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Two‐dimensional liquid chromatography/tandem mass spectrometry analysis of Gradiflow™ fractionated native human plasma
Author(s) -
Wasinger Valerie C.,
Locke Vicki L.,
Raftery Mark J.,
Larance Mark,
Rothemund Deborah,
Liew Audrey,
Bate Irene,
Guilhaus Michael
Publication year - 2005
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.200401160
Subject(s) - chromatography , fractionation , chemistry , proteome , albumin , blood proteins , tandem mass spectrometry , mass spectrometry , sample preparation , human plasma , proteomics , trypsin , biochemistry , enzyme , gene
One of the major challenges facing protein analysis is the dynamic range of protein expression within massively complex samples (Corthals, G. L. et al.., Electrophoresis 2000, 21 , 1104–1115). In plasma this difference is as great as ten orders of magnitude, and this is currently beyond the range of detection achievable by any of the analytical techniques. Plasma has the additional challenge of having a few highly abundant proteins, such as albumin, which mask the detection of lower abundance and biologically significant proteins. The use of the Gradiflow BF400 as a fractionation tool to deplete highly abundant albumin from human plasma is reported here. A sequential three‐step protocol was performed on five plasma samples as part of the International Plasma Proteome Project organised by the HUPO; four containing different anticoagulants: EDTA, citrate, heparin and a control sample (NIBSC); and a serum sample. Plasma from an alternate source also underwent fractionation and served as an in‐house control. Time modulation between 1 and 7 h was observed for the depletion of albumin from these samples. Following albumin depletion, each fraction was trypsin‐digested and the peptides were fractionated further using a 2‐D LC‐MS/MS. Differences in the total number of proteins identified for each sample were also noted.