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Depletion of the highly abundant protein albumin from human plasma using the Gradiflow
Author(s) -
Rothemund Deborah L.,
Locke Vicki L.,
Liew Audrey,
Thomas Theresa M.,
Wasinger Valerie,
Rylatt Dennis B.
Publication year - 2003
Publication title -
proteomics
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.26
H-Index - 167
eISSN - 1615-9861
pISSN - 1615-9853
DOI - 10.1002/pmic.200390041
Subject(s) - chromatography , albumin , chemistry , isoelectric point , isoelectric focusing , mass spectrometry , electrophoresis , blood proteins , protein mass spectrometry , serum albumin , gel electrophoresis , tandem mass spectrometry , biochemistry , enzyme
Analysis of complex protein samples by two‐dimensional electrophoresis (2‐DE) is often more difficult in the presence of a few predominant proteins. In plasma, proteins such as albumin mask proteins of lower abundance, as well as significantly limiting the amount of protein that can be loaded onto the immobilized pH gradient strip. In this paper the Gradiflow, a preparative electrophoresis system, has been used to deplete human plasma of the highly abundant protein albumin under native and denatured conditions. A three step protocol incorporating a charge separation to collect proteins with an isoelectric point greater than albumin and two size separations to isolate proteins larger and smaller than albumin, was used. When the albumin depleted fractions were analysed on pH 3–10 2‐DE gels, proteins that were masked by albumin were revealed and proteins not seen in the unfractionated plasma sample were visualised. Matrix‐assisted laser desorption/ionisation‐time of flight mass spectrometry analysis confirmed the identification of the protein that lies beneath albumin to be C4B‐binding protein α chain. The liquid fractions from the Gradiflow separations were also analysed by liquid chromatography‐tandem mass spectrometry to confirm the proteins were separated according to their size and charge mobility in an electric field.