Optimal conditions and the advantages of using laser microdissection and liquid chromatography tandem mass spectrometry for diagnosing renal amyloidosis

Abstract

Background Liquid chromatography-tandem mass spectrometry (LC-MS/MS) has recently been utilized to accurately detect the amyloid proteins of renal amyloidosis. The present study investigated the optimal procedures for analyzing samples by LCMS/MS, and the advantage of using this technique to diagnosis renal amyloidosis. Methods To detect amyloid proteins, laser microdissected glomeruli from AL (n = 13) or AA (n = 10) renal amyloidosis patients were digested and analyzed by LCMS/MS. To determine the best procedures for analyzing samples by LCMS/MS, we examined the suitability of tissue samples, frozen or formalin-fixed paraffin-embedded (FFPE), the number of dissected glomeruli required for analysis (2, 10, or 50 glomeruli), and the amount of trypsin with or without dithiothreitol (DTT). We additionally compared the detection of amyloid proteins between immunostaining and LCMS/MS. Results Examining 10 dissected glomeruli from FFPE sections digested with trypsin 3 µL (0.1 mg/mL) without DDT made it possible to detect amyloid protein in all 10 AA and in 10 out of 12 AL amyloidosis cases. All AA amyloidosis cases were diagnosed using immunohistochemistry for amyloid A. With immunostaining, however, there were several inconclusive immunoglobulin and/or their light chain staining noted in the AA or AL amyloidosis cases. Even so, LCMS/MS was able to accurately detect amyloid protein in renal amyloidosis. Conclusion The use of 10 laser microdissected glomeruli (170,000-220,000 µm 2 ) with amyloid deposition from FFPE sections digested with trypsin 3 µL (0.1 mg/mL) allowed the accurate detection of amyloid protein in AA and AL amyloidosis.

Amyloid Typing by Mass Spectrometry in Clinical Practice: a Comprehensive Review of 16,175 Samples

“…Hence, wide implementation of this assay could be challenging. Nonetheless, when coupled with a robust quality control and quality assurance system 25 and advanced bioinformatics, 31,36 it can be applied to routine reference laboratory practice as shown in this study and in other published studies. 32,[37][38][39][40][41][42][43] In this study of routine clinical samples sent for amyloid typing to our reference laboratory, we identified 21 different amyloid types.…”

“…Using an approach similar to that applied to detecting amino acid substitutions in hereditary amyloidosis, a database containing immunoglobulin light chain variable family member amino acid sequences can be constructed. 36 With this, one can determine the light chain variable family of AL amyloid deposits. 66 When all of the light chain in an AL case is from a single light chain family, it implies that the AL amyloid is derived from a clonal B-cell/plasma cell process.…”

Proteomics of human glomerulonephritis by laser microdissection and liquid chromatography‐tandem mass spectrometry

“…We think that this number depends on the purpose of the study, the amount of the targeted protein, and the sensitivity of mass spectrometer used. Aoki et al used a 6 × 10 6 μm 3 glomerular volume for LC‐MS/MS, we used three sizes of glomerular volume: 3 × 10 6 μm 3 (which yielded ~12 glomeruli), 6 × 10 6 and 9 × 10 6 μm 3 , dissected from 10‐μm‐thick sections. There were no significant differences in the number of proteins detected by LC‐MS/MS from the three dissected volumes.…”

Characteristic electron-microscopic features of cryofibrinogen-associated glomerulonephritis: a case report

“…Furthermore, LC-MS/MS on laser microdissected glomeruli from paraffin sections was performed as previously described [9], and revealed increased levels of fibrinogen α, β, and γ chains, fibronectin, filamin-A, and C3 ( Fig. 4).…”