Separation of low molecular weight serum proteins using acetonitrile precipitation assessed by one dimensional gel electrophoresis

In human serum, low molecular weight (LMW) proteins play an important role in major biological processes which were masked by high molecular weight (HMW) proteins. Separations and identification of LMW proteins of interest from serum is very intricate task. The objective of the present study was to develop in-house acetonitrile (ACN) precipitation method for separation of LMW proteins from high abundance and HMW proteins in serum samples. Blood Samples (n=94) were collected from area of Vidharbha region of Maharashtra, India. Serum precipitation was done by using different dilutions of ACN. After standardization of ACN, 1:1 dilution was used and accordingly serum, supernatant and precipitate was used for one-dimensional sodium dodecyl sulfate/polyacrylamide gel electrophoresis (1D SDS-PAGE) for separation of proteins. Gel image was analyzed by using Gel analyzer 2010 software. It was noted that LMW proteins were present in supernatant in the molecular range of 42 kDa, 32 kDa, 24 kDa, 19 kDa, 5kDa whereas HMW proteins were found in precipitates at the range of 156kDa, 115 kDa, 102 kDa, 95 kDa, 72 kDa, 69 kDa, 58 kDa, 43 kDa. Results showed that HMW proteins present in precipitated and LMW proteins were concentrated in supernatant. In conclusion, a rapid ACN precipitation method has been developed that reproducibly depletes high abundance and HMW proteins from serum prior to mass spectrometry based proteomic approaches.