How can platelet proteomics best be used to interrogate disease?

Abstract Various modifications of proteins and the resulting proteoforms of a protein can associate with many diseases and are also significantly involved in the rapid regulation of hemostasis and thrombosis. For example, the release of prostacyclin from the intact endothelium and the consequent following phosphorylation of VASP in platelets is a post-translational regulation to keep them in a quiescent state. In Alzheimer’s disease, proteoforms arise from the altered cleavage of the amyloid precursor protein, which finally causes amyloid plaques in the brain. This changed processing of the amyloid precursor protein can also be detected in platelets, making them an attractive source of biomarkers for this neurodegenerative disease. Age-related or prothrombotic disorders can have multiple origins, including genomic, transcriptional, and translational factors, which together can be mapped at the proteome level. Hence, recording these dynamic protein changes under physiological and pathophysiological conditions is paramount in platelet proteomics. To effectively study diseases through platelet proteomics, it is crucial to consider platelets’ primary regulatory mechanism and thoroughly evaluate the disparities between the two leading proteomics technologies, top-down and bottom-up approaches. This commentary provides insights into the differences between these two technologies, which are particularly noticeable in detecting the different proteoforms of a protein. Plain Language Summary What is the context? The repertoire of all proteins in a biological sample is the proteome. Proteomics refers to different biochemical technologies that detect and quantify the proteins in a biological sample, such as platelets. If proteome analyses are carried out on a representative number of samples from a specific patient group and compared to a matched control group, disease-dependent changes in proteins can be found that indicate unknown causes of the disease or also be used as biomarkers for diagnosis and prognosis. It is also essential to consider that the proteins in biological samples can occur in various variations, the proteoforms. These proteoforms of a protein can arise, for example, through genetically-based variations or regulatory post-translational protein modifications. What’s new? There are two fundamentally different methods in proteomics technology: top-down and bottom-up. For bottom-up proteomics, the proteins must be digested into peptides for technical reasons, whereas top-down proteomics analyzes intact proteins. These different sample processing steps significantly impact the resulting data set. This is particularly crucial for platelet proteomics studies, as primary hemostasis is mainly carried out through post-translational modifications of proteins, resulting in various proteoforms that regulate platelet reactivity and thrombus formation. What’s the impact? Bottom-up proteomics can quickly and automatically identify an extensive repertoire of proteins from a platelet sample. This is much more cumbersome with top-down proteomics. In contrast, here, various intact proteoforms of intact proteins can be unbiasedly detected and directly quantified, which is particularly important for examining the global proteome of platelets in clinical samples. This qualitative and quantitative relative assignment to the respective proteoforms of a protein is not possible in bottom-up proteomics.