Many basic
proteins (pI>7) and putative disease
biomarkers are not identified using conventional proteomic methods. This study applied a new method to improve the identification of such
proteins. Prefractionated basic
proteins were compared with total tissue lysates from human
ductal carcinoma in situ tissue loaded on basic immobilized pH gradient strips prior to two-dimensional gel electrophoresis (2-DE). Extraction of alkaline
proteins was achieved in less than 20 min using a chromatofocusing resin and two
buffers in a microcentrifuge tube. Prefractionation showed improved resolution and visualization of low-abundance
proteins on 2-DE
gels, allowing
proteins to be excised, accumulated,
trypsin-digested, and identified by liquid chromatography-tandem mass spectrometry.
Proteins identified in the prefractionated samples had a higher number of
peptides and three times the number of unique basic
proteins when compared with total lysates. Low-molecular-weight (LMW, <26kDa) unique alkaline
proteins comprise 75% of those identified in prefractionated samples compared with 25% identified in total lysates, representing a 9-fold increase of LMW
proteins due to prefractionation. Prefractionation ultimately increases loading capacity of samples onto the 2-DE gel and leads to better resolution, visualization, and identification of
proteins with pI values greater than 7.