The development history of 4D Label Free technology
1. Early exploration of proteomics
The origins of proteomics can be traced back to the 1990s. With the rapid advancement of genome sequencing technology, scientists realized that merely deciphering gene sequences was far from sufficient for a comprehensive understanding of the essence of life activities. Genes are merely carriers of genetic information; proteins are the true executors of life activities. Thus, proteomics emerged, aiming to study the expression, structure, and function of all proteins in an organism.
Early proteomics research methods primarily relied on the combination of two-dimensional gel electrophoresis (2-DE) and mass spectrometry (MS). 2-DE can separate proteins based on their isoelectric points and molecular weights, followed by identification through MS. However, these early methods had many limitations. The resolution of 2-DE was limited, making it difficult to effectively separate low-abundance, highly acidic or basic, and proteins with excessively large or small molecular weights; moreover, the procedures were cumbersome, time-consuming, and lacked reproducibility. At that time, the sensitivity and accuracy of mass spectrometry also needed improvement, which restricted in-depth analysis of complex protein mixtures. Despite this, these early explorations laid the foundation for the development of proteomics and opened the door to a comprehensive understanding of the protein world.
2. The birth of 4D Label Free technology
In the process of deepening proteomics research, the limitations of traditional technologies are becoming more and more prominent. There is an urgent need for a more efficient and accurate technology to meet the growing research needs. 4D Label Free technology comes into being under such circumstances.
The key breakthrough of this technology lies in the introduction of ion mobility (mobility) as an additional dimension. Building on traditional 3D retention time (retention time), mass-to-charge ratio (m/z), and ion strength (intensity), it adds a fourth dimension: the collision cross-sectional area (CCS) of the —— peptide segment, redefining the analytical standards for proteomics. Based on PASEF technology developed at timsTOF Pro, 4D Label Free technology has significantly enhanced both the speed of mass spectrometry scanning and detection sensitivity. Research findings indicate that in the analysis of complex biological samples, traditional techniques often only identify a limited number of proteins. In contrast, 4D Label Free technology, with its innovative ion mobility separation and rapid scanning capabilities, can identify more proteins, greatly expanding the depth and breadth of proteomics research and providing powerful tools for unraveling the mysteries of proteins.
3. The gradual improvement of technology
Since its inception, 4D Label Free technology has been continuously improved and optimized in subsequent research. In terms of identification accuracy, through fine-tuning the ion mobility separation parameters and continuous updates and improvements to the mass spectrometry database, it can more accurately distinguish similar peptide segments, reducing misidentifications and enhancing the reliability of protein identification. For example, in the analysis of a complex tissue sample, the optimized technology reduced the error rate of protein identification by nearly 30%.
In terms of throughput improvement, researchers continuously refine data acquisition and processing algorithms, achieving faster scanning speeds and higher data collection efficiency. For instance, adopting new scanning modes allows for the analysis of more samples within a unit time, significantly shortening the experimental cycle. At the same time, notable progress has been made in quantitative stability. By optimizing experimental procedures and data analysis methods, when continuously injecting 96 needles, the correlation coefficient R²> 0.95, the coefficient of variation CV <5%, and the quantitation range exceeds three orders of magnitude, providing a solid foundation for accurate protein quantification. These significant improvements have made 4D Label Free technology increasingly important in proteomics research, driving continuous development in this field.
The core principle of 4D Label Free technology
1. The mystery of ion flow separation
Ion mobility separation is based on the characteristic of ions migrating in an electric field. In 4D Label Free technology, ion mobility separation plays a crucial role. When ions enter the mobility analyzer, they migrate towards the detector under the influence of the electric field. Different ions have varying migration rates in the electric field due to their size, shape, and charge, which is known as ion mobility. Smaller, more compact ions with higher charge migrate faster, while larger, irregularly shaped ions with lower charge migrate more slowly. In this way, ions are separated over time.
Ion mobility separation is of great significance in 4D Label Free technology. In complex biological samples, proteins undergo enzymatic hydrolysis to produce a large number of peptides. These peptides may overlap in traditional retention times, mass-to-charge ratios, and ion strengths, making them difficult to distinguish precisely. Ion mobility separation, as the fourth dimension, adds another layer of separation, effectively distinguishing co-eluting peptides and enhancing the accuracy and resolution of protein identification, providing a foundation for subsequent precise analysis. (A simple schematic diagram of the principle of ion mobility separation can be inserted here, such as an illustration of different migration rates of ions in an electric field)
2. Data acquisition mode analysis
Data-dependent acquisition-synchronous accumulation continuous fragmentation (ddaPASEF) scanning mode is a critical component of 4D Label Free technology. The workflow is as follows: in the first stage of mass spectrometry, the instrument rapidly scans and analyzes ions from the sample, monitoring their intensity in real time. When higher-intensity ions are detected, the system automatically selects these ions for secondary mass spectrometry analysis. Meanwhile, PASEF technology uses ion mobility separation to simultaneously accumulate ion signals during migration, achieving continuous fragmentation and obtaining more comprehensive ion information.
