Label-free LC-MS/MS strategy for comprehensive proteomic profiling of human islets collected using laser capture microdissection from frozen pancreata

Lina Zhang, Giacomo Lanzoni, Matteo Battarra, Luca Inverardi, Qibin Zhang

Research output: Chapter in Book/Report/Conference proceedingChapter

1 Scopus citations

Abstract

Diabetes mellitus is caused by either loss of pancreatic islets β-cells (Type 1 Diabetes, T1D), insufficient insulin release in the islet β-cells coupled with insulin resistance in target tissues (Type 2 Diabetes, T2D), or impaired insulin release (genetic forms of diabetes and, possibly, T1D subtypes). The investigation of the islet proteome could elucidate facets of the pathogenesis of diabetes. Enzymatically isolated and cultured (EIC) islets are frequently used to investigate biochemical signaling pathways that could trigger β-cell changes and death in diabetes. However, they cannot fully reflect the natural protein composition and disease process of in vivo islets due to the stress from isolation procedures and in vitro culture. The laser capture microdissection method employs a high-energy laser source to separate the desired cells from the remaining tissue section in an environment which is well conserved and close to the natural condition. Here, we describe a label-free proteomic workflow of laser capture microdissected (LCM) human islets from fresh-frozen pancreas sections of cadaveric donors to obtain an accurate and unbiased profile of the pancreatic islet proteome. The workflow includes preparation of frozen tissue section, staining and dehydration, LCM islets collection, islet protein digestion, label-free Liquid Chromatography-Tandem Mass Spectrometry (LC-MS/MS), database search, and statistical analysis.

Original languageEnglish (US)
Title of host publicationMethods in Molecular Biology
PublisherHumana Press Inc.
Pages253-264
Number of pages12
DOIs
StatePublished - 2019

Publication series

NameMethods in Molecular Biology
Volume1871
ISSN (Print)1064-3745

Keywords

  • LC-MS/MS
  • LCM human pancreatic islets
  • Label-free proteomics
  • MaxQuant
  • Perseus

ASJC Scopus subject areas

  • Molecular Biology
  • Genetics

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