Standardized workflows for precise high-throughput proteomics of blood biofluids

Standardized workflows for precise high-throughput proteomics of blood biofluids

Innovations in proteomics methodologies have emerged in recent years, but there are still obstacles inhibiting the translation of biomarkers into clinical use. Streamlined preanalytical proteomic workflows that achieves sensitive and robust analysis of multiple complex blood-based matrices with sufficient reliability for large population-based screening are therefore needed.

A new paper by the Van Eyk group from Cedars-Sinai Medical Center in Los Angeles describes the development of such a workflow for three blood-based proteomes: naive plasma, plasma depleted of the 14 most abundant proteins, and whole blood utilizing the 60 samples per day methods on the Evosep One coupled to a Thermo Orbitrap Exploris 480 MS.

Commercially available biofluids were subjected to protein denaturation, reduction, alkylation and digestion using a Beckman Biomek i7 automated workstation. Initially, they assessed the impact of temperature and time on trypsin proteolytic efficiency and found a 4-hour digestion at 42°C in the presence of 5% TFE to be most suited for maximizing throughput and efficiency.

Optimal parameters for DIA acquisition parameters for plasma

To ensure reproducibility throughout the entire workflow, they also established optimal conditions for DIA analysis in plasma and adapted this to depleted plasma and whole blood. They tested eight different MS methods covering four isolation window widths and two resolution settings, as the instrument cycle time is influenced by resolution settings and number of mass windows which in turn impact the number of identifications and reproducibility.

They found the number of protein identifications across all parameters to be comparable with nearly 200 proteins identified. While one method supported the highest number of peptide identifications compared to the other methods, it performed worst in terms of reproducibility on protein level. Thus, they decided to base their workflow on the method providing the highest number of quantified peptides with a coefficient of variation below 30%, namely the 21 Da method and 50 windows with a resolution of 15,000. Importantly, data acquired using their method were associated with more than 8 data points across the curve, a well-established benchmark for acceptable quantification. 

Precision of standardized workflow

They applied their optimized protocol and compared the number of identifications along with the intra- and inter-day reliability of the workflow on three commonly analyzed biofluids: naive plasma, depleted plasma, and whole blood. To determine the analytical precision, five replicate samples of each biofluid were digested once a day for three consecutive days and analyzed. This analysis revealed that most peptides were identified with a coefficient of variation below 30% on individual days. Comparing across all three days, plasma showed the greatest drop in peptide intensity reproducibility with only 74% of all peptides achieving a coefficient of variation below 30%, compared to 93% and 87% of peptides in depleted plasma and blood, respectively.

Collectively, this paper describes the development of a streamlined process of blood biofluids covering a broad dynamic range associated with minimal hands-on time, fast turnaround, and excellent intra- and inter-day stability. Interestingly, the inter-day precision of depleted plasma was the most stable with more than 90% of peptides reproducibly detected across days. The implementation of standardized workflows like this on a large scale will facilitate the translation of candidate markers into clinical use.
Link: https://www.biorxiv.org/content/10.1101/2021.03.26.437268v1.full.pdf

More research on plasma

Here you can see publications available on plasma research featuring Evosep One. For a full overview of publications published using the Evosep One Technology visit our Literature room here

TitleSubjectMaterialYearSummaryEvosep methodMS instrumentationLearn More
Plasma proteomics with Evosep One2021In this webinar you will learn how the Evosep One, with high reproducibility and robustness, is ideally suited for analysis of large sample cohorts of blood plasma.

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Immunophenotyping assessment in a COVID-19 cohort (IMPACC): A prospective longitudinal study, 2021This resource by the IMmunoPhenotyping Assessment in a Covid-19 cohort (IMPACC) describes a longitudinal study designed to enroll 1000 hospitalized patients with Covid-19. The Evosep One will be used to analyze depleted plasma samples.

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A blood atlas of COVID-19 defines hallmarks of disease severity and specificity, , 2021This publication is a major collaboration led by the Knight group at University of Oxford describing the hallmarks of Covid-19 severity and specificity. It is an integrative multi-omics analysis, where the Evosep One was used for measuring 340 plasma proteomes providing excellent patient stratification.

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Standardized workflow for precise mid- and high-throughput proteomics of blood biofluids, , 2021In this seminar from the Canadian National Proteomics Network (CNPN) meeting 2021, Angela McArdle presents a standardized workflow for precise high-throughput proteomics of blood biofluids. They established optimal conditions for sample preparation and DIA analysis in plasma, then automated and adapted this for depleted plasma and whole blood.