Compared to other acquisition modes, the ddaPASEF scanning mode has significant advantages. The traditional data-dependent acquisition (DDA) mode can only collect a limited number of high-intensity ions in each time window, making it prone to miss low-intensity but important ion information. In contrast, the ddaPASEF scanning mode, with its ion mobility separation and synchronous accumulation of continuous fragmentation, can more efficiently collect ion signals, improving ion utilization and significantly enhancing detection sensitivity and depth. In the analysis of complex samples, this mode can identify more proteins and peptides, providing richer data for proteomics research.
3. Differences from traditional technologies
| Compare projects | 4D Label Free technology | Traditional proteomics techniques |
|---|---|---|
| Separate dimensions | The ion mobility dimension is added on the basis of traditional 3D (retention time, mass charge ratio, ion strength) to realize 4D separation | It mainly relies on 3D separation and has relatively few dimensions |
| Data collection mode | Using ddaPASEF scanning mode, continuous fragmentation is accumulated synchronously, and ion signals are collected efficiently | For example, in DDA mode, each time window collects limited high intensity ions, which is easy to miss information |
| Sample requirements | The sample size requirement is low, each sample only needs 10-20μg protein, plasma as low as 10μl | Usually a larger sample size is required |
| Accuracy of identification | Ion mobility separation can effectively distinguish co-eluting peptides and improve the accuracy of identification | The co-elution problem affects the accuracy of identification |
| flux | There is no upper limit on the number of samples and high throughput | For example, the number of samples is limited in traditional iTRAQ/TMT labeled quantitative proteomics |
From the perspective of principle, 4D Label Free technology shows unique advantages in sample demand, identification accuracy and throughput by increasing the dimension of ion flow and unique data acquisition mode, bringing new breakthroughs to proteomics research.
Study design of the (A) prm-PASEF method creation workflow. (Alexander Brzhozovskiy et al,.2022)
The significant advantages of 4D Label Free technology
1. High sensitivity and adaptability to micro sample
4D Label Free technology achieves high sensitivity, thanks to its advanced ion mobility separation and optimized detection system. Ion mobility separation focuses ions in both time and space, reducing ion loss and enhancing detection efficiency. Additionally, the instruments high resolution and low noise performance further improve its ability to capture weak signals.
In the detection of trace samples, this technology excels. Only 10-20 μg of protein per sample, or as little as 10 μl of plasma, can achieve high-quality proteomic analysis. For instance, in early cancer diagnosis studies, trace exosome samples obtained from patient blood successfully identified multiple protein markers related to tumor development using 4D Label Free technology.
Its practical application scenarios are extensive. In clinical diagnosis, it can be used to analyze micro samples such as biopsy tissue and cerebrospinal fluid, and assist in early detection and accurate diagnosis of diseases; in the field of biopharmaceuticals, it can be used to detect precious cell culture supernatant, and help drug research and development and quality control.
2. Flux and repeatability improved
In terms of throughput, 4D Label Free technology has made a qualitative leap compared to traditional methods. Traditional iTRAQ/TMT labeling quantitative proteomics have certain limitations on sample volume, whereas 4D Label Free technology has no upper limit on sample quantity, making it more suitable for comparative analysis of large cohort samples. For example, in a proteomics study involving thousands of tumor samples, 4D Label Free technology can efficiently analyze all samples, significantly shortening the research cycle.
In terms of reproducibility, the technology performs excellently. Experimental data shows that with continuous injection of 96 needles, the correlation coefficient R²> 0.95 and the coefficient of variation CV <5%. This means that over multiple experiments, the technology can consistently yield similar results, providing reliable data support for research. In clinical drug trials, protein group analysis of patient samples from different batches has been conducted using the 4D Label Free technology, which, thanks to its excellent reproducibility, accurately reveals the mechanisms of drug action and therapeutic differences, offering strong evidence for drug development and optimization.
3. Breakthrough in quantitative accuracy
4D Label Free technology has significantly improved quantitative accuracy. The improvement mainly stems from multi-dimensional separation and precise data acquisition. Ion mobility separation increases the number of separation dimensions, reducing interference from co-eluting peptides, thus allowing mass spectrometry signals to more accurately correspond to specific proteins. Meanwhile, ddaPASEF scanning mode can comprehensively and stably collect ion signals, providing a rich and reliable data foundation for quantitative analysis.
