We propose to use cRFP (common Repository of FBS Proteins) in the MS (mass spectrometry) raw data search of cell secretomes. cRFP is a small supplementary sequence list of highly abundant fetal bovine serum proteins added to the reference database in use. The aim behind using cRFP is to prevent the contaminant FBS proteins from being misidentified as other proteins in the reference database, just as we would use cRAP (common Repository of Adventitious Proteins) to prevent contaminant proteins present either by accident or through unavoidable contacts from being misidentified as other proteins. We expect it to be widely used in experiments where the proteins are obtained from serum-free media after thorough washing of the cells, or from a complex media such as SILAC, or from extracellular vesicles directly.

In the hospital, blood samples are collected to monitor patients' health states, and thus various protein‐based clinical methods have been developed. However, some proteins are found to change in abundances during the process of blood collection and storage. In order to account such pre‐analytical effects, we performed liquid chromatography multiple reaction monitoring mass spectrometry (LC‐MRM‐MS) on 15 selected proteins in plasma samples prepared by varying storage time and temperature of whole blood prior to plasma isolation. Two cytosolic proteins, profilin‐1 (PFN1) and thymosin beta‐4 (TMSB4X), were absolutely quantified using 15N‐labeled recombinant proteins spiked externally. The other 13 proteins were quantified in a relative way compared with the two reference proteins. Triplicated LC‐MRM‐MS measurements showed that the median CV of MRM peak areas was 5.7%. The amounts of PFN1 and TMSB4X increased rapidly depending on the storage time between blood collection and plasma preparation. It indicates the leakage of cellular components into the plasma fraction. Relative quantification further revealed that five proteins including PFN1, S10A8, S10A9, S10A11, and TMSB4X showed significant difference (P < 0.05). We further monitored PFN1 and TMSB4X on 40 samples collected for protein diagnostics under a typical clinical study condition. Compared with the plasma samples prepared within a day, the level of both PFN1 and TMSB4X increased in the plasma samples prepared from the blood collected the day before and kept overnight at 4°C (0.51 to 3.11 μg/mL for PFN1 and 0.98 to 5.36 μg/mL for TMSB4X in average). Our result suggests an effort of assuring plasma quality for accurate protein‐based diagnosis or biomarker discovery and validation.