A RAGHUBAR1,2,3,4, X WANG3,4, A KASSIANOS2,3,4, MS NG2,3, KA DAVE5, E NORRIS5, MJ HEADLAM5, HG HEALY2,3,4, AJ MALLETT2,3,4
1Anatomical Pathology, Pathology Queensland, RBWH, Herston, Australia, 2The University of Queensland, St Lucia, Australia, 3Kidney Health Service, RBWH Metro North HHS , Herston, Australia, 4Conjoint Kidney Research Laboratory, Chemical Pathology, Pathology Queensland, RBWH, Herston, Australia, 5Proteomics Facility, QIMR Berghofer Medical Research Institute, Herston, Australia
Aim: To develop a robust and reproducible micro-proteomic workflow for glomerular proteome profiling.
Background: Current renal histopathological assessment cannot identify the clinically significant molecular pathobiology that discriminates various kidney diseases with similar histomorphology. Here we optimised a mass spectrometry based micro-proteomic workflow with high sensitivity that can potentially enhance current diagnostic precision.
Methods: We investigated the following six variables on 10μm frozen sections of renal biopsy; fixation time – 1min vs 5mins, slide properties – plain vs adhesive vs membrane slides, laser micro-dissection (LMD) instrumentation – UV laser configuration, capture caps – adhesive vs plain caps, “one pot” sample preparation protocol – single-step lysis, reduction and alkylation with subsequent trypsin digestion vs lysis and digestion only vs sequential-step protocol with protein precipitation preceding digestion and finally tissue area – 1.5 x 10⁶μm² vs 3.5 x 10⁶μm².
Results: We found that 5mins fixation in graded alcohol and H&E staining with protease inhibitors minimized protein degradation and removed polyethylene glycol contamination. Membrane slides were identified as highly efficient in LMD, due to the supportive layer between tissue and glass. LMD instrument with the UV laser position on top and capture tube at bottom reduced loss of catapulted tissue, eliminated the need for expensive adhesive caps and allowed direct capture into lysis buffer. Plain caps eliminated protein loss caused by the trapping of tissue within the polymer filling of the adhesive cap. Sequential “one pot” sample preparation protocol with 3.5 x 10⁶μm² of tissue produced consistent, reproducible and in-depth proteome profiles.
Conclusion: Our work is proof of concept of a highly-sensitive micro-proteomic workflow. This mass spectrometry-based profiling of glomeruli in human kidney diseases enhances precision of histopathological diagnoses.
Arti Raghubar works as a medical scientist at Pathology Queensland, Royal Brisbane and Women’s Hospital. Her specialities are transmission and scanning electron microscopy, neuromuscular histology, immunohistochemistry and routine histology. She has completed a Bachelor of Applied Science (Medical Science) from Queensland University of Technology 2002. She is currently completing a Master of Molecular Biology Research Extensive with the University of Queensland. Her primary research interest is in identifying diagnostic molecular markers that can be utilised by histology laboratories in renal pathology.