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.
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
|Title||Subject||Material||Year||Summary||Evosep method||MS instrumentation||Learn More|
|Standardized workflow for precise mid- and high-throughput proteomics of blood biofluids||Clinical research, DIA, Plasma||Publication||2021||The Van Eyk group presents a standardized workflow for precise high-throughput proteomics of blood biofluids using the 60 SPD method. They established optimal conditions for sample preparation and DIA analysis in plasma, then automated and adapted this for depleted plasma and whole blood.||60 SPD||Thermo Orbitrap Exploris 480|
|The COVIDome Explorer Researcher Portal||Clinical research, Covid-19, DDA, Plasma||Publication||2021||Researchers 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.||30 SPD||Bruker timsTOF Pro|
|A robust mass spectrometer for precision medicine – the Orbitrap Exploris 240 mass spectrometer for large-scale plasma protein profiling||DDA, Plasma||Application note||2021||This 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.||60 SPD||Thermo Orbitrap Exploris 240|
|Integrative analysis of cell state changes in lung fibrosis with peripheral protein biomarkers||Clinical research, DIA, Fibrosis, Plasma||Publication||2021||In 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.||100 SPD, 60 SPD||Thermo Q Exactive HF|
|Seroconversion stages COVID19 into distinct pathophysiological states||Clinical research, Covid-19, Plasma||Publication||2020||In 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.||30 SPD||Bruker timsTOF Pro|
|Gradient off-set focusing HPLC insturment for robust and high throughput clinical proteomics||Clinical research, Plasma, Technology||Application note||2017||Evosep One for clinical analysis|
|A Novel LC System Embeds Analytes in Pre-formed Gradients for Rapid, Ultra-Robust Proteomics||DDA, DIA, Plasma, Technology||Publication||2018||Description of Evosep||60 SPD||Thermo Q Exactive HF-X|
|Robust and Reprodusible Protein Quantification in Plasma Using the Evosep One and the Agilent 6495 Triple Quadrupole LC/MS||Plasma, Targeted workflow, Technology||Application note||2020||Agilent feasibility study||60 SPD||Agilent 6495 Triple Quadrupole|
|High dynamic range proteome analysis with BoxCar DIA and super-resolution Orbitrap mass spectrometry||DIA, Plasma, Technology||Poster||2020||BoxCar DIA||100 SPD, 30 SPD||Thermo Orbitrap Exploris 480|
|Clinical proteomics||Clinical research, Plasma||Poster||2018||Clinical proteomics poster||Thermo|
|Development of a Novel LC Concept for Clinical Proteomics||DDA, Plasma, Technology||Poster||2018||Description of Evosep One||60 SPD||Thermo|
|MaxQuant software for ion moiblity enhanced shotgun proteomics||DDA, Plasma, Technology||Publication||2020||MaxQuant for timsTOF analysis||100 SPD||Bruker timsTOF Pro|
|Plasma proteomics goes high-throughput – timsTOF Pro with PASEF and 4D feature alignment to quantify 500 plasma proteins in 11.5 min||Clinical research, DDA, Plasma||Application note||2019||The 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).||100 SPD||Bruker timsTOF Pro|
|Consistency, consistency – Automated Sample Prep for Translational Proteomics||Clinical research, Plasma||Video||2020||In 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.||60 SPD||Thermo|
|An Ultra High-Throughput Plasma Protein Profiling (uHTPPP) Workflow Using a Modified Quadrupole-Orbitrap Mass Spectrometer||Clinical research, DDA, Lung, Plasma, Serum, Tissue||Application note||2020||In 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.||30 SPD, 60 SPD||Thermo Orbitrap Exploris 240|
|Scalable and Automated Plasma Workflow Based on the Thermo Scientific Q Exactive HF-X MS Platform||Clinical research, DDA, Lung, Plasma, Serum||Application note||2019||In 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.||100 SPD, 30 SPD, 60 SPD||Thermo Q Exactive HF-X|
|A paired liver biopsy and plasma proteomics study reveals circulating biomarkers for alcohol-related liver disease||Clinical research, DIA, Liver, Plasma||Publication||2020||This 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, …||30 SPD||Thermo Q Exactive HF-X|
|Fast Confirmatory Analysis of Rhuepos in Plasma for Horseracing Doping Control||Plasma, rHuEPO, Targeted workflow||Application note||2019||This 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.||100 SPD||Thermo Q Exactive HF|
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