Fast photochemical oxidation of proteins (FPOP) employs laser photolysis of hydrogen peroxide to give OH radicals that label amino acid side-chains of proteins on the microsecond time scale. A method for quantitation of hydroxyl radicals after laser photolysis is of importance to FPOP because it establishes a means to adjust the yield of •OH, offers the opportunity of tunable modifications, and provides a basis for kinetic measurements. The initial concentration of OH radicals has yet to be measured experimentally. We report here an approach using isotope dilution gas chromatography/mass spectrometry (GC/MS) to determine quantitatively the initial •OH concentration (we found ~ 0.95 mM from 15 mM H2O2) from laser photolysis and to investigate the quenching efficiencies for various •OH scavengers.
The small GTPase KRAS is localized at the plasma membrane where it functions as a molecular switch, coupling extracellular growth factor stimulation to intracellular signaling networks. In this process, KRAS recruits effectors, such as RAF kinase, to the plasma membrane where they are activated by a series of complex molecular steps. Defining the membrane-bound state of KRAS is fundamental to understanding the activation of RAF kinase and in evaluating novel therapeutic opportunities for the inhibition of oncogenic KRAS-mediated signaling. We combined multiple biophysical measurements and computational methodologies to generate a consensus model for authentically processed, membrane-anchored KRAS. In contrast to the two membrane-proximal conformations previously reported, we identify a third significantly populated state using a combination of neutron reflectivity, fast photochemical oxidation of proteins (FPOP), and NMR. In this highly populated state, which we refer to as “membrane-distal” and estimate to comprise ∼90% of the ensemble, the G-domain does not directly contact the membrane but is tethered via its C-terminal hypervariable region and carboxymethylated farnesyl moiety, as shown by FPOP. Subsequent interaction of the RAF1 RAS binding domain with KRAS does not significantly change G-domain configurations on the membrane but affects their relative populations. Overall, our results are consistent with a directional fly-casting mechanism for KRAS, in which the membrane-distal state of the G-domain can effectively recruit RAF kinase from the cytoplasm for activation at the membrane.
The Multi-Attribute
Method (MAM) Consortium was initially formed
as a venue to harmonize best practices, share experiences, and generate
innovative methodologies to facilitate widespread integration of the
MAM platform, which is an emerging ultra-high-performance liquid chromatography–mass
spectrometry application. Successful implementation of MAM as a purity-indicating
assay requires new peak detection (NPD) of potential process- and/or
product-related impurities. The NPD interlaboratory study described
herein was carried out by the MAM Consortium to report on the industry-wide
performance of NPD using predigested samples of the NISTmAb Reference
Material 8671. Results from 28 participating laboratories show that
the NPD parameters being utilized across the industry are representative
of high-resolution MS performance capabilities. Certain elements of
NPD, including common sources of variability in the number of new
peaks detected, that are critical to the performance of the purity
function of MAM were identified in this study and are reported here
as a means to further refine the methodology and accelerate adoption
into manufacturer-specific protein therapeutic product life cycles.
Incorporation of a reporter peptide in solutions submitted to fast photochemical oxidation of proteins (FPOP) allows for the correction of adventitious scavengers and enables the normalization and comparison of time-dependent results. Reporters will also be useful in differential experiments to control for the inclusion of a radical-reactive species. This incorporation provides a simple and quick check of radical dosage and allows comparison of FPOP results from day-to-day and lab-to-lab. Use of a reporter peptide in the FPOP workflow requires no additional measurements or spectrometers while building a more quantitative FPOP platform. It requires only measurement of the extent of reporter-peptide modification in a LC/MS/MS run, which is performed by using either data-dependent scanning or an inclusion list.
Blocking interactions between PD-1 and PD-L1 opens a new era of cancer treatment involving immunity modulation. Although most immunotherapies use monoclonal antibodies, smallmolecule inhibitors offer advantages. To facilitate development of small-molecule therapeutics, we implemented a rapid approach to characterize the binding interfaces of small molecule inhibitors with PD-L1. We determined its interaction with a synthetic macrocyclic peptide by using two mass-spectrometry-based approaches, hydrogen-deuterium exchange and fast photochemical oxidation of proteins or FPOP and corroborated the findings with our X-ray structure of the PD-L1-macrocycle complex. Although all three approaches show that macrocycle binds directly to PD-L1 over regions 46-87 and 114-125, the two protein footprinting approaches show additional binding at the N-terminus of PD-L1, and FPOP reveals some critical binding residues. The outcomes not only show the binding regions but also demonstrate the utility of MS-based footprinting to probe of protein-ligand inhibitory interactions in cancer immunotherapy.
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