Simultaneous Phenotyping and Quantification of α-1-antitrypsin by Liquid Chromatography-tandem Mass Spectrometry

Background: α-1-Antitrypsin (A1AT) deficiency results from a genetic disorder at 2 common loci. Diagnosis requires quantification 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 quantification (nephelometry), genotyping, and/or phenotyping. We developed a multiple reaction monitoring liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for simultaneous quantification of A1AT and identification of the 2 most common deficiency alleles present in 95% of the patients with A1AT deficiency.

Methods: Serum samples (n = 40) were digested with trypsin, and appropriate ¹³C/¹⁵N-labeled standard peptides were added. We performed LC-MS/MS analysis with a 0.5- by 150-mm C18 column and H₂O:acetonitrile:n-propanol:formic acid (A:98:1:1:0.2 and B:10:80:10:0.2; flow 12 μL/min) mobile phase in positive ion mode on a TSQ Quantum triple quadrupole MS system. We measured the A1AT concentration by comparison to a calibration curve and determined the phenotype by the presence or absence of variant peptides. We compared the results to the current phenotyping assay by isoelectric focusing (IEF) and the immunonephelometry quantitative assay.

Results: For A1AT allele detection, in 39 of 40 samples the LC-MS/MS results were identical to those obtained by IEF gel electrophoresis. The single discrepant result was rerun by IEF at a lower dilution, and the results were in concordance. The A1AT quantification by LC-MS/MS also compared favorably with nephelometry.

Conclusions: The LC-MS/MS method correlates well with current phenotyping and nephelometric assays and has the potential to improve the laboratory diagnosis of genetic A1AT deficiency.