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Chromatographic Separation of Unfolded Proteins for Protein Quantification


NIST is developing methods for the quantification of protein biomarkers by mass spectrometry (MS). Stable isotope labeling of proteins/peptides followed by MS analysis allows relative quantification of protein samples and could be an important analytical approach in assessing pre-analytical variables and protein biomarkers. However, the application of this powerful approach faces technical and conceptual challenges because of high dynamic range of proteins in a biological sample and inability to detect low abundant proteins. Relative quantification by stable isotope labeling in vitro includes the unfolding of proteins with SDS or urea, introduction of an isotopic label, and mixing the two samples to be compared in a particular ratio. Any chromatographic separations before labeling/mixing will cause quantitative variations and increase the uncertainty in the results. It is safe to perform separations after labeling/mixing since the protein ratios are established however the proteins are already unfolded and the available procedures to separate unfolded proteins have low recovering efficiency. NIST is working to develop chromatographic separations of unfolded proteins suitable for protein quantification.


Pre-analytical variables, such as collection, processing, storage and transportation can alter the proteome of serum/plasma and other biological samples used in clinical diagnoses. To define conditions for proper sample handling, analytical methods that can quantify many proteins simultaneously are needed to evaluate potential impact of the pre-analytical variables. Through the development of robust and inexpensive techniques that allow quantification of many proteins simultaneously, better knowledge of sample handling can be achieved. These techniques are also necessary for discovery and validation of potential biomarkers of human diseases.

Additional Technical Details:

A novel strategy for the quantitative profiling of serum proteome was developed. 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 MALDI MS. Labeling of unfolded proteins was performed using normal, D0-acrylamide and deuterated, D3-acrylamide. The workflow for separating the unfolded proteins includes whole gel elution and cation-exchange liquid chromatography and combines electrophoretic separation based on the protein molecular weight followed by chromatographic separation in the presence of 8 mol/L urea based on protein charge. Another study was designed to improve the understanding of proteomic changes that underlie senescence of retinal pigment epithelial (RPE) cells. Senescence of retinal pigment epithelial (RPE) cells is a crucial event in the pathogenesis of age-related macular degeneration. Anion-exchange chromatography was adopted for separation of stable isotope labeled unfolded proteins. Several large fragments of typical cytosolic proteins, such as GAPDH, triosephosphate isomerase, and M2-type pyruvate kinase were found ≈2-3 fold increased in the senescent RPE cells. These data point to a new type of a “waste” material in post-mitotic cells that may contribute to the senescent phenotype.  

Major Accomplishments:

  • Cation-exchange chromatography was applied to separate unfolded proteins labeled with D0- and D3-acrylamides.
  • This new workflow allowed quantification of a large number of serum proteins, including those with abundance less than 10-5 than albumi
  • Anion-exchange chromatography was used to separate stable isotope labeled unfolded proteins from RPE cells.
  • This study is the first demonstration that large fragments of cytosolic proteins can be accumulated in the senescent cells.