Well-Plate μFASP for Proteomic Analysis of Single Pancreatic Islets

Abstract

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.

Zero-Degree Celsius Capillary Electrophoresis Electrospray Ionization for Hydrogen Exchange Mass Spectrometry

“…Peptic peptides were sequenced with PLGS 3.0.3 (Waters Corporation, Manchester, United Kingdom), as previously described, , with a modification of setting enzymes to “non-specific.” CE–MS data obtained with UDMS E were searched against a database of UNIPROT peptide sequence entries for HBA_BOVIN, HBB_BOVIN, and PEPA_PIG. Complete data were further processed with the HX-DEAL module in Mass Spec Studio for HDX-analysis …”

“…Unlabeled peptides were analyzed in data independent acquisition (DIA) mode using the UDMS E method with instrument settings previously reported for this instrument, unless otherwise stated. 36 , 37 …”

“…The instrument was calibrated on the day of use using an appropriate dilution of sodium iodide (18600791-3, Waters Corporation, Manchester, United Kingdom) in 50% MeOH and 0.1% formic acid delivered to the electrospray emitter at 500 nL/min through the sheath liquid capillary. Unlabeled peptides were analyzed in data independent acquisition (DIA) mode using the UDMS E method with instrument settings previously reported for this instrument, unless otherwise stated. , …”

“…Unlabeled peptides were analyzed in data independent acquisition (DIA) mode using the UDMS E method with instrument settings previously reported for this instrument, unless otherwise stated. 36,37 Hydrogen-Deuterium Exchange Sample Preparation and Workflow. For HDX−MS, stock solutions of either 2 mg/mL HPLC peptide standard, 4.4 mM ATII with 4.4 mM BK or 120 mg/mL intact bovine Hb were prepared in 50 mM ammonium bicarbonate in H 2 O, pH 6.79.…”

Bottom-Up Proteomics: Advancements in Sample Preparation

“…The authors have also developed a 96-well plate version of the micro-FASP method, which showed high reproducibility (R 2 > 0.99) when processing Xenopus laevis lysate (12 replicates, 1 μg each). Similarly, Sandbaumhüter et al developed a flexible well-plate μFASP device suitable for a small amount of protein (1 μg) [ 113 ]. The filter area for the centrifugal filter units of the conventional FASP was reduced from approximately 119 mm 2 to 0.785 mm 2 for the μFASP device.…”