Qproteome murine albumin depletion kit

Albumin and immunoglobulins were separately removed from pooled sera of each gender subgroup using the Qproteome Murine Albumin Depletion Kit (Qiagen Inc., Valencia, CA) according to the manufacturer's protocol. Briefly, aliquots of 25 μL serum were applied to each depletion column and incubated for 5 min at room temperature. The depleted serum was collected by centrifugation at 500 ×g for 10 sec. Protein concentrations of the depleted serum samples were measured with a Bradford assay (Bio-Rad Laboratories Inc., Hercules, CA).

Plasma samples (150 μg) were depleted for albumin using the Qproteome Murine Albumin Depletion Kit with spin columns as described by the manufacturer (Qiagen, Germantown, MD). Depleted samples (average 61 μg) were desalted using four volumes of ice-cold acetone and incubated O/N at −20 °C. Protein concentrations in platelet and plasma samples were measured using the BCA Protein Assay Kit (Product # 23225, Pierce, Thermo Scientific, Rockford, IL) using a SpectraMax 96 well plate reader, SoftMax Pro Protein Quantitation and BCA software (Sunnyvale, CA). Platelet proteins (10 μg total in SDS-sample buffer) were separated on a 10% SDS-polyacrylamide gel. The gel was sliced crosswise into 23 bands and digested in-gel with trypsin^{25 (link)}. The sample preparation is described in detail in the supplementary methods section (S5).

PMs were lysed with RIPA buffer and then centrifuged at 15,000 rpm for 5 min at 4 °C. The supernatant was immunoblotted with anti-C1q antibody (Abcam, Clone: 9A7, ab71089). For the serum analysis, the serum was treated with the Qproteome Murine Albumin Depletion Kit (Qiagen). The samples were immunoblotted with anti-C1q antibody (Abcam, Clone: 9A7, ab71089).