Incorporation of a Reporter Peptide in FPOP Compensates for Adventitious Scavengers and Permits Time-Dependent Measurements

Niu, Ben; Mackness, Brian C.; Rempel, Don L.; Zhang, Hao; Cui, Weidong; Matthews, C. Robert; Zitzewitz, Jill A.; Gross, Michael L.

doi:10.1007/s13361-016-1552-4

Cited by 34 publications

(39 citation statements)

References 22 publications

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“…Although small changes in protein concentration usually will not affect oxidation, large variations in ·OH may occur from sample to sample when the solution contains adventitious scavengers (e.g., TCEP, ATP, enzyme cofactors, lipids, DMSO, unexpected proteins). One approach to this problem is to add a short peptide (e.g., leu-enkephalin, YGGFL) as a reporter to calibrate oxidative potential (Figure 6A) [47]. The reporter peptide must have exposed reactive residues but should not bind to the protein.…”

Section: Expansion Of the Fpop Platformmentioning

confidence: 99%

“…Longer times of reaction give higher levels of reporter oxidation. Plotting extents of protein modification vs. those of the reporter gives a time-dependent, dose-response curve that clearly pinpoints when differences in solvent accessibility occur in protein states (Figure 6B) [47]. The reporter peptide approach achieves spatial resolution at the peptide and sometimes the residue levels.…”

Section: Expansion Of the Fpop Platformmentioning

confidence: 99%

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Implementing fast photochemical oxidation of proteins (FPOP) as a footprinting approach to solve diverse problems in structural biology

Zhang

Cheng

Rempel

et al. 2018

Methods

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Fast photochemical oxidation of proteins (FPOP) is a footprinting technique used in mass spectrometry-based structural proteomics. It has been applied to solve a variety of problems in different areas of biology. A FPOP platform requires a laser, optics, and sample flow path properly assembled to enable fast footprinting. Sample preparation, buffer conditions, and reagent concentrations are essential to obtain reasonable oxidations on proteins. FPOP samples can be analyzed by LC-MS methods to measure the modification extent, which is a function of the solvent-accessible surface area of the protein. The platform can be expanded to accommodate several new approaches, including dose-response studies, new footprinting reagents, and two-laser pump-probe experiments. Here, we briefly review FPOP applications and in a detailed manner describe the procedures to set up an FPOP protein footprinting platform.

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Section: Expansion Of the Fpop Platformmentioning

confidence: 99%

Section: Expansion Of the Fpop Platformmentioning

confidence: 99%

Implementing fast photochemical oxidation of proteins (FPOP) as a footprinting approach to solve diverse problems in structural biology

Zhang

Cheng

Rempel

et al. 2018

Methods

Self Cite

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show abstract

“…Dosimetry measurements also consider the differences in laser energy between both intra-and inter-day experiments. A few approaches have been used to measure a radical dose, including using adenine as a dosimeter with UV absorption detection (96), derivatized phenylalanine with isotope dilution GC/MS detection (97), and a reporter peptide that does not require additional detection (98). An adoption of one dosimetry method across all FPOP labs would better standardize the method and make it more widely applicable.…”

Section: Outlook: Development Of New Footprinting Methods and In Vivomentioning

confidence: 99%

Fast photochemical oxidation of proteins (FPOP): A powerful mass spectrometry–based structural proteomics tool

Johnson

Stefano

Jones

2019

Journal of Biological Chemistry

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Edited by Wolfgang Peti Fast photochemical oxidation of proteins (FPOP) is a MSbased method that has proved useful in studies of protein structures, interactions, conformations, and protein folding. The success of this method relies on the irreversible labeling of solvent-exposed amino acid side chains by hydroxyl radicals. FPOP generates these radicals through laser-induced photolysis of hydrogen peroxide. The data obtained provide residue-level resolution of protein structures and interactions on the microsecond timescale, enabling investigations of fast processes such as protein folding and weak protein-protein interactions. An extensive comparison between FPOP and other footprinting techniques gives insight on their complementarity as well as the robustness of FPOP to provide unique structural information once unattainable. The versatility of this method is evidenced by both the heterogeneity of samples that can be analyzed by FPOP and the myriad of applications for which the method has been successfully used: from proteins of varying size to intact cells. This review discusses the wide applications of this technique and highlights its high potential. Applications including, but not limited to, protein folding, membrane proteins, structure elucidation, and epitope mapping are showcased. Furthermore, the use of FPOP has been extended to probing proteins in cells and in vivo. These promising developments are also presented herein.

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“…Scavengers (glutamine or histidine) are also added to the solution at concentrations that limit the lifetime of ˙ OH to less than ∼1 µs (i.e., the labeling pulse is faster than protein folding/unfolding) ( Hambly and Gross 2005 ; Gau et al, 2013 ; Yan et al, 2014 ), although recent evidence suggests that radicals may be longer-lived ( Vahidi and Konermann 2016 ). Dose-response experiments can also be performed by increasing/decreasing the scavenger concentration to alter the length of the labeling pulse ( Niu et al, 2017 ). In this example, samples were taken at periodic intervals over a 48 h time course of Aβ 1–42 aggregation.…”

Section: Footprinting Mass Spectrometrymentioning

confidence: 99%

Structural Proteomics Methods to Interrogate the Conformations and Dynamics of Intrinsically Disordered Proteins

Beveridge

Calabrese

2021

Front. Chem.

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Intrinsically disordered proteins (IDPs) and regions of intrinsic disorder (IDRs) are abundant in proteomes and are essential for many biological processes. Thus, they are often implicated in disease mechanisms, including neurodegeneration and cancer. The flexible nature of IDPs and IDRs provides many advantages, including (but not limited to) overcoming steric restrictions in binding, facilitating posttranslational modifications, and achieving high binding specificity with low affinity. IDPs adopt a heterogeneous structural ensemble, in contrast to typical folded proteins, making it challenging to interrogate their structure using conventional tools. Structural mass spectrometry (MS) methods are playing an increasingly important role in characterizing the structure and function of IDPs and IDRs, enabled by advances in the design of instrumentation and the development of new workflows, including in native MS, ion mobility MS, top-down MS, hydrogen-deuterium exchange MS, crosslinking MS, and covalent labeling. Here, we describe the advantages of these methods that make them ideal to study IDPs and highlight recent applications where these tools have underpinned new insights into IDP structure and function that would be difficult to elucidate using other methods.

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Incorporation of a Reporter Peptide in FPOP Compensates for Adventitious Scavengers and Permits Time-Dependent Measurements

Cited by 34 publications

References 22 publications

Implementing fast photochemical oxidation of proteins (FPOP) as a footprinting approach to solve diverse problems in structural biology

Implementing fast photochemical oxidation of proteins (FPOP) as a footprinting approach to solve diverse problems in structural biology

Fast photochemical oxidation of proteins (FPOP): A powerful mass spectrometry–based structural proteomics tool

Structural Proteomics Methods to Interrogate the Conformations and Dynamics of Intrinsically Disordered Proteins

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