Strategy combining separation of isotope-labeled unfolded proteins and matrix-assisted laser desorption/ionization mass spectrometry analysis enables quantification of a wide range of serum proteins.
A novel strategy for the quantitative profiling of serum proteome is described. It includes an ammonium sulfate depletion of the serum, an affordable stable isotope labeling chemistry for samples with a large amount of protein, separation of the unfolded proteins, and relative quantification by matrix-assisted laser desorption/ionization (MALDI) mass spectrometry (MS). Labeling of unfolded proteins was performed using normal (D(0)) acrylamide and deuterated (D(3)) acrylamide. The workflow for separating the unfolded proteins includes whole gel elution and ion exchange liquid chromatography, and it combines electrophoretic separation based on the protein molecular weight followed by chromatographic separation in the presence of 8M urea based on protein charge. This was followed by trypsinolysis and MALDI MS analysis, leading to the quantification of a large number of serum proteins, including those with an abundance of 10(-5) less than albumin. This robust and inexpensive workflow is suitable for the quantitative profiling of protein changes in serum associated with preanalytical variables.
Liao
Wei-Li
WL
Center for Advanced Research in Biotechnology, National Institute of Standards and Technology, University of Maryland Biotechnology Institute, Rockville, MD 20850, USA.
Turko
Illarion V
IV
D000595:Amino Acid Sequence; D000645:Ammonium Sulfate / Q000737:chemistry; D001774:Blood Chemical Analysis / Q000379:methods*; D001797:Blood Protein Electrophoresis; D001798:Blood Proteins / Q000032:analysis* / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D011232:Chemical Precipitation; D002852:Chromatography, Ion Exchange; D015180:Electrophoresis, Gel, Two-Dimensional; D007554:Isotopes; D011489:Protein Denaturation; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
D001798:Blood Proteins; D007554:Isotopes; D000645:Ammonium Sulfate