An Improved Fast Photochemical Oxidation of Proteins (FPOP) Platform for Protein Therapeutics

Zhang, Ying; Rempel, Don L.; Zhang, Hao; Gross, Michael L.

doi:10.1007/s13361-014-1055-0

Cited by 35 publications

(39 citation statements)

References 23 publications

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“…The unmodified protein is the most abundant followed by a singly oxidized species (+15.9949 Da), a doubly oxidized species (+31.9898 Da), etc. (Figure 1A) according to the fraction modified [18]. The kinetics of oxidative modification at the protein level are relative to the oxidative modification of reporter leu-enkephalin (M + 15.9949), determined from the extracted ion chromatograms (EICs) of both unmodified and modified leu-enkephalin (mass tolerance of 15 ppm; see Figure S2).…”

Section: Resultsmentioning

confidence: 99%

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

Niu

Mackness

Rempel

et al. 2016

J. Am. Soc. Mass Spectrom.

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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.

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Section: Resultsmentioning

confidence: 99%

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

Niu

Mackness

Rempel

et al. 2016

J. Am. Soc. Mass Spectrom.

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“…FPOP was performed as described previously [10]. A 248 nm KrF excimer laser (GAM Laser Inc., Orlando, FL) adjusted to approximately 30 mJ/pulse was used to irradiate the flowing sample solution.…”

Section: Methodsmentioning

confidence: 99%

“…For quantitative analysis of oxidative modification at the peptide level, only the intensities of the Extracted Ion Chromatogram (XIC) that were identified as corresponding to either unmodified peptides or related modified peptides from VEGF or Fab-1 were used to report extent of modification [10]. …”

Section: Methodsmentioning

confidence: 99%

“…Here, we report epitope and paratope mapping by determining the extent of labeling for the unbound Fab-1 and VEGF, and compare this information to the Fab-1:VEGF complex, using our recently improved FPOP format [10]. We observed several tryptic peptides with significantly reduced modification in the bound state and used high-resolution MS 2 to obtain residue-level information.…”

Section: Introductionmentioning

confidence: 99%

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Mapping the Binding Interface of VEGF and a Monoclonal Antibody Fab-1 Fragment with Fast Photochemical Oxidation of Proteins (FPOP) and Mass Spectrometry

Zhang

Wecksler²,

Molina³

et al. 2017

J. Am. Soc. Mass Spectrom.

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We previously analyzed the Fab-1:VEGF (vascular endothelial growth factor) system described in this work, with both native top-down mass spectrometry and bottom-up mass spectrometry (carboxyl-group or GEE footprinting) techniques. This work continues bottom-up mass spectrometry analysis using a Fast Photochemical Oxidation of Proteins (FPOP) platform to map the solution binding-interface of VEGF and a fragment antigen-binding region of an antibody (Fab-1). In this study, we use FPOP to compare the changes in solvent accessibility by quantitating the extent of oxidative modification in the unbound versus bound states. Determining the changes in solvent accessibility enables the inference of the protein binding sites (epitope and paratopes) and a comparison to the previously published Fab-1:VEGF crystal structure. Using this method, we investigated peptide-level and residue-level changes in solvent accessibility between the unbound proteins and bound complex. Mapping these data onto the Fab-1:VEGF crystal structure enabled successful characterization of both the binding region and regions of remote conformation changes. These data, coupled with our previous higher order structure (HOS) studies, demonstrate the value of a comprehensive toolbox of methods for identifying the putative epitopes and paratopes for biotherapeutic antibodies.

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“…11,14-16 FPOP-MS has been applied to analyze the structure of a variety of purified proteins, in solution, using flow systems. [17][18][19][20] However its application for analyzing membrane transporters has been hampered by their difficult preparation and structural complications. 21 A physiologically important example of membrane transporters is cystic fibrosis transmembrane conductance regulator (CFTR).…”

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confidence: 99%

Development of Structural Marker Peptides for Cystic Fibrosis Transmembrane Conductance Regulator in Cell Plasma Membrane by Reversed-Footprinting Mass Spectrometry

Farrokhi¹,

Bajrami²,

Nemati³

et al. 2015

Anal. Chem.

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A targeted mass spectrometry-based method is presented that adopts the fast photochemical oxidation of proteins (FPOP) for footprinting of cystic fibrosis transmembrane conductance regulator (CFTR) membrane transporter at its original plasma membrane location. Two analytical imperatives were sought: (1) overall simplification in data acquisition and analysis and (2) lower quantitation limits, which enabled direct analysis of intrinsically low-abundance transmembrane proteins. These goals were achieved by using a reversed-footprinting technique that monitored the unoxidized peptides remaining after the FPOP treatment. In searching for structurally informative peptides, a workflow was designed for accurate and precise quantitation of CFTR peptides produced from proteolytically digesting the plasma membrane subproteome of cells. This sample preparation strategy mitigated the need for challenging purification of large quantities of structurally intact CFTR. On the basis of the interrogated peptides, it was proposed a concept of the structural marker peptide that could report CFTR structure and function in cells. The reversed-footprinting mass spectrometry extends the FPOP technology to study conformation and interaction changes of low-abundance proteins directly in their endogenous cellular locations.

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An Improved Fast Photochemical Oxidation of Proteins (FPOP) Platform for Protein Therapeutics

Cited by 35 publications

References 23 publications

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

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

Mapping the Binding Interface of VEGF and a Monoclonal Antibody Fab-1 Fragment with Fast Photochemical Oxidation of Proteins (FPOP) and Mass Spectrometry

Development of Structural Marker Peptides for Cystic Fibrosis Transmembrane Conductance Regulator in Cell Plasma Membrane by Reversed-Footprinting Mass Spectrometry

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