Well-Plate muFASP for Proteomic Analysis of Single Pancreatic Islets

• Filter-aided sample preparation (FASP) is widely used in bottom-upproteomics for tryptic digestion. However, the sample recovery yield of this methodis limited by the amount of the starting material. While similar to 100 ng of digested protein issufficient for thorough protein identification, proteomic information gets lost with aprotein content <10 mu g due to incomplete peptide recovery from thefilter. Wedeveloped and optimized aflexible well-plate mu FASP device and protocol that issuitable for an similar to 1 mu g protein sample. In 1 mu g of HeLa digest, we identified 1295 +/- 10proteins with mu FASP followed by analysis with liquid chromatography-massspectrometry. In contrast, only 524 +/- 5 proteins were identified with the standardFASP protocol, while 1395 +/- 4 proteins were identified in 20 mu g after standard FASPas a benchmark. Furthermore, we conducted a combined peptidomic and proteomicstudy of single pancreatic islets with well-plate mu FASP. Here, we separated neuropeptides and digested the remaining on-filterproteins for bottom-up proteomic analysis. Our results indicate inter-islet heterogeneity for the expression of proteins involved inglucose catabolism, pancreatic hormone processing, and secreted peptide hormones. We consider our method to provide a usefultool for proteomic characterization of samples where the biological material is scarce. All proteomic data are available under DOI:10.6019/PXD029039

Ämnesord

NATURVETENSKAP  -- Biologi -- Biokemi och molekylärbiologi (hsv//swe)

NATURAL SCIENCES  -- Biological Sciences -- Biochemistry and Molecular Biology (hsv//eng)

Nyckelord

filter-aided sample preparation

islets of Langerhans

liquid chromatography-mass spectrometry

peptidomics

proteomics

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