In Cell Footprinting Coupled with Mass Spectrometry for the Structural Analysis of Proteins in Live Cells

Abstract

Protein footprinting coupled with mass spectrometry has become a widely used tool for the study of protein-protein and protein-ligand interactions and protein conformational change. These methods provide residue-level analysis on protein interaction sites and have been successful in studying proteins in vitro. The extension of these methods for in cell footprinting would open an avenue to study proteins that are not amenable for in vitro studies and would probe proteins in their native environment. Here we describe the application of an oxidative-based footprinting approach inside cells in which hydroxyl radicals are used to oxidatively modify proteins. Mass spectrometry is used to detect modification sites and to calculate modification levels. The method is probing biologically relevant proteins in live cells, and proteins in various cellular compartments can be oxdiatively modified. Several different amino acid residues are modified making the method a general labeling strategy for the study of a variety of proteins. Further, comparison of the extent of oxidative modification with solvent accessible surface area reveals the method successfully probes solvent accessibility. This marks the first time protein footprinting has been performed in live cells.

Probing the Time Scale of FPOP (Fast Photochemical Oxidation of Proteins): Radical Reactions Extend Over Tens of Milliseconds

“…A GAM EX50 excimer laser (Orlando, FL, USA) generating 18 ns laser pulses at 64 Hz, 248 nm, and 37.5 mJ was used for · OH production 40 cm downstream of the mixer. These experimental parameters are well within the range of earlier FPOP experiments [30,34,36,37,39,40,53]. The width of the laser beam at the irradiation spot was 1 mm, as determined from paper burn patterns; 160 μL capillary outflow aliquots were collected 1 s after labeling in microcentrifuge tubes containing 80 μL of 100 mM phosphate buffer, 5 μM Met, and 1 μM catalase prior to flash freezing in liquid nitrogen and lyophilization.…”

“…Secondly, FPOP holds promise for examining short-lived protein folding intermediates [33]. FPOP has been adopted by various researchers, and it has been applied to a range of protein systems [34][35][36][37][38][39][40][41][42][43][44][45].…”

The Application of Fluorine‐Containing Reagents in Structural Proteomics

“…A goal in structural proteomics is the determination of endogenous protein structure in live cells . The efficient isolation of the targeted peptide subset from the crude sample is crucial to success in reaching that goal.…”

Fluorierte Reagenzien in der Strukturproteomik

“…Ein Ziel in der Strukturproteomik ist die Bestimmung der endogenen Proteinstruktur in lebenden Zellen . Die Effizienz, mit der Zielpeptide aus der biologischen Probe isoliert werden können, ist für den Erfolg dieser Strategie entscheidend.…”