Poster Presentation HUPO 2019 - 18th Human Proteome Organization World Congress

In-depth proteomics at a single glomerulus level in human kidneys using MALDI Imaging Mass Spectrometry and shotgun proteomics on paraffin embedded biopsy tissue section (#738)

Yume Mukasa 1 , Takashi Nirasawa 2 , Ryo Kajita 2 , Marion Rabant 3 , Jean-Paul Duong Van Huyen 3 , Patrick Bruneval 4 , Hatsue Ishibashi-Ueda 5 , Nobuto Kakuda 1 , Masaya Ikegawa 1
  1. Department of Life and Medical Systems, Doshisha University, Kyotanabe, Kyoto, Japan
  2. Bruker Japan K. K., Yokohama, Kanagawa, Japan
  3. Necker-Enfants malads Hospital, Paris, France
  4. Georges-Pambidou European Hospital, Paris, France
  5. National Cerebral and Cardiovascular Center Research Institute, Suita, Osaka, Japan

The molecular characterization at the level of individual glomerulus in kidneys remains a technological challenge that needs to be addressed in order to better understand pathological mechanisms. Here, we developed and applied a mass spectrometry-based methodology to investigate heterogeneity of proteomes from in situ tissue sections from human biopsied samples with or without kidney disease.

In this study, we adopted paraffin-embedded tissue sections fixed in formalin-acetic acid-alcohol (FAA) fixative, using matrix-assisted laser desorption/ionization imaging mass spectrometry (MALDI-IMS). Furthermore, we established a workflow that integrates MALDI-IMS and shotgun analysis (timsTOF Pro system). FAA fixed paraffin embedded tissue samples were from Necker Children‘s Hospital. After HE staining, serial slide thickness of 10 μm was made from the same block of the sample on ITO coated slide glass. Dewaxing, antigen retrieval, pH adjustment on tissue samples and on-tissue digestion with trypsin, deposition of α-cyano-4-hydroxycinnamic acid as a matrix using TM-Sprayer. MALDI-IMS was done by rapifleX with a spatial resolution of 20 or 50 μm. Ions were detected in mass range of m/z 800 to 3000. Statistical analysis was performed with SCiLS Lab 2019 software. Shotgun Proteomics from serial sections of MALDI-IMS were attempted using timsTOF Pro with nanoElute system.

Here we have established an integrated workflow using MALDI-IMS method and shotgun proteomics analysis from FAA-fixed kidney biopsies from non-pathological cases. Of note, we have succeeded in obtaining IMS data with 20 mm resolution and was able to obtain MS ion images at a single glomerulus level from FAA-fixed renal biopsies. Thousands of proteins from the serial sections with shotgun proteomics were also annotated which were analyzed with Protein ID and Proteinscape ver 3.0. We will further apply this method to elucidate and validate its efficacy with a variety of renal diseases such as diabetic nephropathy and renal amyloidosis.