Phenotyping of α-1-Antitrypsin by liquid chromatography-high resolution mass spectrometry

Abstract

More than seventy-five isotypes of α-1-antitrypsin (AAT) have been described. To assess risks associated with AAT deficiency, isotype identification is necessary. Isoelectric focusing (IEF) is traditionally used for isotype differentiation, however, IEF has limited scope since it is a manual procedure that is not suitable for automation, and antitrypsin variants must differ in net charge in order to be resolved. In comparison, mass spectrometric assays are easily automated and offer a more complete solution for characterization of proteins. To capitalize on these advantages, we have developed a qualitative top-down liquid chromatography-mass spectrometry (LC-MS) method for selective phenotyping of AAT. This technique requires no sample pretreatment, and has the potential for use in routine clinical diagnostics. We have validated our LC-MS results against both DNA sequencing and IEF. Thus far, this method has identified the AAT variants P_Lowell, S and Z, as well as unique fragments shared by different M alleles. Its high selectivity is indirectly illustrated by the detection of a variant carrying the amino acid substitution p.Ala308Ser, which cannot be visualized by IEF.

Keywords: Alpha 1-antitrypsin; Collision induced dissociation; Isoelectric focusing; LC–MS; Pi typing; SERPINA1.

Fig. 1

The complete amino acid sequence of AAT Uniprot P01009-1 shown with four fragment peptides (bold or boxed text) containing the p.ArgR125His, p.Asp280Val, p.Glu288Val, p.Ala308Ser, p.Glu366Lys and p.Glu400Asp shifts. The three glycosylation sites (N) and the cysteinylation site are grey-boxed. Typical results obtained using the LC–MS method: a total ion count (TIC) chromatogram (B); full scan spectra of chromatographic peak at 7.59 min (C).

Fig. 2

Mass spectra in the range m/z 994-999.5 of 288Glu(wt) homozygous (A), 288Val homozygous (B), 288Glu(wt)/288Val heterozygous (C) samples. Selected mass numbers unique for 288Glu(wt) (filled boxes) and for 288Val (empty boxes) are indicated.

Fig. 3

Mass spectra in the range m/z 740–741 of 366Glu(wt) homozygous (A), 366Lys homozygous (B), and 366 Glu(wt)/366Lys heterozygous (C) samples. Selected mass numbers unique for 366Glu(wt)(filled boxes) and for 366Lys (empty boxes) are indicated.

Fig. 4

Dot plots of ratios between indicated intensities for non-wild-type fragments and wild-type fragments. The ratios are plotted against categorized heterozygous (he), homozygous (ho) and wild-type (wt) samples. Dark grey circles represent DNA-sequenced samples and light grey diamonds represents non-sequenced samples. Overlaid are box plots with further information. The box represents the first and third quartiles, the band inside the box indicates the median and the whiskers indicate the lowest or highest ratio still within 1.5 times interquartile range of the lower or higher quartile.