A Comprehensive Workflow for 4D Label-Free Quantitative Proteomics Analysis

Quantitative proteomics has emerged as a powerful tool in life sciences and translational medicine for elucidating molecular mechanisms, identifying biomarkers, and evaluating pharmacological effects. Although labeling-based strategies can improve quantitative accuracy, they are often limited by high costs and restricted sample scalability. The 4D label-free quantitative proteomics approach, integrating ion mobility (TIMS), data-independent acquisition (DIA), tandem mass spectrometry (MS/MS), and advanced computational algorithms, has effectively overcome bottlenecks in sensitivity, depth, and throughput. This technology has been widely applied in tumor biology, neurodegenerative disorders, immune regulation, and pharmacodynamic studies.

What Is 4D Proteomics? Decoding the Fourth Dimension

At the core of 4D proteomics lies the incorporation of ion mobility as a fourth dimension in mass spectrometry, enhancing protein identification resolution and quantification precision by complementing conventional three-dimensional MS data (mass-to-charge ratio [m/z], retention time [RT], and signal intensity).

Key Technologies: TIMS + PASEF

4D proteomics typically utilizes the Bruker timsTOF Pro platform, which integrates:

• TIMS (Trapped Ion Mobility Spectrometry): Enables ion separation based on mobility, enhancing resolution of isomeric species.

• PASEF (Parallel Accumulation–Serial Fragmentation): Increases MS/MS acquisition speed while maintaining sensitivity.

This synergistic combination significantly boosts proteome depth (>7,000 proteins), reproducibility (coefficient of variation <15%), and throughput (>100 samples/week).

Standard Workflow of 4D Label-Free Quantitative Proteomics

To ensure maximum data accuracy and reproducibility, a standard 4D label-free quantitative proteomics pipeline should consist of the following critical steps:

1. Sample Preparation: Ensuring Quality from the Source

(1) Common Sample Types

Cell lines, tissues, serum, plasma, cerebrospinal fluid (CSF), urine, and more.

(2) Standardized Procedures

• Protein extraction and quantification: Efficient protein extraction using lysis buffers (e.g., RIPA, 8M urea), followed by quantification via the BCA assay.

• Enzymatic digestion: Proteins are digested using Trypsin/LysC to ensure high-quality peptide generation.

• Peptide cleanup: Purification with C18 columns or StageTips to eliminate interfering ions and reduce background noise.

2. LC-MS/MS Analysis: Core Data Acquisition

(1) Chromatographic Configuration

A nanoLC system is coupled with the timsTOF Pro, using a 75 µm ID × 25 cm column packed with C18 resin at a flow rate of 300 nL/min.

(2) Mass Spectrometry Optimization

• DIA mode with customizable isolation windows (commonly 32 or 64 windows);

• PASEF mode enabling MS/MS acquisition rates >100 Hz;

• Automated calibration ensures high precision in both m/z and ion mobility dimensions.

This configuration enables high proteome coverage and low missing value rates from a single injection.

3. Data Analysis: From Spectra to Biological Insight

Data analysis is the most critical step in determining the biological value of the experiment. 4D label-free datasets are commonly processed with the following platforms:

(1) Data Processing Tools

• Spectronaut: Supports DIA analysis and integrates m/z, RT, and IM data.

• DIA-NN: Neural network–based software that enhances peptide identification sensitivity.

• FragPipe with MSFragger: Open-source solution suited for custom workflows and advanced users.

(2) Analysis Workflow Overview

Spectrum matching → Protein identification → Intensity normalization → Differential expression analysis → Functional enrichment → Visualization and interpretation.

Applications of 4D Proteomics Technology

1. Tumor Heterogeneity Profiling

Identification of subtype- or stage-specific differential proteins in cancer.

2. Mechanistic Studies of Drug Action

Assessment of pathway activation or inhibition following therapeutic intervention.

3. Clinical Biomarker Discovery

Mining candidate proteins associated with disease progression and prognosis.

4. Multi-Omics Integration

Combination with transcriptomics or metabolomics to generate a comprehensive molecular landscape.

MtoZ Biolabs: Technical Advantages and Service Highlights

As a leading proteomics service provider, MtoZ Biolabs offers high-throughput, standardized solutions for 4D label-free quantitative proteomics. Our platform provides the following key advantages:

• High-resolution instrumentation: Bruker timsTOF Pro 2 enables acquisition at the million-spectra scale.

• Standardized front-end processing: Compatible with a wide range of samples, including blood, tissue, and cultured cells.

• Flexible data analysis pipelines: Tailored workflows for mechanism-oriented research and clinical discovery studies.

• Stringent quality control and project management: Ensures data stability and reproducibility throughout the project lifecycle.

Whether your goal is to dissect fundamental biological mechanisms or to identify clinically actionable biomarkers, MtoZ Biolabs is dedicated to supporting research with scientific rigor.

4D label-free quantitative proteomics represents a significant milestone in analytical biology, offering unparalleled depth, reproducibility, and throughput. By pushing the limits of proteome resolution, this technique is transforming our understanding of biological systems. For projects involving complex sample analyses or translational biomarker discovery, MtoZ Biolabs invites you to explore the proteome in unprecedented detail.

MtoZ Biolabs, an integrated chromatography and mass spectrometry (MS) services provider.

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4D Proteomics Service