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The COVIDome Explorer Researcher Portal, , , 2021Researchers from University of Colorado led by Joaquin M. Espinosa, have created a user-friendly researcher portal enabling easy access and real-time analysis of matched multi-omics datasets for COVID-19, termed the COVIDome Explorer. They illustrate the use through a multi-omics analysis of biosignatures associated with C-reactive protein (CRP), an established marker of poor prognosis in COVID-19.

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A robust mass spectrometer for precision medicine – the Orbitrap Exploris 240 mass spectrometer for large-scale plasma protein profiling, 2021This application note from Thermo Fisher Scientific Precision Medicine Science Center demonstrates the reliability and robustness of the Evosep One coupled with the Orbitrap Exploris 240 MS for large-scale, untargeted plasma protein profiling. They observed excellent reproducibility of protein and peptide identifications over 100 injections of human serum performed five weeks apart.

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Integrative analysis of cell state changes in lung fibrosis with peripheral protein biomarkers, , , 2021In this study, the Theis and Schiller groups at the Helmholtz Zentrum München, investigated the correspondence of cell state changes in diseased organs to peripheral protein signatures in pulmonary fibrosis patient cohorts. From plasma proteome profiling of more than 122 patients, they propose CRTAC1 protein levels in plasma as a novel biomarker.,

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Seroconversion stages COVID19 into distinct pathophysiological states, , 2020In this study, researchers from University of Colorado led by Joaquin M. Espinosa, found highly variable seroconversion status among hospitalized Covid-19 patients. Their results support the existence of distinct pathophysiological states, with seroconversion status being potentially useful as a surrogate marker of underlying processes.

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Gradient off-set focusing HPLC insturment for robust and high throughput clinical proteomics, , 2017Evosep One for clinical analysis

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A Novel LC System Embeds Analytes in Pre-formed Gradients for Rapid, Ultra-Robust Proteomics, , , 2018Description of Evosep

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Robust and Reprodusible Protein Quantification in Plasma Using the Evosep One and the Agilent 6495 Triple Quadrupole LC/MS, , 2020Agilent feasibility study

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High dynamic range proteome analysis with BoxCar DIA and super-resolution Orbitrap mass spectrometry, , 2020BoxCar DIA,

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Clinical proteomics, 2018Clinical proteomics poster

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Development of a Novel LC Concept for Clinical Proteomics, , 2018Description of Evosep One

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MaxQuant software for ion moiblity enhanced shotgun proteomics, , 2020MaxQuant for timsTOF analysis

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Plasma proteomics goes high-throughput – timsTOF Pro with PASEF and 4D feature alignment to quantify 500 plasma proteins in 11.5 min, , 2019The timsTOF Pro with PASEF and the Evosep One for biomarker discovery in large sample cohorts of human blood plasma (blood plasma from 192 severe infection patients).

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Consistency, consistency – Automated Sample Prep for Translational Proteomics, 2020In this webinar, Emily Chen, Sr. Director at the Thermo Fisher Precision Medicine Science Center presents an automated, robust and scalable sample preparation pipeline for large-scale clinical research samples.

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An Ultra High-Throughput Plasma Protein Profiling (uHTPPP) Workflow Using a Modified Quadrupole-Orbitrap Mass Spectrometer, , , , , 2020In this application note, the group of Emily Chen at the Thermo Fisher Precision Medicine Science Center describes a high-throughput plasma and serum proteomics analysis workflow for large population cohort studies that utilizes a standardized sample preparation method, high-throughput data acquisition, and easy to implement QC standard.,

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Scalable and Automated Plasma Workflow Based on the Thermo Scientific Q Exactive HF-X MS Platform, , , , 2019In this application note, the group of Emily Chen at PMSC describes a high-throughput plasma and serum proteomics analysis workflow for large population cohort studies that utilizes a standardized sample preparation method, high-throughput data acquisition, and easy to implement QC standard., ,

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A paired liver biopsy and plasma proteomics study reveals circulating biomarkers for alcohol-related liver disease, , , 2020This publication from Matthias Mann groups in Copenhagen and Münich describes a paired liver biopsy and plasma proteomics study, which make use of Boxcar DIA scanning for a large clinical cohort of nearly 600 patients. They have developed a machine learning model based on their biomarker panel, which for the first time outperforms existing tests, …

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Fast Confirmatory Analysis of Rhuepos in Plasma for Horseracing Doping Control, , 2019This application note describes a rapid targeted analysis monitoring two tryptic rHuEPO peptides in plasma for horseracing doping control. The results illustrate the promising capabilities of the Evosep One innovative technology for the analysis of protein/peptide-based drugs in the context of drug testing.

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High throughput plasma analysis

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THE 60 SAMPLES PER DAY METHOD

Among our five standard methods this is the most popular method with an excellent balance of throughput and depth. 

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