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4D-DIA Quantitative Proteomics

State-of-the-art Combination of 4D Proteomics and DIA Technology

Entering New Era of Mass Spectrometry-based Proteomics

A new generation of 4D-DIA proteomics technology combines 4D proteomics, which added ion mobility as the fourth separation dimension to traditional LC-MS/MS (retention time, m/z and MS/MS fingerprint), with data independent acquisition (DIA) strategy, which avoids data imbalance caused by randomness by realizing "lossless acquisition" of all possible data.

4D-DIA Proteomics


What is 4D Proteomics?

Classic LC-MS/MS-based bottom-up proteomics separate peptides based on three dimensions:  chromatographic retention time (RT); mass-to-charge ratio (m/z) and ion intensity.

Ion mobility analysis is a gas-phase technique allowing the separation of ions based on their mobility through an inert gas (typically helium or nitrogen) under the influence of an electric field. Adding ion mobility as an additional dimension of separation for peptide ions to LC-MS/MS analysis make MS-based proteomics enter a new era. 

It significantly improves the scanning speed and detection sensitivity, as well as enhances proteomic analysis performance in terms of identification depth, detection cycle, and quantitative accuracy.


Data Dependent/Independent Acquisition (DDA/DIA)

There are two data acquisition strategies in tandem mass spectrometry (MS/MS) data acquisition:

DDA mode:
The mass spectrometer selects a fixed number of most intense precursor ions. They are then fragmented and analyzed, in the second stage of tandem mass spectrometry.

DIA mode: The mass spectrometer divides the full scan range of mass spectrometry into several windows and selects, fragments, and collects all ions in each window.

DIA strategy avoids data imbalance caused by randomness by realizing "lossless acquisition" of all possible data. However, the "all-acquisition" strategy produces a highly complex spectrogram that poses a great challenge to data analysis. 



Technical Strengths of 4D-DIA Quantitative Proteomics

Improved Detection Accuracy and Reliability


The greatest challenge in traditional DIA without ion mobility separation lies in the difficulty of reliably analyzing mixed spectra. During quantification, matching is mainly based on chromatographic elution time (retention time). However, due to co-elution, the retention time alone is not enough, and many other interfering signals will affect the detection accuracy.

4D technology provides ion-mobility separation and adds an extra dimension for calibration. It can accurately discriminate the specific peptide signals from the mixed spectra of DIA, effectively reducing spectra complexity and improving the detection accuracy and reliability of DIA.

Fig. The chromatograms of EVGSHFDDFVTNLIEK peptide after calibration in traditional DIA (left) and 4D-DIA (right). There is a high interference signal in the traditional DIA method (left), while the signal background in the 4D-DIA (right) is very clean.


Nearly 100% Ion Utilization, Maximized Detection Sensitivity


In the 4D-diaPASEF scanning mode, the ion-mobility-related CCS value correlates well with m/z. This feature allows the Quadrupole to scan gradually to collect nearly 100% of the ion signals, greatly enhancing the sensitivity and depth of detection.

In comparison, in the traditional LC-MS/MS-based proteomics without ion mobility separation, only limited signals can be collected.


Fig. 4D-DIA based on the 4D platform can reach nearly 100% ion utilization. Left, Data acquisition in 4D-DIA mode; Right, Comparison of ion utilization among 4D-DIA, DDA, and DIA. (Source: Florian Meier, et al. Nature Methods.)

Significant Improvement in Detection Depth


4D-DIA identifies and quantifies low-abundant proteins more accurately than traditional methods, thereby increasing the depth of proteomics detection.


Fig. >7,500 proteins can be identified from a single injection of 200 ng of HeLa lysate (120-minute run time), and 6,974 proteins can be quantified with 96% data completeness in triplicate runs. In comparison, conventional proteomics usually requires μg-level samples to detect about 5,000 proteins.


Better quantitative integrity


4D-DIA technology can further push the limits of sensitivity with advanced instrument performance and upgraded acquisition methods. Even at this trace level, 4D-DIA showed 85% data integrity for protein quantification.


Fig. >3,000 protein can be identified from a single injection of 10 ng HeLa lysate (120-minute run time), and 3,323 proteins can be identified by triplicated runs. While under the same study conditions, the 4D-DDA mode could only identify 2,723 proteins.

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