A method for MSE differential proteomic analysis of archival formalin-fixed celloidin-embedded human inner ear tissue

Abstract

Proteomic analysis of cadaveric formalin-fixed, celloidin-embedded (FFCE) temporal bone tissue has the potential to provide new insights into inner ear disorders. We have developed a liquid chromatography-mass spectrometry (LC-MS) method for tissue sections embedded with celloidin. Q-TOF (Quadripole-time of flight mass spectrometry) MS E (mass spectrometry where E represents collision energy) and Identity E ™ were used in conjunction with nano-UPLC (capillary ultrahigh pressure liquid chromatography) for robust identification and quantification of a large number of proteins.Formalin fixed paraffin-embedded (FFPE) mouse liver sections were used to evaluate formalin decross-linking by five different methods. Unfixed fresh mouse liver tissue was used as a control. Five different methods for preparation of FFPE tissue for MS analysis were compared, as well as four methods for celloidin removal with FFCE mouse liver tissue. The methods judged best were applied to FFCE 20 μm sections of mouse inner ear samples, and FFCE 20 μm human inner ear and human otic capsule bone sections.Three of the five tissue extraction methods worked equally in detecting peptides and proteins from FFPE mouse liver tissue. The modified liquid tissue kit protocol was chosen for further studies. Four different celloidin removal methods were compared and the acetone removal method was chosen for further analysis. These two methods were applied to the analysis of FFCE inner ear and otic capsule sections. Proteins from all major cellular components were detected in the FFCE archival human temporal bone sections. This newly developed technique enables the use of FFCE tissues for proteomic studies.

Identification of Target Proteins Involved in Cochlear Otosclerosis

“…17 However, it appears that the protein identification rate is lowered by factors inherent in the use of celloidin-embedded tissues that increase sample complexity. Particularly, the methods required to remove celloidin can lead to protein degradation and/or modification.…”

“…Sections were pre-treated as previously described, 17 using a bibulous paper soaked in formalin to stick onto Superfrost® Plus slides (Thermo Fisher Scientific, Waltham, MA). Celloidin was removed with acetone for 30 minutes 20 and then hydrated with decreasing concentrations of ethanol.…”

“…Celloidin was removed with acetone for 30 minutes 20 and then hydrated with decreasing concentrations of ethanol. 17 Otosclerotic lesions were microdissected using scalpel under an operating microscope from the hTB with otosclerosis, and similar areas of otic capsule were dissected from the control case, in order to eliminate other structures (e.g., Organ of Corti, modiolus, and nonotic capsule surrounding bone) from the analysis. Microdissected specimens likely contained some adjacent spiral ligament tissue, including hyalinized spiral ligament in some cases of otosclerosis, since the spiral ligament was closely adherent to adjacent otic capsule endosteum.…”

“…Microdissected specimens likely contained some adjacent spiral ligament tissue, including hyalinized spiral ligament in some cases of otosclerosis, since the spiral ligament was closely adherent to adjacent otic capsule endosteum. Nine dissected areas of interest were pooled for each sample and transferred to a collection tube to be treated using the modified protocol previously described by Aarnisolo et al 17 Briefly, extraction buffer from the Liquid Tissue kit® (MS Protein Prep Kit, Expression Pathology, Rockville, MD) was added and samples were incubated at 95°C for 90 minutes, with 5-seconds centrifugations every 20 minutes. Then the samples were centrifuged at 10,000 rcf for 1 minute and cooled on ice before being processed as described in the modified protocol of Aarnisolo et al 17 …”

“…Recently, a reliable method for liquid chromatography-mass spectrometry (LC-MS) analysis on formalin fixed celloidin embedded tissues was developed. 17, 18 We employed this method to study otosclerotic lesions in our FFCE collection, in order to identify proteins involved in the otosclerotic process.…”