Proteomics of specific treatment-related alterations in Fabry disease: a strategy to identify biological abnormalities

Abstract

Fabry disease is inherited as an X-linked disorder secondary to deficiency of alpha-galactosidase A, resulting in abnormal metabolism of substances containing alpha-d-galactosyl moieties. As a consequence, a multisystem disorder develops, culminating in strokes, progressive renal, and cardiac dysfunction. Signs and symptoms of Fabry disease become manifest in childhood, but diagnosis is often delayed. Thirteen children with Fabry disease (age range, 6.5-17 years) were studied as part of a 6-month open-label study of enzyme replacement therapy (ERT) with agalsidase alfa. Paired serum samples were drawn at the start of the study and after 6 months of ERT. Global protein changes in paired samples were compared by using differential stable isotope labeling of peptide lysine residues with O-methylisourea and subsequent nanoHPLC-tandem MS. Statistically significant decreases were observed for five proteins following ERT: alpha(2)-HS glycoprotein, vitamin D-binding protein, transferrin, Ig-alpha-2 C chain, and alpha-2-antiplasmin. The presence of low levels of alpha-2-antiplasmin and plasminogen was confirmed by alternate means in 34 consecutive patients, including four of five ERT-naïve subjects. Decreased alpha-2-antiplasmin was associated with a parallel increase in circulating VEGF. Soluble VEGF receptor-2 was significantly elevated in plasma of patients compared with pediatric controls and decreased with ERT. These results suggest previously unknown abnormalities of fibrinolysis and angiogenesis factors in Fabry disease. We demonstrated the feasibility of identifying treatment-specific alterations in a small number of subjects that point to previously unsuspected disease-related biological abnormalities.

Fig. 1.

A representative MS/MS analysis of differentially labeled peptide mixtures derived from baseline and treated sera samples. (A) Portion of a survey MS scan showing doubly charged ions corresponding to (344–355) TFTCTAAYPESK peptide from Ig-α-I chain C-region (P01876); calculated masses 1,416.63 and 1,419.63 Da for light and heavy label, respectively. (B) Corresponding MS/MS spectrum of parent ion at m/z 709.32 showing one of the pairs of singly charged y-ions. (C) Protein identification and quantitation by using the Global Proteome Machine search engine.

Fig. 2.

Validation of the α-2-antiplasmin abnormality (A) and plasminogen abnormality (B) in Fabry disease. (A) α-2-Antiplasmin in 34 consecutive patients, five of whom were ERT-naïve (open triangles), with Fabry disease, 10–55 years of age. The gray area is the reference range, the dashed line is the mean of the reference population, and the horizontal solid line is the mean of the Fabry patient group. As a group, α-2-antiplasmin levels in Fabry patients were lower than normal (P < 0.0001). (B) Graph of the correlation between plasma levels of α-2-antiplasmin and plasminogen in Fabry patients with low or borderline levels.