. High-Resolution Mass Spectrometry

Samples are analyzed using Orbitrap high-resolution MS combined with ETD/ECD fragmentation to precisely identify peptide sequences and modification sites.

 

4. PTM Quantification

The PRM approach specifically monitors targeted peptides, quantifying the relative abundances of PTM sites, ensuring accurate and reliable quantitative results.

 

5. Data Analysis and Reporting

Specialized bioinformatics software processes the MS data, systematically identifying PTM patterns, combinatorial states, and dynamic trends. A comprehensive and clear analytical report is then provided.

 

Service Advantages

1. Accurate PTM Site Identification: Middle-down proteomics retains combinational PTM information, accurately revealing complex modification networks.

2. High Sensitivity and Precision: Utilizing high-resolution MS and PRM significantly improves quantitative accuracy, particularly effective for low-abundance PTM analyses.

3. Comprehensive Combinational PTM Analysis: Overcomes limitations of traditional methods in distinguishing co-occurring PTMs, clearly defining combinational modification patterns.

4. Wide Application Scope: Suitable for analyzing PTMs on membrane proteins, transcription factors, key signaling proteins, and more.

 

Applications

Middle-Down MS-Based PTM Analysis Service supports diverse research and applications, including but not limited to:

1. Signaling Pathway Studies: Identifying complex PTM patterns of critical signaling proteins to elucidate signaling mechanisms.

2. Drug Mechanism Elucidation: Analyzing PTM dynamics in response to drug treatment to evaluate drug efficacy and target activity.

3. Epigenetic Research: Decoding histone modification patterns to explore biological functions of epigenetic regulation.

4. Disease Biomarker Discovery: Identifying disease-specific PTM isoforms, aiding biomarker identification and drug target development.

 

Case Study

1. Middle-Down Mass Spectrometry Reveals Activity-Modifying Phosphorylation Barcode in a Class C G Protein-Coupled Receptor

This study proposes a novel strategy combining middle-down proteomics with parallel reaction monitoring (PRM) mass spectrometry to quantitatively analyze the phosphorylation states of the C-terminal tail of a class C GPCR—metabotropic glutamate receptor 2 (mGluR2). The results revealed complex phosphorylation patterns of mGluR2 under basal and agonist-induced conditions, identified specific phosphorylation sites linked to receptor activity, and confirmed the regulatory role of phosphorylation in receptor signaling. Middle-Down MS-Based PTM Analysis Service employs a middle-down proteomic strategy integrated with high-resolution mass spectrometry, providing precise quantitative analyses of complex post-translational modifications (PTMs) such as phosphorylation. It is ideal for membrane proteins, GPCRs, and critical signaling pathway proteins, enabling accurate localization of modification sites and assessment of their dynamic changes and relative abundances.

 



Ives, AN. et al. J Am Chem Soc. 2022.

Figure 3. Middle-Down Proteomic analysis of mGluR2 Phosphorylation States

 

2. Middle-Down Proteomic Analyses with Ion Mobility Separations of Endogenous Isomeric Proteoforms

This study presents a strategy combining middle-down proteomics with ion mobility spectrometry (IMS) to effectively resolve endogenous histone proteoform isomers. Through initial separation by liquid chromatography, refined resolution via field asymmetric waveform IMS (FAIMS), and accurate localization using Orbitrap mass spectrometry coupled with electron transfer dissociation (ETD), the researchers successfully identified specific modification patterns and sites of histone isomers in mouse embryonic stem cells. Middle-Down MS-Based PTM Analysis Service employs a middle-down proteomics approach integrated with ion mobility and high-resolution mass spectrometry to precisely differentiate and identify protein isoforms bearing complex post-translational modifications (PTMs), enabling effective isomer separation, site localization, and accurate qualitative analysis.

 



Shliaha, PV. et al. Anal Chem. 2020.

Figure 4. Normalized Reconstructed FAIMS Scans for the Five Top Isomers within Select Compositions

 

Deliverables

1. Detailed PTM Mapping Report

2. Quantitative PTM Data

3. Graphical PTM Profiles

4. Supporting Raw Data Files

5. Customized Bioinformatics Analysis

 

Through the Middle-Down MS-Based PTM Analysis Service, MtoZ Biolabs is committed to delivering high-quality PTM analyses, supporting deeper investigation into protein biological functions and disease mechanisms. For further questions or collaboration, please feel free to contact our technical team for professional consultation and tailored service solutions.