Relevant research data shows that the quantitative range of this technology exceeds three orders of magnitude and can be highly consistent with western blot results. In a study on disease biomarker screening, the 4D Label Free technique accurately quantified changes in protein expression levels across different samples, identifying multiple biomarkers with potential diagnostic value. The quantitative results are highly consistent with traditional western blot methods and offer advantages such as high throughput and high efficiency, providing more powerful tools for early diagnosis and treatment monitoring of diseases.
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4D Label Free Technical data analysis
1. Standard data analysis process
The standard data analysis process of 4D Label Free technology covers multiple critical stages, providing comprehensive and in-depth information for proteomics research. The identification and analysis stage aims to determine the types of proteins in the sample. By comparing peptide data obtained from mass spectrometry with known protein databases, algorithms match the sequence information of peptides to accurately identify the proteins present in the sample. This process lays the foundation for subsequent analyses and clarifies the research subject.
Differential expression analysis focuses on the changes in protein expression levels between different samples. By statistically analyzing quantitative information of proteins in each sample and using appropriate statistical methods such as t-tests and ANOVA, it identifies proteins that show significant differences in expression under different conditions. These differentially expressed proteins may be closely related to specific physiological or pathological processes and are key targets for further research.
Quality control analysis runs throughout the entire process. By monitoring indicators such as the distribution of peptide ion mass deviations, peptide ion scores, and protein abundance ratios, it evaluates the reliability and accuracy of data. This ensures the quality of experimental data, providing a solid foundation for subsequent analyses. These standard data analysis steps work together to offer a systematic and comprehensive analytical framework for proteomics research.
2. Advanced data analysis methods
In addition to the standard analysis process, 4D Label Free technology also involves various advanced data analysis methods. GSEA enrichment analysis, or gene set enrichment analysis, can map differentially expressed proteins to specific biological pathways or functional categories, determining whether these pathways or functions are significantly enriched across different samples. For example, in cancer research, GSEA enrichment analysis has revealed that certain pathways related to cell proliferation and apoptosis are significantly enriched in tumor samples, shedding light on the potential mechanisms of tumor development.
Marker screening is also an important advanced analytical method. By integrating factors such as protein expression levels, biological functions, and clinical information, it identifies biomarkers with diagnostic, prognostic evaluation, or therapeutic guidance value. In cardiovascular disease research, through the combination of 4D Label Free technology and advanced analytical methods, multiple protein markers closely related to disease severity and prognosis have been successfully identified, providing evidence for early diagnosis and precise treatment. These advanced analytical methods offer powerful tools for delving into the biological significance behind proteomics data.
3. Personalized data analysis services
For different research needs, the 4D Label Free technology also provides personalized data analysis services. The processing method typically involves customizing specific analytical strategies and algorithms based on the researchers specific questions and objectives. Common directions for personalized analysis include constructing specific protein interaction networks, aimed at revealing the relationships between proteins and understanding their functional regulatory mechanisms within cells. In the study of neurological diseases, building interaction networks of specific neural proteins helps identify new neural signaling pathways and potential therapeutic targets.
Another direction is the analysis of protein expression trends. By analyzing the dynamic changes in protein expression levels at different time points or under different treatment conditions, we can reveal the patterns and trends of protein expression. In plant growth and development studies, analyzing the expression trends of specific proteins at different growth stages helps understand the molecular mechanisms of plant growth regulation. Personalized data analysis services meet diverse research needs, driving proteomics research towards deeper and more precise directions.
Application fields of 4D Label Free technology
1. Application in drug development
In the field of drug development, 4D Label Free technology plays an indispensable role. In terms of drug response, by analyzing changes in the proteome of cells or tissues before and after drug treatment, it is possible to clearly understand the impact of drugs on protein expression within cells, thereby evaluating drug efficacy and mechanisms of action. For example, a research team used this technology to study the effects of an anticancer drug on cancer cells, finding that multiple proteins related to cell proliferation and apoptosis underwent significant changes after drug treatment, providing key clues for a deeper understanding of the drugs anti-cancer mechanism.
In target identification, 4D Label Free technology also demonstrates its prowess. It can accurately identify the target proteins for drug action, providing clear direction for drug development. For instance, in the research and development of a new cardiovascular drug, this technology successfully identified the key target protein, accelerating the drugs development process. Research findings indicate that 4D Label Free technology offers more comprehensive and accurate information for drug development, helping to improve efficiency, reduce costs, and expedite the market launch of new drugs.
2. The contribution of biomedical research
4D Label Free technology has brought numerous benefits to biomedical research. In the discovery of disease biomarkers, its high sensitivity and precise quantification capabilities enable the screening of protein markers closely related to disease onset and progression from complex biological samples. Taking Alzheimers disease as an example, researchers used this technology to analyze cerebrospinal fluid samples from patients and healthy controls, successfully identifying several new disease-related protein markers, providing possibilities for early diagnosis.
