M-protein and monoclonal anti-PF4/polyanion antibody light and heavy chains have identical mass spectrometry profiles. (A) Displayed are LC-ESI-QTOF MS light chain (LC) +11 (m/z) distributions of serum proteins and (B) anti-PF4/polyanion antibodies isolated from the patient’s serum using PF4-treated heparin Sepharose beads, as described. In the spectra, red represents the +11 m/z distribution of all κ containing immunoglobulins, and blue represents the +11 m/z LC distribution of κ chains associated with an IgG heavy chain (HC). The number above each peak indicates the identified chain’s +11 m/z ratio. The x-axis depicts m/z ratios, and the height of the peak shows the identified antibodies’ relative abundance. Each subpanel contains an inset depicting the deconvoluted spectra with the molecular mass of the identified immunoglobulin LC and a table summarizing the major LC m/z values and deconvoluted masses. Similar to (A) and (B), (C) and (D) display the +37 m/z HC distribution and deconvoluted spectra for the immunoglobulin γ (G) HC spectra from MGTS patient serum and anti-PF4 antibody enriched eluate, respectively. The various peaks in (C) and (D) depict differentially glycosylated forms of the HC immunoglobulin. (E) PF4-dependent binding of patient IgG HC (γ) and κ/λ LCs to normal donor platelets were evaluated. Mean and 1 SD of triplicate measurements are presented. (F) Platelet counts (y-axis) were correlated with M-protein levels (x-axis). M-protein intervals were qualitatively grouped into 4 intervals and the percentage of normal (≥150 × 109/L) and low (<150 × 109/L) platelet counts are displayed for each interval. M-protein and platelet count represented by each data point were obtained on the same day of testing, except on 2 occasions when they were drawn within 48 hours of each other.
