Building Standards for Proteomics: A Targeted Mass Spectrometry Approach for Quantification of Cardiovascular Disease Biomarker in Human Blood Albert Sickmann (Presenter) ISAS Presenter Bio: Albert Sickmann is the director of the Leibniz Institute for Analytical Sciences (ISAS e.V.) and head of the department “Bioanalytics“. Furthermore, he is professor at the Ruhr-University Bochum (Faculty of Medicine) and the University of Aberdeen. His research interests are the highly complex signalling and metabolic pathways in cells and cell layers in the human body at different levels by developing and using techniques to measure signalling pathways with spatial and temporal resolution.

Authors: Christin Lorenz, Sebastian Malchow, Christina Looße and Albert Sickmann
Leibniz-Institut für Analytische Wissenschaften – ISAS – e.V.

Platelets are primarily known to be key players in thrombosis and hemostasis. Targeting platelet function and signaling may represent novel therapeutic strategies in prevention of cardiovascular diseases. The absolute quantification of phosphorylation sites in actived platelets can direct the establishment of new diagnostic assays by characterization of samples with clinical relevance, including quantification of platelet receptors and signaling proteins and their regulation via posttranslational modifications in human blood.

Introduction

Today, only a few protein biomarkers are used clinically in routine diagnostic strategies to detect e.g. cardiovascular diseases. When casting valid clinical biomarkers, the appropriate study design, sample type, and analytical method is of high relevance. To reveal the molecular mechanism of cardiovascular diseases in detail and to develop new diagnostic strategies, a thorough identification and characterization of potential biomarkers in human blood is required. Over the last years, mass spectrometry has gained significant attention for relative and absolute quantification of proteins in different matrices, as well as post-translational modifications (PTMs).

Methods

We applied a standardized workflow for quantification of cardiovascular disease biomarker in human platelets (Malchow et al. 2017). In detail, platelets are isolated and lyzed, followed by a tryptic protein digest. Subsequently, a mix of stable isotope-labeled peptides of interesting biomarker proteins in concentrations ranging from 0.1 to 100 fmol is added to 3 µg digest. These peptides are used as an internal calibration curve to accurately quantify endogenous peptides and corresponding proteins by nanoLC-MS/MS. In order to assure a valid quantification, limit of detection (LOD) and limit of quantification (LOQ), as well as linear range, were determined.

Results

Through advances in sample preparation, mass spectrometry instrumentation, and data analysis software, the multiplexed absolute quantification of proteins via proteotypic peptides in large sample cohorts became feasible. In collaboration with clinicians, well-characterized samples (i.e., of known gender, age, diseases, treatments/daily medications, and lifestyle) as well as appropriate controls need to be selected. Furthermore, the number of tested samples needs to be defined properly to gain statistical relevance and to detect changes confidently. The mass spectrometry-based quantification of proteins needs to be an important methodology to elucidate quantitative changes in health and disease. Hundreds of proteins can be quantified by using a targeted mass spectrometry approach. In order to gain reproducible and reliable results, similar requirements are demanded of the technical part, e.g., used methods and workflows need to be standardized. Setting up a standardized method includes method validation to ensure accuracy, precision, sensitivity, specificity, and linearity. Defining and characterizing these parameters is the key to constant quality and validity, which is crucial to a routine laboratory.

Conclusions & Discussion

This study aims to establish targeted mass spectrometry based assays for quantitative analysis of protein quantities and phosphorylation level in resting and activated platelets The absolute quantification of proteins and their phosphorylation allows the characterization of potential marker proteins representing an activation state of platelets associated with disease mechanisms. Thus, supporting the development of more sensitive and more selective mass spectrometry based diagnostic tests.

Malchow S., Loosse C., Sickmann A., Lorenz C. Quantification of Cardiovascular Disease Biomarkers in Human Platelets by Targeted Mass Spectrometry. Proteomes 2017, 15;5(4).

Peng B, Geue S, Coman C, Münzer P, Kopczynski D, Has C, Hoffmann N, Manke MC, Lang F, Sickmann A, Gawaz M, Borst O, Ahrends R. Identification of key lipids critical for platelet activation by comprehensive analysis of the platelet lipidome.

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