Achieve Exceptional Longitudinal Performance in Clinical Plasma Proteomics

WHITE PAPER info@evotec.com | www.evotec.com 1 Evotec International GmbH, Anna-Sigmund-Str. 5, 82061 Neuried, Germany 2 Metabolomics and Proteomics Core, Helmholtz Zentrum München, Heidemannstraße 1, 80939 München, Germany Evotec’s industrialized Mass Spectrometry platform shows exceptional longitudinal performance for neat human plasma Authors: Oliver Kardell1, Till Kindel1, Frank Rolfs1, Barbara Kracher1, Andreas Tebbe1, Stefanie M. Hauck2, Carleen Kluger1 Executive summary Here, we highlight the robustness and excellent performance of our industrialized mass spectrometry platform for measuring neat plasma samples. We demonstrate the platform’s capability to monitor clinically relevant protein biomarkers longitudinally with high reproducibility and precision. By tracking FDA-approved biomarker proteins, we observed Abstract Enhancing mass spectrometry (MS)-based proteomics for clinical applications focuses on developing standardized, comprehensive, and purpose-specific workflows for clinical specimens. In this pursuit determining and optimizing various bioanalytical parameters such as selectivity, sensitivity, accuracy, and precision are imperative. In this study, we present a longitudinal quality assessment of MS-based proteomics measurements of plasma using our highly industrialized platform. We monitored key performance indicators, including sensitivity, reproducibility, and precision, by measuring an internal control plasma sample and p. 2 Graphical abstract Evotec’s industrialized Mass Spectrometry platform shows exceptional longitudinal performance for neat human plasma exceptional precision, with most markers exhibiting variability below 5% at all time points. These results underscore the robustness of our LC-MS measurements on clinical samples, as well as the long-term stability and reproducibility of our LC-MS platform for neat plasma measurements. Plasma Sample MSCoreSys control Internal control LC/MS platform Data Analysis Benchmarking Pipeline T1 T2 T3 an additional plasma control sample provided by the MSCoreSys consortium [1]. For both plasma control samples, consistent identifications, high reproducibility, and excellent precision were achieved. To demonstrate how this supports analysis of human patient samples, FDA-approved biomarker proteins were tracked, exhibiting an exceptional precision which was even below 5% for most markers at all time points. In conclusion, the results underscore the robustness of our LC-MS measurements on clinical samples as well as the long-term stability and reproducibility of the platform. Introduction Clinical proteomics focuses on analyzing protein profiles in clinical samples like blood, urine, and tissue to uncover disease mechanisms and improve diagnosis and treatment. Mass spectrometry plays a key role in the development of diagnostic tools, the identification of biomarkers and drug targets, and the expansion of knowledge regarding disease pathways. Successful clinical implementation of MS-based assays requires standardized, accurate, and reproducible workflows capable of handling large sample cohorts. Several multicenter studies have been conducted to evaluate the repeatability and reproducibility of MS-based proteomics measurements, both within and across laboratories [2–4]. In such collaborative effort, the MSCoreSys consortium measured plasma and serum samples in a longitudinal fashion and demonstrated excellent reproducibility for essential performance characteristics such as identifications, data completeness and precision. At Evotec, we received Materials and Methods Both the MSCoreSys plasma sample and the internal control plasma sample were prepared using the iST kit from PreOmics (PreOmics GmbH, Martinsried, Germany) according to manufacturer’s specifications. Resulting peptides were purified and evaporated after elution from the cartridge. The peptide pellets were stored at -20° until usage. LC-MS/MS measurements were performed on an Orbitrap Exploris 480 system in Data independent acquisition (DIA) mode. For each timepoint 5 replicate measurements were performed per sample. MS data was analyzed per timepoint and setup with Spectronaut (version 18.1) by searching against a human FASTA database (Swissprot and varsplic version 2022_05). For downstream analysis an internally developed custom R package for benchmarking (version 2.1) was used. Evotec’s industrialized Mass Spectrometry platform shows exceptional longitudinal performance for neat human plasma p. 3 the study’s plasma control sample and applied a similar methodology to monitor key performance indicators on our industrialized high-throughput LC-MS platform. Additionally, we measured an internal plasma control sample obtained from pooled healthy human donors on the same LC-MS system. To mimic a longitudinal plasma proteomics study, in which either visits from individual patients occur over time or sub-parts of a clinical cohort can arrive at multiple consecutive timepoints, samples were measured at three different time points spaced 3–4 months apart. Furthermore, both plasma control samples were prepared without any enrichment, depletion, or fractionation workflow. All generated MS data was analyzed per timepoint and plasma control sample, respectively. Overall, the experimental design allowed us to assess the consistency and reliability of our LC-MS platform for plasma measurements as well as the stability of the plasma samples over an extended period of time up to one year. Results The longitudinal study was performed using Evotec’s high-throughput Mass Spectrometry platform. To evaluate robustness of the platform, two