4D Label-free Quantitative Proteomics Analysis Service

What Is 4D Label-Free Quantitative Proteomics?

4D Label-Free Quantitative Proteomics combines label-free relative quantification with TIMS (Trapped Ion Mobility Spectrometry). During LC-MS/MS analysis, TIMS separates peptide ions in the gas phase according to ion mobility and can provide collisional cross section (CCS) information as an additional ion property.

PASEF (Parallel Accumulation-Serial Fragmentation) allows ions to be accumulated in parallel and released sequentially for fragmentation, increasing MS/MS sampling efficiency. The resulting 4D data are commonly described by retention time, m/z, ion mobility, and signal intensity. Protein abundance is still evaluated using a label-free relative quantification strategy. TIMS-PASEF adds ion mobility separation and an additional acquisition dimension without introducing isotopic or chemical labels.

Services at MtoZ Biolabs

Using a high-resolution LC-MS/MS platform, MtoZ Biolabs integrates TIMS separation and PASEF acquisition into 4D Label-Free Quantitative Proteomics projects for global protein identification and relative quantification. MtoZ Biolabs can analyze tissue, cell, microorganism, biofluid, and protein extract samples and provide protein identification, relative abundance, differential protein results, and functional annotation when included in the project analysis plan.

When This Service Is a Good Fit

4D Label-Free Quantitative Proteomics is suitable for projects that need to:

• Compare global protein abundance across treatment groups, time points, genotypes, or other experimental conditions.
• Add ion mobility separation to improve peptide separation in complex proteomic samples.
• Analyze multiple sample groups without the channel-number constraints associated with isotopic or chemical labeling.
• Obtain protein identification, relative quantification, differential protein results, and project-specific functional analysis within one proteomics project.

4D Label-Free Quantitative Proteomics is primarily used for discovery and comparative proteomics. Targeted quantification of predefined proteins and multiplexed isobaric-label quantification require different quantitative approaches.

Workflow of 4D Label-Free Quantitative Proteomics

1. Sample Preparation and Protein Extraction

Prepare samples, extract proteins, and perform quality assessment according to sample type and project requirements.

2. Protein Digestion and Peptide Processing

Digest proteins and prepare peptides for LC-MS/MS analysis, including cleanup as required by the sample.

3. LC and TIMS Separation

Separate peptides by liquid chromatography and then by ion mobility in TIMS.

4. PASEF LC-MS/MS Acquisition

Acquire high-resolution MS and MS/MS data using PASEF acquisition.

5. Protein Identification and Label-Free Quantification

Identify peptides and proteins and calculate relative protein abundance from label-free mass spectrometric signals.

6. Differential and Functional Analysis

Compare protein abundance between experimental groups and perform differential protein statistics. Functional annotation and pathway analysis are included when specified in the project analysis plan.

Figure 1. 4D Label-Free Quantitative Proteomics Workflow.

Why Choose MtoZ Biolabs?

1. TIMS-PASEF Analysis

TIMS ion mobility separation and PASEF acquisition are integrated into the 4D Label-Free LC-MS/MS workflow.

2. End-to-End Proteomics Workflow

MtoZ Biolabs supports sample processing, LC-TIMS-PASEF acquisition, protein identification, relative quantification, and differential analysis within one project.

3. Multiple Sample Types

Projects can be evaluated for animal tissue, plant tissue, microorganisms, cells, serum/plasma, urine, and protein extracts.

4. Project Data Delivery

Deliverables can include protein identification and quantification results, differential analysis, mass spectrometry parameters, project-specific functional analysis, and raw mass spectrometry data.

Sample Submission Suggestion

The following values are suggested submission amounts. Final requirements may vary with species, sample characteristics, sample preparation status, and project design.

Sample Type	Suggested Amount
Animal Tissue	≥20 mg
Plant Tissue	≥2 g
Microorganisms	≥50 mg
Cells	≥1×10⁷
Serum/Plasma	≥10 μL
Urine	≥1 mL
Protein Extract	≥20 μg

 

For limited sample amounts, complex samples, or samples in which protein extraction has already been completed, provide the species, sample type, group design, biological replicates, sample amount, and existing sample-processing information before submission. The MtoZ Biolabs technical team will evaluate the sample conditions and project requirements.

Applications

1. Differential Protein Expression Studies

Compare relative protein abundance across treatment groups, disease models, developmental stages, or other experimental conditions.

2. Drug Response Studies

Evaluate protein abundance changes associated with drug treatment and identify proteins or pathways for subsequent investigation.

3. Genetic Perturbation Studies

Compare proteomic changes following gene knockout, overexpression, or other genetic perturbations.

4. Plant and Microbial Proteomics

Analyze protein abundance changes under different growth conditions, environmental stimuli, or stress treatments.

5. Biomarker Candidate Screening

Screen differential proteins as candidates for subsequent targeted validation or further research.

Deliverables

1. Experimental and Analytical Report

2. Mass Spectrometry Analysis Parameters and Related Data

3. Protein Identification and Relative Quantification Results

4. Differential Protein Statistical Analysis Results

5. Functional Annotation and Pathway Analysis Results (If Applicable)

6. Raw Mass Spectrometry Data

FAQ

Q1: What is the difference between 4D Label-Free and conventional Label-Free Quantitative Proteomics?

Both approaches use label-free relative quantification. 4D Label-Free adds TIMS ion mobility separation to LC-MS/MS and uses PASEF acquisition. The 4D data are commonly represented by retention time, m/z, ion mobility, and signal intensity.

Q2: How many sample groups can be analyzed by 4D Label-Free?

Label-free quantification is not constrained by isobaric labeling channel numbers. The appropriate number of groups and biological replicates depends on the research question, sample source, and statistical analysis requirements.

Q3: Can 4D Label-Free be used directly for post-translational modification quantification?

Standard 4D Label-Free projects focus on global protein identification and relative quantification. Quantification of PTMs such as phosphorylation, acetylation, or ubiquitination generally requires modification-specific enrichment and dedicated mass spectrometry workflows.

Q4: What information should be provided before project consultation?

Provide the species, sample type, group design, biological replicates, sample amount, existing sample-processing information, and research objective so that the technical team can evaluate sample preparation and the 4D Label-Free analysis plan.

For project consultation, provide species, sample type, group design, biological replicates, sample amount, sample-processing status, and research objective.