Identification Of Systemic Amyloidosis By Laser Microdissection Combined With Mass Spectrometry Proteomics

Background and Objectives:Combining formalin-fixed paraffin-embedded (FFPE) specimens with mass spectrometry (MS) based proteomics provides a promising method to study disease-related protein differences. However, there is still no international standard in the protein extraction procedures. In the current study, we attempted to compare5extraction buffers for the protein analysis in different kinds of rat FFPE tissues, to confirm the optimized extraction method for accurate identification of the largest number of proteins.Amyloidosis is a group of diseases characterized by extracellular deposition of misfolded proteins in an insoluble β-pleated conformation. Accurate typing of amyloidosis has implications for prognosis, genetic counseling and treatment, thus is of paramount importance. Immunohistochemistry (IHC), as the most common method, is problematic in amyloidosis typing due to limited antibody categories, epitope loss caused by formalin fixation and background staining from serum contamination. Laser micro-dissection followed by MS (LMD/MS) is an emerging approach for describing the composition of amyloid deposition, therefore representing a more accurate typing strategy. In this report, we described the LMD/MS results of amyloidosis cases in our center, evaluating the success rate of subtyping among different kinds of FFPE specimens. We also tried to investigate the value of immunofixation electrophoresis (IFE) and free light chain (FLC) in diagnosis of immunoglobulin light-chain amyloidosis (AL amyloidosis) compared with LMD/MS.Methods:We applied Zwittergent3-16based buffer, SDS-containing buffer with/without PEG20000, urea-containing buffer and FFPE-FASP protein preparation kit for protein extraction from different kinds of rat FFPE micro-dissected tissues, including heart, brain, liver, lung and kidney. The most efficient buffer on the basis of peptide count, protein count, spectra count and protein abundance was further utilized in MS typing for amyloidosis.We collected133biopsy samples from patients who were diagnosed with systemic amyloidosis in Peking Union Medical College Hospital from January,2000to May,2014. For each case, a10-μm section was stained with congo-red, and amyloid deposits were identified under fluorescent light, followed by micro-dissection and protein extraction by the optimal buffer from the first part of study. Afterwards, protein solution was digested into tryptic peptides and analyzed by MS. The amyloidosis subtype was confirmed based on the most abundant amyloidgenic protein.Results:Zwittergent-containing buffer yielded largest number of peptides, proteins and the highest protein abundance in FFPE LMD specimens. Moreover, Zwittergent3-16did not impair the enzymatic digestion and was well compatible with MS analysis. Therefore we chose Zwittergent-containing buffer for protein extraction of amyloidosis-involved tissues.The tissue types of133biopsy specimens were as follows, subcutaneous abdominal fat (27%), tongue (20%), gingiva (11%), kidney (11%), intestine (8%), heart (6%), bone marrow (5%), liver (5%) and others (9%). Specific types of amyloid were accurately detected in116(87%) cases, including52(39%) AL-X,,49(37%) AL-κ,7(5%) ATTR,2(2%) AA,2(2%) AH/AL+AH,2(2%) AFib,1(1%) AApoA-II and1(1%) ALys. Diagnosis of amyloidosis was ruled out in5patients and was indeterminate in4cases. Besides, we were unable to type8cases of amyloidosis. On the whole, LMD/MS reached91%sensitivity in amyloid typing. To predict AL amyloidosis, the sensitivity of serum protein electrophoresis (SPEP), IFE, FLC and IFE+FLC was23.6%,55.0%,63.9%and69.4%, respectively, while the specificity was92.3%,70.6%,77.8%and55.6%. Commonly involved organs like kidney, heart, liver, tongue and intestine turned out to be satisfactory sources of FFPE samples with typing success rate of almost100%. In contrast, MS analysis was successful in only86%of subcutaneous abdominal fat samples.Conclusions:Zwittergent-containing buffer is the most effective and efficient extraction buffer for micro-dissected FFPE tissues.Compared with IHC, IFE and FLC, LMD/MS method provided a more direct technique for accurate typing of amyloidosis in a single procedure. As tissue sources for FFPE specimens, involved organs are better choices than subcutaneous fat tissues for MS analysis.