Simultaneous Phenotyping and Quantitation of α-1-Antitrypsin by LC-MS/MS
Wed 11:00 AM - Track 2: Targeted Proteomics
Bob Bergen
Mayo Proteomics Research Center
Yuhong Chen, Division of Clinical Pharmacology, Department of Molecular and Experimental Therapeutics
Melissa R. Snyder, Division of Clinical Pharmacology, Department of Molecular and Experimental Therapeutics
Linda M. Benson, Mayo Proteomics Research Center
Jerry A. Katzmann, Division of Clinical Biochemistry & Immunology, Department of Laboratory Medicine & Pathology, Division of Hematology, Department of Internal Medicine
H. Robert Bergen, III, Mayo Proteomics Research Center
α-1-antitrypsin (A1AT) is a plasma glycoprotein secreted by the liver. A1AT is a member of the serine protease inhibitor family. One of its main functions is to inhibit neutrophil elastase protease activity in the lungs. Any deficiency results in uncontrolled proteolytic lung tissue damage. The A1AT protein is very polymorphic with over 100 polymorphisms identified. However, deficiency results from a genetic disorder at two common loci accounting for 95% of all recognized disease. Diagnosis requires quantitation of A1AT and subsequent identification of the specific variant. The current algorithm of laboratory testing for the diagnosis of A1AT deficiency uses a combination of initial quantitation (nephelometry) and subsequent genotyping and/or phenotyping (isoelectric focusing). Because A1AT is one of the top 10 most abundant plasma proteins it is an easy target for characterization by mass spectrometry. We developed a multiple reaction monitoring LC-MS/MS method for simultaneous quantification of A1AT and the identification of the Z and S alleles which account for 95% of the disease. The methodology is comprised of trypsin digestion, addition of isotopically labeled peptides containing the S and Z variants and a proteotypic peptide for quantitation. Comparison of the proteotypic peptide to a standard curve yields quantitative information while the presence of the peptides containing the two common deficiency alleles indicates the Z or S disease as well as zygosity.

The LC-MS/MS method correlates well with current phenotyping and genotyping assays. It is a promising method with the potential to improve the laboratory diagnosis of genetic A1AT deficiency.

Supported by the Mayo Clinic College of Medicine, the Mayo Clinic Center for Individualized Medicine and the Mayo Clinic Research Committee for financial support. Y. Chen was supported by a NIH Training Grant in Clinical Pharmacology (T32 GM08685). This work is supported by generous gifts from Mr. and Mrs. Gordon C. Gilroy and the David Woods Kemper Memorial Foundation.