Two-dimensional liquid chromatography/tandem mass spectrometry analysis of Gradiflow fractionated native human plasma.

Abstract:

: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.

journal_name

Proteomics

journal_title

Proteomics

authors

Wasinger VC,Locke VL,Raftery MJ,Larance M,Rothemund D,Liew A,Bate I,Guilhaus M

doi

10.1002/pmic.200401160

subject

Has Abstract

pub_date

2005-08-01 00:00:00

pages

3397-401

issue

13

eissn

1615-9853

issn

1615-9861

journal_volume

5

pub_type

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