Well-Plate μFASP for Proteomic Analysis of Single Pancreatic Islets

J Proteome Res. 2022 Apr 1;21(4):1167-1174. doi: 10.1021/acs.jproteome.2c00047. Epub 2022 Mar 16.


Filter-aided sample preparation (FASP) is widely used in bottom-up proteomics for tryptic digestion. However, the sample recovery yield of this method is limited by the amount of the starting material. While ∼100 ng of digested protein is sufficient for thorough protein identification, proteomic information gets lost with a protein content <10 μg due to incomplete peptide recovery from the filter. We developed and optimized a flexible well-plate μFASP device and protocol that is suitable for an ∼1 μg protein sample. In 1 μg of HeLa digest, we identified 1295 ± 10 proteins with μFASP followed by analysis with liquid chromatography-mass spectrometry. In contrast, only 524 ± 5 proteins were identified with the standard FASP protocol, while 1395 ± 4 proteins were identified in 20 μg after standard FASP as a benchmark. Furthermore, we conducted a combined peptidomic and proteomic study of single pancreatic islets with well-plate μFASP. Here, we separated neuropeptides and digested the remaining on-filter proteins for bottom-up proteomic analysis. Our results indicate inter-islet heterogeneity for the expression of proteins involved in glucose catabolism, pancreatic hormone processing, and secreted peptide hormones. We consider our method to provide a useful tool for proteomic characterization of samples where the biological material is scarce. All proteomic data are available under DOI: 10.6019/PXD029039.

Keywords: filter-aided sample preparation; islets of Langerhans; liquid chromatography−mass spectrometry; peptidomics; proteomics.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Chromatography, Liquid / methods
  • Humans
  • Islets of Langerhans* / chemistry
  • Mass Spectrometry
  • Proteins / analysis
  • Proteomics* / methods


  • Proteins