Middle-throughput LC-MS-based Platelet Proteomics with Minute Sample Amounts Using Semiautomated Positive Pressure FASP in 384-Well Format

Stefan Loroch; Eleftherios Panagiotidis; Kristoffer Klewe; Frauke Swieringa; Johan W.M. Heemskerk; Jan-Paul Lerch; Andreas Greinacher; Ulrich Walter; Kerstin Jurk; Tobias John; Katalin Barkovits; Thomas Dandekar; Katrin Marcus; Johannes Balkenhol

doi:10.1055/a-2516-1812

Thrombosis and Haemostasis, Table of Contents

Thromb Haemost
DOI: 10.1055/a-2516-1812

New Technologies, Diagnostic Tools and Drugs

Middle-throughput LC-MS-based Platelet Proteomics with Minute Sample Amounts Using Semiautomated Positive Pressure FASP in 384-Well Format

Stefan Loroch ^*

¹Medical Proteome-Center (MPC), Medical Faculty, Ruhr-University Bochum, Bochum, Germany

²Medical Proteome Analysis, Center for Protein Diagnostics (PRODI), Ruhr-University Bochum, Bochum, Germany

³Fa. Dr. Loroch/QC|MS, Biomedizin Zentrum, Dortmund, Dortmund, Germany

⁴Department of Bioanalytics, Leibniz-Institute for Analytical Sciences – ISAS – e.V., Dortmund Germany

,

Eleftherios Panagiotidis^*

⁴Department of Bioanalytics, Leibniz-Institute for Analytical Sciences – ISAS – e.V., Dortmund Germany

,

Kristoffer Klewe

³Fa. Dr. Loroch/QC|MS, Biomedizin Zentrum, Dortmund, Dortmund, Germany

,

Frauke Swieringa

⁵Department of Biochemistry, Cardiovascular Research Institute Maastricht (CARIM), Maastricht University, Maastricht, The Netherlands

⁶Synapse Research Institute, Platelet Pathophysiology, Maastricht, the Netherlands

,

Johan W.M. Heemskerk

⁵Department of Biochemistry, Cardiovascular Research Institute Maastricht (CARIM), Maastricht University, Maastricht, The Netherlands

⁶Synapse Research Institute, Platelet Pathophysiology, Maastricht, the Netherlands

,

Jan-Paul Lerch

⁷Fakultät für Mathematik, Universität Bielefeld, Bielefeld, Germany

,

Andreas Greinacher

⁸Institut für Immunologie und Transfusionsmedizin, Universitätsmedizin Greifswald, Greifswald, Germany

,

Ulrich Walter

⁹Center for Thrombosis and Hemostasis (CTH), Univeristy Medical Center Mainz, Mainz, Germany

,

Kerstin Jurk

⁹Center for Thrombosis and Hemostasis (CTH), Univeristy Medical Center Mainz, Mainz, Germany

,

Tobias John

¹Medical Proteome-Center (MPC), Medical Faculty, Ruhr-University Bochum, Bochum, Germany

²Medical Proteome Analysis, Center for Protein Diagnostics (PRODI), Ruhr-University Bochum, Bochum, Germany

,

Katalin Barkovits

¹Medical Proteome-Center (MPC), Medical Faculty, Ruhr-University Bochum, Bochum, Germany

²Medical Proteome Analysis, Center for Protein Diagnostics (PRODI), Ruhr-University Bochum, Bochum, Germany

,

Thomas Dandekar

¹⁰Department of Bioinformatics, Biocenter, University of Würzburg, Würzburg, Germany

,

Katrin Marcus

¹Medical Proteome-Center (MPC), Medical Faculty, Ruhr-University Bochum, Bochum, Germany

²Medical Proteome Analysis, Center for Protein Diagnostics (PRODI), Ruhr-University Bochum, Bochum, Germany

,

Johannes Balkenhol

¹⁰Department of Bioinformatics, Biocenter, University of Würzburg, Würzburg, Germany

› Author Affiliations

Abstract

Background

Comprehensive characterization of platelets requires various functional assays and analytical techniques, including omics disciplines, each demanding a separate aliquot of the given sample. Consequently, sample material for each assay is often highly limited, necessitating the downscaling of methods to work with just a few micrograms of platelet protein.

Materials and Methods

Here, we present a novel sample preparation platform for proteomics analysis using only 3 μg of purified platelet protein, corresponding to 2 × 10⁶ platelets, which can be obtained from approximately 2 to 8 μL of blood from a healthy individual (1.5 × 10⁵–4.5 × 10⁵ platelets/μL) or approximately 100 μL of blood from a patient with severe thrombocytopenia (<2 × 10⁴ platelets/µL).

Results

Using this platform, we detected a significant fraction of key players in the platelet activation cascade and, most importantly, identified 36 clinically relevant platelet disease markers even with a non-state-of-the art instrument. This makes LC-MS-based proteomics a highly attractive alternative to conventional assays, which often require milliliters of blood. Our platform transitions from our previously established 96-well proteomics workflow (PF96), which has been successfully employed in numerous platelet proteomics studies, into the 384-well format. This transition is accompanied by (1) a more than two-fold increase in sensitivity, (2) improved reproducibility, (3) a four-fold increase in throughput, allowing 1,536 samples to be processed per lab worker per week, and (4) reduced sample preparation costs.

Conclusion

Thus, LC-MS-based platelet proteomics offers a compelling alternative to immunoaffinity assays (which depend on antibody availability and quality), as well as to genomic assays (which can only reveal genotypes). In summary, in conjunction with recent advances in LC-MS instrumentation, our platform represents a highly valuable tool for rapid phenotyping of platelets in research with extraordinary potential for future employment in companion or routine diagnostics.

Keywords

proteomics - platelets - platelet disease markers - platelet signaling - throughput - PF96 - PF384 - 96-well format - 384-well format

Full Text

References

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Supplementary Material

Supplementary Material