Generation of serum fractions

After collection of the whole blood in standard serum tubes, the blood was allowed to clot by leaving it undisturbed at room temperature for 20 min. The clot was separated from the serum by centrifuging at 2000×g for 10 min in a refrigerated centrifuge. Serum was carefully removed by pipetting and stored at − 80 °C until further fractioning. 100 kDa, 50 kDa and 10 kDa Amicon Ultra-0.5 mL Centrifugal Filters (Merck) were pre-rinsed with 0.5 ml 0.1 M NaOH at 14,000×g for 30 min to avoid glycerine interference in the analysis. Next, filters were rinsed with distilled water by spinning 0.5 mL distilled water for 30 min at 14,000×g. Every 30 min wash was followed by spinning the device in the inverted position at 1000×g for 2 min to remove the residual solution contained in the filter. 0.5 mL serum was transferred to the 100 kDa filter and centrifuged at 14,000×g for 30 min. The filtrate of this centrifugation step contained molecules with < 100 kDa. The concentrate (remainder of the serum in the filter) was collected by placing the filter device upside down and spinning for 1000×g for 2 min. The concentrate contained molecules with > 100 kDa. The filtrate of the 100 kDa filter was then transferred to the 50 kDa filter and centrifuged at 14,000×g for 30 min. The filtrate of this centrifugation step contained molecules with < 50 kDa. The concentrate of the 50 kDa filter was collected by placing the filter device upside down and spinning for 1000×g for 2 min. The concentrate contained molecules with 50–100 kDa. The filtrate of the 50 kDa filter was transferred to the 10 kDa filter and centrifuged at 14,000×g for 30 min. The filtrate of this centrifugation step contained molecules with < 10 kDa. The concentrate of the 10 kDa filter was collected by placing the filter device upside down and spinning for 1000×g for 2 min. The concentrate contained molecules with 10–50 kDa and was stored at − 80 °C until Raman spectroscopy.