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A Novel Strategy Combining FPLC Separation of Stable Isotope-Labeled Unfolded Proteins and MALDI MS Analysis Enables Quantification of a Wide Range of Serum Proteins
Published
Author(s)
Wei-Li Liao, Illarion Turko
Abstract
A novel strategy for quantitative profiling of serum or plasma proteomes is described. This strategy includes scaling up and ammonium sulfate depletion of original sample, affordable chemistry of stable isotope labeling for big protein sample, separation of labeled unfolded proteins, and quantification by MALDI MS. Serum proteins were first unfolded in 8 M urea and labeled with a pair of acrylamides, none-deuterated or D0-acrylamide and deuterated or D3-acrylamide. Three workflows for separating labeled unfolded proteins were then compared that is (1) liquid-based IEF/2D-PAGE; (2) FPLC/2D-PAGE; and (3) whole gel elution/FPLC. Whole gel elution/FPLC which combines electrophoretic separation based on protein molecular weight with the following chromatographic separation in the presence of 8 M urea based on protein charge resulted in quantification of a highest number of serum protein including those which abundance falls in a category of at least 10-5 to albumin. It makes this robust workflow suitable for quantitative profiling of protein changes in serum or plasma associated with pre-analytical variables. For example, quantitative pattern of 20 randomly picked up proteins did not reveal changes in serum after its exposure to room temperature for 24 hours.
Liao, W.
and Turko, I.
(2021),
A Novel Strategy Combining FPLC Separation of Stable Isotope-Labeled Unfolded Proteins and MALDI MS Analysis Enables Quantification of a Wide Range of Serum Proteins, Journal of Proteome Research
(Accessed October 9, 2024)