The plasma proteome is a complex sample matrix that contains a wealth of information and serves as a vast source for biomarker discovery. Analysis of the complex plasma proteome by techniques such as two-dimensional (2-D) gel electrophoresis and mass spectrometry is hindered by the presence of highly abundant proteins including albumin and immunoglobulins. Ultrafiltration (UF), with centrifugal devices, has been used to prepare low molecular weight (LMW, < 30kDa) samples for biomarker analysis.

In this report we describe the use of centrifugal UF devices to fractionate both, LMW and HMW, fractions from serum. Protein separation was accomplished by serial filtration through decreasing molecular weight cutoff (MWCO) devices from 100K, 50K, 30K, and 10K MWCO. This serial fractionation strategy enabled the enrichment of proteins by size as well as increasing throughput speed as compared to directly preparing samples using a lower MWCO device. Additionally, the relative purity of samples prepared by the serial fractionation strategy was greater than the relative purity of samples prepared using a single low MWCO device.

Analysis of the LMW proteome by 2-D gel electrophoresis can be improved by using proteins fractionated by size. The serial fractionations method enabled the enrichment of low abundance proteins in the LMW proteome. Further analysis may be done by peptide digestion and mass spectrometry. In conclusion, protein fractionation using centrifugal UF devices is a simple and rapid method to reduce sample complexity and enrich for the low abundance proteins based on molecular weight.

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