1. Core Strategies for Quantitative Proteomics: Label-Free, TMT and DIA

Three mainstream approaches dominate quantitative proteomics analysis: label-free quantification, TMT labeling and data-independent acquisition (DIA).

Label-free quantification relies on data-dependent acquisition (DDA) and calculates protein abundance using MS1 peak intensity, and typically selects the top N precursor ions for secondary fragmentation. It features a straightforward workflow, low experimental cost and universal sample compatibility. However, this method suffers from frequent missing values and suboptimal reproducibility, particularly for low-abundance proteins, limiting its application in large-scale quantitative studies.

TMT/iTRAQ isobaric multiplex labeling enables parallel quantification of multiple samples, typically coupled with DDA scanning. This technique supports high-throughput batch sample analysis but has inherent limitations, including ratio compression effects, high reagent costs and compromised sensitivity for low-abundance molecular components.

As an advanced mass spectrometry acquisition mode, DIA systematically fragments all precursor ions within consecutive mass-to-charge (m/z) windows. It delivers superior reproducibility, minimal missing values, stable quantitative performance and complete raw data archiving, and is widely used for large-scale, long-term and repeatable proteomic profiling.

2. Technological Iteration of DIA Proteomics

DIA technology has undergone continuous optimization to overcome historical bottlenecks in scanning speed, detection sensitivity and operational workflow. Traditional DIA depends on pre-established DDA spectral libraries and adopts broad ion isolation windows, which easily induce co-fragmentation interference. Its scanning speed also restricts its performance in high-throughput experimental scenarios.

Direct-DIA (DDA-free DIA) removes the requirement for pre-constructed DDA spectral libraries. Leveraging deep learning algorithms, it generates de novo spectral libraries directly from DIA raw data, effectively simplifying experimental workflows, shortening analysis cycles and reducing overall research costs.

4D-DIA further integrates ion mobility (IM) as a fourth analytical dimension, complementing the conventional three dimensions of m/z ratio, retention time and signal intensity. This additional dimension efficiently eliminates background interference, improves spectral purity, and enhances analytical performance for complex biological samples and post-translational modification identification.

Astral-DIA represents the latest DIA technology, powered by the Orbitrap Astral mass spectrometer. This high-resolution mass spectrometry platform was launched by Thermo Fisher Scientific in 2023. It integrates quadrupole, Orbitrap and the brand-new Astral (Asymmetric Ion Mirrors) mass detector. It realizes genuine parallelization of high-resolution MS1 scanning and ultra-fast MS/MS acquisition, fundamentally breaking the efficiency and depth limitations of conventional DIA platforms.

3. Core Technologies and Revolutionary Advantages of Astral-DIA

Astral-DIA adopts a unique dual-analyzer parallel operating architecture. The Orbitrap analyzer performs high-resolution, high-mass-accuracy full-scan MS1 detection to guarantee reliable and precise protein quantification. Equipped with an asymmetric lossless multi-reflection design, the Astral analyzer delivers ultra-fast high-resolution accurate-mass (HRAM) MS/MS scanning at a rate of ~200 Hz. Intelligent ion splitting and routing enable synchronous, independent operation of the two analyzers.

This innovative architecture brings comprehensive performance upgrades for proteomic analysis. In terms of throughput, Astral-DIA supports the analysis of 180–300 samples per day, and can identify over 8,000 proteins within an 8-minute chromatographic gradient. For proteome coverage, it achieves the identification of more than 10,000 protein groups in a 24-minute gradient, significantly improving the detection capacity of low-abundance regulatory proteins that are critical for biological function.

Benefiting from ultra-high sensitivity and single-ion detection capability, Astral-DIA performs robustly with low-input samples. It supports narrow-window DIA (nDIA) with isolation windows as narrow as 2 Th, in contrast to the 10–25 Th windows used in conventional DIA. This drastic narrowing of isolation windows minimizes co-fragmentation interference and substantially improves spectral purity. In large-cohort studies, the median coefficient of variation (CV) of Astral-DIA quantification is below 10%, ensuring exceptional quantitative stability for large-scale and long-term reproducible research. Additionally, full compatibility with Direct-DIA enables library-free analysis, further simplifying workflows and improving experimental efficiency.

4. BMKGENE: Full-Process Professional Astral-DIA Proteomics Services

As a leading global omics solution provider, BMKGENE has fully deployed the advanced Orbitrap Astral platform and launched standardized, end-to-end Astral-DIA proteomics services for global researchers. Our service covers a wide range of sample types, including tissues, cells, biological fluids, exosomes and low-input samples. We provide full-process technical support, encompassing protein extraction, quantification, enzymatic digestion, desalting, Astral-DIA data acquisition and in-depth bioinformatics analysis.

Our comprehensive service portfolio includes protein identification and quantification, differential expression screening, and functional enrichment analysis. We also support multi-omics integrative analysis, with standardized data reports and publication-level visual outputs.

Combining the cutting-edge Orbitrap Astral platform, optimized and validated analytical protocols, strict full-process quality control and high-throughput processing capacity, BMKGENE can deliver stable, high-quality Astral-DIA services for diverse research fields, including plant science, animal breeding, microbiology and biomedical research.

References

1. Stewart, Hamish I. et al. “Parallelized Acquisition of Orbitrap and Astral Analyzers Enables High-Throughput Quantitative Analysis.” Analytical Chemistry 95(42) (2023): 15656–15664. DOI: 10.1021/acs.analchem.3c02856
2. Heil, Lilian R. et al. “Evaluating the Performance of the Astral Mass Analyzer for Quantitative Proteomics Using Data-Independent Acquisition.” Journal of Proteome Research 22(10) (2023): 3290–3300. DOI: 10.1021/acs.jproteome.3c00357
3. Guzman, Ulises H. et al. “Ultra-Fast Label-Free Quantification and Comprehensive Proteome Coverage with Narrow-Window Data-Independent Acquisition.” Nature Biotechnology 42(12) (2024): 1855–1866. DOI: 10.1038/s41587-023-02099-7