Abstract DP041: Proteomics of Ischemic Stroke Thrombi: Optimizing Minimum Sample Input Requirements from Rat Models to Human Clots

Background: Proteomics of clots retrieved during mechanical thrombectomy (MT) enables biomarker discovery, insight into clot composition and formation, clot etiology differentiation, and development of clot removal or resolving treatments targeted to molecular signatures. Because clots are heterogeneous and tissue availability is limited, strategic allocation is essential. Establishing minimum protein input for proteomics is critical to reduce heterogeneity, prevent sample waste, and maximize clot samples for parallel omics investigations. Objective: To define minimum clot sample input for robust thrombus proteomics using protein estimation and mass spectrometry of rat blood thrombi, creating a standardized workflow for optimal sample prep and use. Methods: Blood from male Sprague Dawley rats (8-10 wk) was collected from cardiac puncture under approved ACUC protocol. Thrombi were formed in centrifuge tubes by mixing blood with 0.06% CaCl2, incubated at 37°C for 5 h, and stored at 4°C overnight , followed by storage in -80°C in RNA solution until use. Rat thrombi are stratified by weight with rat thrombi smaller than human thrombi, so we employed: small < 5 mg, medium 5-15 mg, large >15 mg. Samples are homogenized and lysed using a detergent-containing buffer, followed by centrifugation for protein extraction. Protein concentration was estimated by densitometry against a BSA standard curve, enzymatically digested, followed by detergent removal and iTRAQ mass spectrometry (MS). Adequacy thresholds are defined a priori based on protein yield, signal-to-noise ratios, and reproducibility metrics. Results: Minimum protein yield and tissue mass for reproducible proteomic coverage were established. Average protein concentrations for small, medium, and large samples were 3.39 mg/mL, 8.82 mg/mL , and 10.30 mg/mL, respectively. iTRAQ MS confirms minimal concentration and thresholds for proteome depth, spectral match reproducibility, and signal-to-noise ratios across varying input sizes. These findings will provide the first input–output curve for clot proteomics establishing reproducibility applicable to human samples. Conclusions: Establishing minimum input requirements enables reproducible clot proteomics and preserves material. We will shortly apply this standardized protocol to our current human clot library (retrieved via MT from ~20 varied etiology stroke patients) ensuring reproducible proteomics and biomarker discovery with optimal sample use for multi-omics.