Well-Plate μFASP for Proteomic Analysis of Single Pancreatic Islets

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.