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Plasma Proteomics for Epidemiology: Increasing Throughput With Standard-Flow Rates.
Xiaoke Yin1, Ferheen Baig1, Eloi Haudebourg1
1From the King's British Heart Foundation Centre, King's College London, United Kingdom (X.Y., F.B., E.H., H.H., M.M.); Agilent Technologies Ltd, Cheadle, United Kingdom (R.T.B., A.S.); Biognosys AG, Schlieren, Switzerland (T.G., S.M., L.R.); Department of Neurology, Medical University of Innsbruck, Austria (R.P., J.W., S.K.); School of Medicine, University of California San Diego (S.T., J.L.W.); and Department of Laboratory Medicine, Bruneck Hospital, Italy (P.S.).
Standard-flow liquid chromatography–mass spectrometry (LC-MS) enhances throughput for large-scale plasma proteomics in epidemiological studies. This method offers improved reproducibility over nanoflow LC-MS but requires more sample volume for comparable sensitivity.
Area of Science:
- Proteomics
- Analytical Chemistry
- Epidemiology
Background:
- Mass spectrometry (MS) enables sensitive and selective quantification of plasma proteins.
- Nanoflow liquid chromatography (LC) coupled with MS is common but limits sample throughput, reproducibility, and robustness, restricting study sizes in plasma proteomics.
- Large-scale epidemiological studies require high-throughput, reproducible methods for plasma protein quantification.
Purpose of the Study:
- To evaluate a standard-flow LC-MS method using multiple reaction monitoring (MRM) for large-scale epidemiological cohorts.
- To compare the performance of standard-flow LC-MS with nanoflow LC-MS in terms of run time, reproducibility, and quantification accuracy.
- To assess the suitability of standard-flow LC-MS for high-throughput plasma proteomics.
Main Methods:
- Standard-flow LC-MS with multiple reaction monitoring (MRM) was employed.
- LC-MS run times were reduced significantly compared to nanoflow LC-MS.
- Quantification of 100 plasma proteins was performed in over 1500 LC-MS runs.
- Retention time stability and protein measurement variation were compared between standard-flow and nanoflow LC-MS.
- Apolipoprotein measurements were compared using two commercial MRM kits and an antibody-based multiplexing kit.
Main Results:
- Standard-flow LC-MS reduced run times by approximately two-thirds compared to nanoflow LC-MS.
- Retention time stability was substantially improved with standard-flow LC-MS (SD < 0.05 min) versus nanoflow LC-MS (0.26–0.44 min).
- Standard-flow LC-MS exhibited less variation in protein measurements.
- Achieving similar sensitivity with standard-flow LC-MS required five times more sample volume.
- Good agreement was found between two MRM kits, but some MRM measurements differed from antibody-based assays.
Conclusions:
- Standard-flow LC-MS, combined with multiplexing, enhances throughput and reduces costs for large-scale protein measurements in epidemiological studies.
- The method offers improved reproducibility and speed for plasma proteomics.
- Accurate absolute proteoform quantification in large cohorts will necessitate the use of protein standards rather than peptide standards.
