Novel capillary-flow LC HRAM MS platform for fast targeted analysis and robust profiling of complex samples

Significance of the topic

Capillary-flow liquid chromatography mass spectrometry combines high sensitivity with high throughput, reducing solvent consumption and contamination relative to analytical flow, while offering better robustness and ease of use compared to nano-flow. This makes capillary-flow LC-MS appealing for routine high-resolution accurate-mass proteomics and targeted quantification of complex biological samples.

Objectives and study overview

This study describes a novel capillary-flow LC-MS platform that integrates a Thermo Scientific capillary-flow UltiMate 3000 RSLCnano system, a 150 µm ID EASY-Spray column, and a Q Exactive HF-X mass spectrometer. The authors evaluate performance in shotgun proteomics using full-scan and data dependent acquisition, and targeted analysis using parallel reaction monitoring, covering complex samples such as HeLa cell digests and crude plasma.

Methods and instruments

• Chromatography: UltiMate 3000 RSLCnano in pre-concentration or direct injection modes, flow rates of 1.2 µL/min for sensitivity and 3 µL/min for throughput, 150 µm x 150 mm C18 EASY-Spray column with 2 µm particles.
• Mass spectrometry: Q Exactive HF-X with EASY-Spray source, full-scan MS1 resolution 60 000, injection time 100 ms; MS2 resolution 7500, injection time 35 ms, TopN up to 40 for DDA; PRM settings optimized for trastuzumab peptides.
• Data analysis: Thermo Scientific Xcalibur, TraceFinder, Skyline for PRM; Proteome Discoverer for DDA data processing.

Key results and discussion

• Sensitivity gains: capillary-flow operation yields up to 40-fold higher signal than analytical flow at 450 µL/min and is within 2 to 4 times the sensitivity of nano-flow at 300 nL/min when loading equal sample amounts.
• Chromatographic performance: peak widths at half maximum of about 3 seconds at 3 µL/min and less than 15 seconds at 1.2 µL/min, translating to peak capacities around 300 for both fast and longer gradients.
• Robustness: retention time reproducibility with RSD below 1% over 350 consecutive injections and MS1 peak area stability with RSD below 10% over 150 injections without internal standards.
• Shotgun proteomics: identification of over 3500 proteins and 20000 peptides from 1 µg of HeLa digest in 60 minutes, increasing to over 4300 proteins with higher loading (up to 5 µg) without loss of chromatographic quality.
• Depth of coverage: HeLa and plasma proteomes span four orders of magnitude in abundance; crude plasma digest yields over 200 protein groups and almost 2000 peptides in single-shot DDA.
• Targeted quantification: baseline separation of trastuzumab signature peptide and its deamidated isomers within a 12 minute cycle; linear quantification range from 500 ng to 500 µg/mL in plasma, corresponding to a detection limit of about 2 fmol on column.

Benefits and practical applications

The described platform offers a balance of speed, sensitivity, and robustness suited for routine profiling of complex proteomes, high-throughput targeted quantification of biotherapeutics, biomarker monitoring, and quality control in industrial and academic laboratories.

Future trends and applications

• Further reduction of analysis times through higher acquisition rates and multiplexed methods.
• Integration with immunodepletion or orthogonal fractionation for deeper plasma profiling.
• Expansion to other biofluids and post-translational modification analyses.
• Automation and large-scale clinical proteomics using capillary-flow workflows.

Conclusion

The novel capillary-flow LC-MS platform effectively combines high throughput with near-nano-flow sensitivity, delivers excellent robustness and reproducibility, and supports both comprehensive shotgun proteomics and targeted PRM quantification, making it a versatile tool for modern analytical laboratories.

References

1. Boychenko A, Meding S, Samonig M, Swart R. Sensitive, Fast and Robust Quantification of Antibodies in Complex Matrices by Capillary Flow UHPLC and High-Resolution Accurate-Mass MS. ASMS 2016, Poster Note PN64787.
2. Boychenko A, Meding S, Decrop W, Baynham M, Ruehl M, Wong JMT, Markus M, Swart R. Deep and Reproducible Human Proteome Profiling with Novel Nano Flow LC Technology and HRAM Mass-Spectrometry. Pittcon 2017, Poster PO72267.
3. Bults P, Bischoff R, Bakker H, Gietema JA, van de Merbel NC. LC-MS/MS-Based Monitoring of In Vivo Protein Biotransformation: Quantitative Determination of Trastuzumab and Its Deamidation Products in Human Plasma. Anal Chem. 2016;88(3):1871–1877.