CREATING REFERENCE MATERIALs FOR CLINICALLY-RELEVANT PROTEINS USING QconCAT PEPTIDESá

 

Tyler A. Zimmerman, Mark S. Lowenthal, and Karen W. Phinney

 

Transferrin is an iron-transport protein, and abnormal levels of serum transferrin are implicated in diseases like iron deficiency anemia, iron overload disorders, and hemochromatosis. When symptoms of such diseases are present, current diagnoses are based on measuring transferrin levels with colorimetric methods that are inaccurate. However, mass spectrometry using known amounts of isotopically labeled peptides as internal standards gives higher quantitative accuracy, in a method called isotope-dilution mass spectrometry (IDMS). For a single target protein, the highest accuracy is obtained by developing a reference material that contains quantities that are extensively validated through an unbroken chain of comparisons. The reference material will then be commercially available from NIST to calibrate instruments and improve clinical diagnosis of iron overload disorders. Here the recently developed QconCAT method for creating isotope-labeled peptides, genetically rather than synthetically, is applied for the first time towards creating a transferrin reference material. For comparison to QconCAT results, transferrin levels were first obtained using IDMS with traditional synthetic isotope labeling. Transferrin levels from individual donor sera averaging ~12 picomoles/ÁL show high precision and fall within the broad range of previously reported literature values for transferrin. Future work will adapt QconCAT labeling to the creation of a reference material for transferrin.