Plasma proteomics goes high throughput – timsTOF Pro with PASEF and 4D feature alignment to quantify 500 plasma proteins in 11.5 min

Importance of the Topic

Clinical blood plasma analysis underpins much of modern diagnostics and biomarker discovery. Traditional immunoassays offer specificity but are constrained in multiplexing capacity and require available antibodies. Mass spectrometry–based proteomics, particularly when scaled for high throughput and robust quantification, promises an unbiased survey of hundreds to thousands of proteins in plasma, enabling the identification of novel biomarkers and monitoring of disease states in large patient cohorts.

Objectives and Study Overview

This study aimed to develop and validate a rapid, high-throughput LC-MS/MS workflow capable of quantifying approximately 500 plasma proteins per 11.5-minute run. By combining the Evosep One liquid chromatography system with the timsTOF Pro mass spectrometer using Parallel Accumulation Serial Fragmentation (PASEF) and four-dimensional feature alignment, the authors evaluated analytical robustness, reproducibility, and depth of coverage in a cohort of 192 patients with severe infections.

Methodology and Instrumentation

Blood plasma samples were depleted of the 12 most abundant proteins, digested with trypsin, and loaded (100 ng per sample) onto disposable Evotips for desalting and partial elution. The Evosep One system operated a 100-run-per-day gradient (11.5 minutes) delivering peptides to the timsTOF Pro, where ions were separated by trapped ion mobility spectrometry (TIMS) and fragmented by PASEF in data-dependent acquisition at ~100 Hz. Data processing in PEAKS Studio X applied a 4D feature matching algorithm across retention time, m/z, ion mobility (1/K0), and intensity to transfer identifications between runs and reduce missing values.

Used Instrumentation

• Evosep One high-throughput LC system with pre-formed gradients and Evotip trap columns.
• timsTOF Pro mass spectrometer (Bruker Daltonik) equipped with a TIMS device and PASEF capability.
• PEAKS Studio X software for feature alignment, identification (1% PSM FDR), and label-free quantification.

Main Results and Discussion

The workflow enabled processing of 192 patient samples plus 20 QC injections in 51 hours of LC-MS/MS time (212 runs). QC samples consistently yielded ~200 protein identifications with high stability (CV of identifications = 3.5%, quantification R² > 0.98, median CV = 9.3%). Patient samples showed larger biological variability (CV of identifications = 20.2%).

Applying four-dimensional alignment increased the median quantified protein groups per run from 188 to 500 in QC and from ~300 to ~478 in patient samples, without introducing drift over the sample sequence. Across all runs, 772 proteins were quantified, covering nearly five orders of magnitude in abundance. Notably, the workflow captured classic abundant plasma proteins (e.g., albumin, apolipoproteins), intermediate-abundance analytes (e.g., C-reactive protein), and low-abundance markers such as prostate-specific antigen and interferon-gamma.

Benefits and Practical Applications

• High throughput: 100 plasma samples per day at 11.5 minutes per run.
• Robustness: stable performance validated by regular QC injections.
• Depth of coverage: ~500 proteins per run without extensive fractionation or labeling.
• Reproducible label-free quantification suitable for large cohort studies.
• Capacity to detect mid- to low-abundance biomarkers for disease monitoring and discovery.

Future Trends and Opportunities

As cohort sizes expand, the four-dimensional feature alignment is expected to further increase proteome coverage and reduce missing data. Integration with targeted MS methods or data-independent acquisition could refine quantification of candidate biomarkers. Ongoing advances in instrument sensitivity, ion mobility separation, and software algorithms will likely enable routine clinical proteomics workflows for personalized diagnostics and longitudinal studies.

Conclusion

This work demonstrates a streamlined, high-throughput plasma proteomics platform combining the Evosep One and timsTOF Pro with PASEF and 4D feature alignment. The approach achieves robust quantification of ~500 proteins in 11.5-minute runs, supporting unbiased biomarker discovery and large-scale clinical studies with minimal sample input and without complex fractionation or labeling.

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