Abstract
Nanoscale or single-cell technologies are critical for biomedical applications. However, current mass spectrometry (MS)-based proteomic approaches require samples comprising a minimum of thousands of cells to provide in-depth profiling. Here, we report the development of a nanoPOTS (nanodroplet processing in one pot for trace samples) platform for small cell population proteomics analysis. NanoPOTS enhances the efficiency and recovery of sample processing by downscaling processing volumes to <200 nL to minimize surface losses. When combined with ultrasensitive liquid chromatography-MS, nanoPOTS allows identification of ~1500 to ~3000 proteins from ~10 to ~140 cells, respectively. By incorporating the Match Between Runs algorithm of MaxQuant, >3000 proteins are consistently identified from as few as 10 cells. Furthermore, we demonstrate quantification of ~2400 proteins from single human pancreatic islet thin sections from type 1 diabetic and control donors, illustrating the application of nanoPOTS for spatially resolved proteome measurements from clinical tissues.
Highlights
Nanoscale or single-cell technologies are critical for biomedical applications
NanoPOTS glass chips were microfabricated with photolithographically patterned hydrophilic pedestals surrounded by a hydrophobic surface to serve as nanodroplet reaction vessels for multi-step proteomic sample processing
The chip consisted of the patterned glass slide (Supplementary Figure 1) and a glass spacer, which was sealed to a membrane-coated glass slide to minimize evaporation of the nanowell contents during the various incubation steps (Fig. 1a, b)
Summary
Nanoscale or single-cell technologies are critical for biomedical applications. current mass spectrometry (MS)-based proteomic approaches require samples comprising a minimum of thousands of cells to provide in-depth profiling. Trifluoroethanol-based protein extraction and denaturation[11], filter-aided sample preparation[12], MS-friendly surfactants[14,15], high-temperature trypsin digestion[13], adaptive focused acousticassisted protein extraction[9], and immobilized digestion protocols[12] have achieved some advances in the processing of small samples Using these methods, a proteome coverage of ~600 was reported when 100 cells were analyzed, and thousands of proteins were identified with samples comprising thousands of cells (Table S1)[9,12,13,14,17]. We demonstrate the reproducible quantification of ~2400 proteins from single human pancreatic islet cross-sections isolated from 10-μm-thick pancreatic tissue slices, illustrating the enabling potential for molecular characterization of tissue cellular heterogeneity and pathology in type 1 or type 2 diabetes
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