In systemic
amyloidoses, widespread deposition of
protein as
amyloid causes severe organ dysfunction. It is necessary to discriminate among the different forms of
amyloid to design an appropriate therapeutic strategy. We developed a proteomics methodology utilizing two-dimensional
polyacrylamide gel electrophoresis followed by matrix-assisted
laser desorption/ionization mass spectrometry and
peptide mass fingerprinting to directly characterize
amyloid deposits in abdominal subcutaneous fat obtained by fine needle aspiration from patients diagnosed as having
amyloidoses typed as
immunoglobulin light chain or
transthyretin. Striking differences in the two-dimensional gel
proteomes of adipose tissue were observed between controls and patients and between the two types of patients with distinct, additional spots present in the patient specimens that could be assigned as the
amyloidogenic proteins in full-length and truncated forms. In patients heterozygotic for
transthyretin mutations, wild-type
peptides and
peptides containing amyloidogenic
transthyretin variants were isolated in roughly equal amounts from the same
protein spots, indicative of incorporation of both species into the deposits. Furthermore novel spots unrelated to the
amyloidogenic proteins appeared in patient samples; some of these were identified as
isoforms of serum
amyloid P and
apolipoprotein E,
proteins that have been described previously to be associated with
amyloid deposits. Finally changes in the normal expression pattern of resident adipose
proteins, such as down-regulation of alphaB-
crystallin,
peroxiredoxin 6, and
aldo-keto reductase I, were observed in apparent association with the presence of
amyloid, although their levels did not strictly correlate with the grade of
amyloid deposition. This proteomics approach not only provides a way to detect and unambiguously type the deposits in abdominal subcutaneous fat aspirates from patients with
amyloidoses but it may also have the capability to generate new insights into the mechanism of the diseases by identifying novel
proteins or
protein post-translational modifications associated with
amyloid infiltration.