Abstract: TH-PO087
Laser Capture Microdissection and Liquid Chromatography Tandem-Mass Spectrometry for Small Amounts of Formalin-Fixed Paraffin Embedded Kidney Tissue
Session Information
- Clinical/Diagnostic Renal Pathology and Lab Medicine - I
November 02, 2017 | Location: Hall H, Morial Convention Center
Abstract Time: 10:00 AM - 10:00 AM
Category: Glomerular
- 1004 Clinical/Diagnostic Renal Pathology and Lab Medicine
Authors
- Nagelkerken, Sophie I., Leiden University Medical Center, Leiden, Netherlands
- Janssen, George, Leiden University Medical Center, Leiden, Netherlands
- Veraar, Kimberley, Leiden University Medical Center, Leiden, Netherlands
- Van veelen, Peter, Leiden University Medical Center, Leiden, Netherlands
- Baelde, H. J., Leiden University Medical Center, Leiden, Netherlands
- Bruijn, Jan A., Leiden University Medical Center, Leiden, Netherlands
- Bajema, Ingeborg M., Leiden University Medical Center, Leiden, Netherlands
Background
Liquid chromatography tandem-mass spectrometry (LC-MS/MS) is a sensitive and specific technique for in depth protein identification but diagnostic kidney material is scarce, limiting the number of protein identifications. Only a few studies have used this technique for protein identification in formalin fixed paraffin embedded (FFPE) glomerular material and only one used a practical amount that can be used for diagnostic biopsies. Therefore, we optimized a work-flow of laser capture microdissection (LCM) of glomeruli, followed by LC-MS/MS on FFPE tissue for a minimal amount of tissue with a maximum protein yield.
Methods
One FFPE tissue block of a normal human kidney was utilised. Cross-sections of randomly selected kidney and glomeruli were collected from 6 µm sections, mounted on a 1.0 PEN membrane, using LCM. Protein extraction and digestion was performed using filter aided sample preparation (FASP) with polyethylene glycol (PEG) as a carrier, adapted from previously described methods for microdissected colon tissue. Samples were purified using mixed anion exchange solid phase extraction (MAX-SPE) and analysed using LC-MS/MS.
Results
Using the PEG-FASP protocol, we established the optimum amount of tissue to be 3 nl, based on 605 protein identifications, compared to 216 and 693 identifications in 1 and 10 nl tissue respectively. In a reproduction analysis of 5 experiments, using both random kidney and glomerular tissue, an average of 457 and 228 proteins were identified respectively. The method was able to distinguish glomerular samples from random kidney samples, as demonstrated by differentially identified proteins and clustering of glomerular and random kidney samples.
Conclusion
The relative number of proteins detected was comparable to or higher than reported in previous studies using FFPE glomerular tissue. The presented work-flow expands the potential for novel protein identification in glomerular diseases, while keeping the amount of tissue small enough for diagnostic practicality. Therefore, the combination of LCM followed by LC-MS/MS, using PEG-FASP and MAX-SPE for sample preparation, is a suitable and promising technique for diagnostic applications, especially when specific proteins are overexpressed or abundant within glomeruli.
Funding
- Private Foundation Support