In the study of protein function mechanisms, this technology can comprehensively analyze the expression regulation and interaction networks of proteins within cells. For example, in cancer research, 4D Label Free technology is used to analyze the differences in proteomes between tumor cells and normal cells, providing deep insights into the mechanisms of key proteins during tumor development, thus offering theoretical support for developing targeted therapeutic strategies. The application of 4D Label Free technology in biomedical research helps reveal the pathogenesis of diseases, offering crucial support for disease diagnosis, treatment, and prevention.
3. A new tool for plant and microbial research
In the field of plant and microbial research, 4D Label Free technology has become a powerful new tool. In studies on plant growth and development, this technology can be used to analyze changes in the plant proteome at different growth stages or under varying environmental conditions, revealing the molecular mechanisms of plant growth regulation. For example, researchers conducted proteomic analyses on plants subjected to drought stress and identified multiple proteins related to drought response, providing genetic resources for breeding drought-resistant crop varieties.
In the field of microbiology, this technology can be used to analyze protein expression changes in microorganisms under different nutritional conditions or stress states, understanding their metabolic regulation and adaptation mechanisms. For instance, in the study of pathogenic bacteria, analyzing the proteomic changes during the infection process using 4D Label Free technology helps identify new virulence factors and drug targets, providing a basis for developing novel antimicrobial drugs. Experimental results have shown that 4D Label Free technology offers deeper and more comprehensive insights into plant and microbial research, driving continuous progress in this field.
Future prospects of 4D Label Free technology
1. Technology development trends
4D Label Free technology has a broad future development prospect. Renowned proteomics expert Professor Matthias Mann once pointed out that technological integration is a crucial direction for the future development of proteomics. 4D Label Free technology holds promise for deep integration with gene editing techniques such as CRISPR-Cas9, enabling gene editing to alter protein expression. This can then be followed by precise analysis of proteomic changes using 4D Label Free technology, allowing for a more in-depth exploration of the relationship between genes and protein functions.
In terms of performance improvement, the resolution and sensitivity of the instrument will be further enhanced. With the advancement of hardware technology, the accuracy of ion mobility separation will increase, allowing for the differentiation of finer ion differences and the identification of more low-abundance proteins. The speed of data acquisition and processing will also significantly improve, reducing experimental time costs. At the same time, integration with artificial intelligence and machine learning algorithms will become tighter, enabling smarter data analysis and result prediction, providing stronger technical support for proteomics research and driving the field to new heights.
2. Profound implications for proteomics
4D Label Free technology has profoundly advanced proteomics research. It significantly enhances the depth and breadth of protein identification, enabling researchers to discover more unknown proteins and fill gaps in the proteomics knowledge map. This makes it possible to comprehensively decipher the functions and interaction networks of proteins, aiding in the construction of more complete protein regulation models.
This technology may also lead to new research directions. For example, the study of protein dynamic modifications will delve deeper, revealing the patterns of protein modification changes under different physiological and pathological conditions. In terms of spatiotemporal specific studies of the proteome, combining high-resolution imaging techniques holds promise for precise localization and dynamic monitoring of proteins within cells and tissues. These breakthroughs will shift proteomics from mere protein identification and quantification to functional mechanisms and dynamic regulation research, bringing fresh perspectives and opportunities to life science research.
3. Potential applications in interdisciplinary fields
In interdisciplinary fields, 4D Label Free technology holds tremendous potential for application. In bioinformatics, the massive amount of proteomic data generated provides rich resources for algorithm development and model training. By integrating bioinformatics algorithms, it is possible to uncover hidden patterns in protein data, predict protein structure and function, construct complex protein interaction network models, and accelerate the discovery of drug targets and disease mechanism research.
In the field of systems biology, 4D Label Free technology can be integrated with multi-omics techniques such as metabolomics and transcriptomics to comprehensively decipher the molecular regulatory networks of biological systems. For example, when studying how plants respond to environmental stresses, combining transcriptomics to understand changes in gene expression, using metabolomics to analyze the accumulation of metabolic products, and then employing 4D Label Free technology to analyze protein dynamics, reveals the molecular mechanisms by which plants adapt to their environment from multiple levels. This provides more comprehensive and in-depth data support for systems biology research, driving interdisciplinary studies to achieve new breakthroughs.
References
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- 4D-MBR and dia-PASEF®: Ushering in the area of 4D-Proteomics. https://www.bruker.com/en/applications/academia-life-science/proteomics/4d-proteomics.html
- Dodds JN, Baker ES. Ion Mobility Spectrometry: Fundamental Concepts, Instrumentation, Applications, and the Road Ahead. J Am Soc Mass Spectrom. 2019 Nov;30(11):2185-2195. doi: 10.1007/s13361-019-02288-2. Epub 2019 Sep 6. PMID: 31493234; PMCID: PMC6832852.
