High-Throughput Proteomics Enabled by a Photocleavable Surfactant

Abstract

Mass spectrometry (MS)-based proteomics provides unprecedented opportunities for understanding the structure and function of proteins in complex biological systems; however, protein solubility and sample preparation before MS remain a bottleneck preventing high-throughput proteomics. Herein, we report a high-throughput bottom-up proteomic method enabled by a newly developed MS-compatible photocleavable surfactant, 4-hexylphenylazosulfonate (Azo) that facilitates robust protein extraction, rapid enzymatic digestion (30 min compared to overnight), and subsequent MS-analysis following UV degradation. Moreover, we developed an Azo-aided bottom-up method for analysis of integral membrane proteins, which are key drug targets and are generally underrepresented in global proteomic studies. Furthermore, we demonstrated the ability of Azo to serve as an "all-in-one" MS-compatible surfactant for both top-down and bottom-up proteomics, with streamlined workflows for high-throughput proteomics amenable to clinical applications.

Keywords: high-throughput proteomics; mass spectrometry; membrane proteins; photocleavable surfactants.

Figure 1.

Enhanced enzymatic digestion and MS analysis using a photocleavable surfactant, Azo. (a) Scheme for Azo-aided bottom-up proteomics. (b) Degradation of Azo by UV irradiation. (c) UV-Vis spectrum for Azo highlighting maximal absorbance at 305 nm. (d) UV-Vis spectrum monitoring the rapid degradation of Azo (0.1% in water) as a function of irradiation time with a 100 W mercury lamp. (d) Digestion of myoglobin [0–1 h, Azo (0–0.2%)] was monitored by SDS-PAGE, stained with Coomassie Blue, and (f) LC-MS analysis using a Q-TOF mass spectrometer. Relative abundances of the extracted ion chromatograms was normalized to 5.5 E8.

Figure 2.

Rapid and reproducible extraction, enzymatic digestion, and LC-MS/MS analysis of proteins from human embryonic kidney 293T (HEK293T) cells (a) SDS-PAGE analysis demonstrates consistent extraction profiles across three extractions (marker (M), 1, 2, 3). Aliquots were taken from each exaction and digested overnight or for 1 h. After LC-MS/MS and MaxQuant identification, a high degree of overlap was observed for both the (b) overnight and (c) 1 h samples. Similarly (d) 1403 of the 1726 combined identified protein groups were observed in both digestion methods. Data collected using Q-TOF.

Figure 3.

Analysis of integral membrane protein enabled by Azo. Proteins were extracted and membrane proteins enriched using a Triton x-114 cloud point extraction procedure, then precipitated, and solubilized again in Azo for in-solution digestion. Additionally, Azo was used for whole cell protein extraction. Using an (a) overnight digestion a significantly higher number of integral membrane proteins were identified using the phase separation enrichment followed by Azo-aided digestion. Similarly a 30 min digestion was performed demonstrating a high-throughput membrane protein analysis methods. (b) A table of representative membrane proteins with a high number of transmembrane domains that were solubilized using Azo. Data represent a single LC-MS experiment.

Figure 4.

Azo-enabled top-down proteomics analysis of cardiac tissue. (a-d) Intact mass spectra of proteoforms analyzed by LC-MS. Data collected using Maxis II Q-TOF.

Scheme 1.

Scheme for azo-enable high-throughput top-down and bottom-up proteomics.