distinct plasma control samples were measured – one provided by the MSCoreSys consortium [1] and one internal control plasma from pooled healthy donor samples. For each sample type, measurements of 5 replicates were conducted 3–4 months apart at three different timepoints. Evotec’s industrialized Mass Spectrometry platform shows exceptional longitudinal performance for neat human plasma p. 4 Figure 1: Identifications (I) and data completeness (II) for the MSCoreSys plasma measurements (a) and for the internal control plasma measurements (b) on protein group- and peptide-level, respectively. Complete profiles refer to features present in all replicate runs, shared with at least 50% to be at least present in 50% of the replicate runs, sparse to be present in more than one run and less than 50% of the runs, and unique to be only present in one replicate run. First, key characteristics such as number of identifications (IDs) and data completeness were evaluated. In detail, the measurements of the MSCoreSys plasma control achieve a median of 531 protein identifications with a data completeness ranging between 85%–99% and the measurements of the internal control plasma sample reach a median of 546 proteins and a data completeness between 95%–99% for the respective timepoints (Fig. 1). MSCoreSys control Internal control Evotec’s industrialized Mass Spectrometry platform shows exceptional longitudinal performance for neat human plasma p. 5 In addition, tracking quantitative precision over time was a crucial aspect in assessing the performance of the setup. As displayed in Figure 2, the median quantitative precision of label-free quantification (LFQ) ranges between 4.5%–5% for the MSCoreSys plasma measurements for the respective timepoints. The coefficient of variation (CV) for the internal Figure 2: Quantitative precision for all quantified protein groups (I) and focused on quantified FDA-approved biomarkers [5] (II) for the MSCoreSys plasma measurements (a) and for the internal control plasma measurements (b). MSCoreSys control Internal control control plasma measurements spans 3.9%–4.8% across the timepoints. Furthermore, all quantified proteins were matched to FDA-approved biomarkers [5] and their quantitative precision was monitored across timepoints. These biomarkers exhibit CV values well below 20%. Evotec’s industrialized Mass Spectrometry platform shows exceptional longitudinal performance for neat human plasma p. 6 Discussion This study aimed to evaluate the performance of plasma measurements over time by examining the variability in key metrics such as ID count, data completeness, and quantitative precision. Additionally, by utilizing different plasma samples, we assessed the robustness of the overall LC-MS plasma measurements and established a baseline for the performance without any enrichment, depletion, or fractionation workflow. Both plasma control measurements demonstrated excellent consistency in their identifications for each timepoint being mostly above 85% up to 99% both on protein group- and peptide-level. Furthermore, protein group detections range between 500–550 protein groups. Focusing on precision, in both cases the measurements exhibited outstanding performance. The Orbitrap Exploris 480 system yielded median precision values of less than or equal 5% across all quantified protein groups at all time points for both plasma control samples. Additionally, known FDA-approved biomarker proteins were tracked and showed excellent performance with CV values significantly below the 20% threshold, which is a recognized requirement for in-vitro diagnostic assays. In total, these results emphasize the robustness and reliability of our LC-MS platform in consistently identifying and quantifying protein groups with high precision in measuring clinical specimens such as plasma. Conclusions We previously showed Evotec’s capabilities to analyze large-cohorts of clinical specimen in unprecedented depth with the Proteograph technology and demonstrated the longitudinal reproducibility of our applied pipeline (Nanoparticle-Based Proteomics Analysis of Human Biofluid Samples From Different Patient Cohorts – Evotec). Here, we focus on the robustness and excellent performance of our industrialized high-throughput LC-MS platform for measuring neat plasma samples and we showcase how we can monitor clinically relevant protein biomarkers in a longitudinal fashion with very high reproducibility and precision. Acknowledgements We would like to explicitly thank our partners from the Metabolomics and Proteomics Core from the Helmholtz Zentrum for providing the samples of the MSCoreSys longitudinal study. Evotec’s industrialized Mass Spectrometry platform shows exceptional longitudinal performance for neat human plasma p. 7 References [1] Kardell et. al, Multicenter longitudinal quality assessment of MS-based prpteomics in plasma and serum, J. Proteome Res. 2025. [2] Bennet et. al, The 2012/2013 ABRF Proteomic Research Group Study: Assessing Longitudinal Intralaboratory Variability in Routine Peptide Liquid Chromatography Tandem Mass Spectrometry Analyses. Mol Cell Proteomics 2015. [3] Collins et. al, Multi-laboratory assessment of reproducibility, qualitative and quantitative performance of SWATH-mass spectrometry, Nature communications 2017. [4] Kardell et. al, Multicenter Collaborative Study to Optimize Mass Spectrometry Workflows of Clinical Specimens, J. Proteome Res. 2023. [5] Anderson N., The clinical plasma proteome: a survey of clinical assays for proteins in plasma and serum, Clinical chemistry 2010. 2025/05 | V1 Evotec SE Manfred Eigen Campus Essener Bogen 7 22419 Hamburg (Germany) info@evotec.com | www.evotec.com Learn more: