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Mass-spectrometric exploration of proteome structure and function : Nature : Nature Research

Mass-spectrometric exploration of proteome structure and function : Nature : Nature Research | Mass Spectrometry Daily | Scoop.it
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Kelleher Lab IDs 5000-plus Proteoforms in Largest Human Top-Down Analysis ... - Proteo Monitor

Kelleher Lab IDs 5000-plus Proteoforms in Largest Human Top-Down Analysis ... - Proteo Monitor | Mass Spectrometry Daily | Scoop.it
Kelleher Lab IDs 5000-plus Proteoforms in Largest Human Top-Down Analysis ...
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Extending the Isotopically Resolved Mass Range of Orbitrap Mass Spectrometers - Analytical Chemistry (ACS Publications)

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Jared B. Shaw and Jennifer S. Brodbelt 

 

Abstract

 

The routine analysis of large biomolecules (greater than 30 kDa) has been a challenge for Orbitrap mass spectrometers due to the relatively high kinetic energy of ions entering and within the Orbitrap mass analyzer. This characteristic results in rapid signal decay for large biomolecules due to energetic collisions with background gas molecules. Here, we report a method to significantly enhance the analysis of large biomolecules in an Orbitrap mass spectrometer. The combination of reduced C-trap and higher energy collisional dissociation (HCD) cell bath gas pressures, using helium as the bath gas and trapping ions in the HCD cell prior to mass analysis, greatly increased sensitivity and reduced signal decay for large protein ions. As a result, isotopic resolution of monoclonal immunoglobulin G was achieved, and we have established a new high-mass record for which accurate mass measurement and isotopic resolution have been achieved.

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Absolute quantification of transcription factors during cellular differentiation using multiplexed targeted proteomics

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Jovan Simicevic, Adrien W Schmid, Paola A Gilardoni, Benjamin Zoller, Sunil K Raghav, Irina Krier, Carine Gubelmann, Frédérique Lisacek, Felix Naef, Marc Moniatte & Bart Deplancke

 

Abstract

 

The cellular abundance of transcription factors (TFs) is an important determinant of their regulatory activities. Deriving TF copy numbers is therefore crucial to understanding how these proteins control gene expression. We describe a sensitive selected reaction monitoring–based mass spectrometry assay that allowed us to determine the copy numbers of up to ten proteins simultaneously. We applied this approach to profile the absolute levels of key TFs, including PPARγand RXRα, during terminal differentiation of mouse 3T3-L1 pre-adipocytes. Our analyses revealed that individual TF abundance differs dramatically (from ~250 to >300,000 copies per nucleus) and that their dynamic range during differentiation can vary up to fivefold. We also formulated a DNA binding model for PPARγ based on TF copy number, binding energetics and local chromatin state. This model explains the increase in PPARγ binding sites during the final differentiation stage that occurs despite a concurrent saturation in PPARγ copy number.

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Improving Low Abundance Protein Identification in Complex Proteome Samples

Search for application articles, scientific posters, methods, techniques and case studies for your laboratory science application on SelectScience.net.
Sasa Miladinovic's insight:

NanoLC coupled with nanospray ionization (NSI) MS has become the standard for identification of low abundance proteins in complex proteomics samples. Although this technique provides sensitivity and resolution, the low flow rates used with this technique often result in long sample loading times, long gradient delays and long system re-equilibration times. The MS only generates useful MSMS spectra during the LC gradient time, resulting in MS utilization rates of only 20-50% of the total run time (inject to inject). This application note presents a method to increase the relative MS utilization time without impacting sensitivity or resolution.

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Simultaneous Quantitative and Qualitative Analysis of Proteolytic Digests of Therapeutic Monoclonal Antibodies using a TripleTOF® System

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In recent years MS has emerged as a superior method for characterizing the heterogeneity of monoclonal antibodies. This application note presents a high-speed LC/MS/MS method for the analysis of enzymatically digested therapeutic antibodies. Sophisticated software tools, that enable more accurate peptide mapping, are also described.

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A MASSive Laboratory Tour. An Interactive Mass Spectrometry Outreach Activity for Children

A MASSive Laboratory Tour. An Interactive Mass Spectrometry Outreach Activity for Children | Mass Spectrometry Daily | Scoop.it
Sasa Miladinovic's insight:

Julia H. Jungmann, Nadine E. Mascini, Andras Kiss, Donald F. Smith, Ivo Klinkert, Gert B. Eijkel, Marc C. Duursma, Berta Cillero Pastor, Kamila Chughtai, Sanaullah Chughtai, Ron M. A. Heeren

 

Abstract

 

It is imperative to fascinate young children at an early stage in their education for the analytical sciences. The exposure of the public to mass spectrometry presently increases rapidly through the common media. Outreach activities can take advantage of this exposure and employ mass spectrometry as an exquisite example of an analytical science in which children can be fascinated. The presented teaching modules introduce children to mass spectrometry and give them the opportunity to experience a modern research laboratory. The modules are highly adaptable and can be applied to young children from the age of 6 to 14 y. In an interactive tour, the students explore three major scientific concepts related to mass spectrometry; the building blocks of matter, charged particle manipulation by electrostatic fields, and analyte identification by mass analysis. Also, the students carry out a mass spectrometry experiment and learn to interpret the resulting mass spectra. The multistage, inquiry-based tour contains flexible methods, which teach the students current-day research techniques and possible applications to real research topics. Besides the scientific concepts, laboratory safety and hygiene are stressed and the students are enthused for the analytical sciences by participating in “hands-on” work. The presented modules have repeatedly been successfully employed during laboratory open days. They are also found to be extremely suitable for (early) high school science classes during laboratory visit-focused field trips.

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Increasing Throughput in Targeted Proteomics Assays: 54-plex Quantitation in a Single Mass Spectrometry Run

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Robert A. Everley , Ryan C Kunz , Fiona E. McAllister , and Steven P. Gygi

 

Abstract

 

Targeted proteomics assays such as those measuring endpoints in activity assays are sensitive and specific but often lack in throughput. In an effort to significantly increase throughput, a comparison was made between the traditional approach which utilizes an internal standard and the multiplexing approach which relies on isobaric tagging. A kinase activity assay was used for proof of concept, and experiments included 3 biological replicates for every condition. Results from the two approaches were highly similar with the multiplexing showing greater throughput. Two novel 6-plex isobaric tags were added for a total of three 6-plex experiments (18-plex) in a single run. Next, three mass variants of the target peptide were labeled with the three isobaric tags giving nine 6-plex reactions for 54-plex quantitation in a single run. Since the multiplexing approach allows all samples to be combined prior to purification and acquisition, the 54-plex approach resulted in a significant reduction in purification resources (time, reagents etc.) and a ~50 fold improvement in acquisition throughput. We demonstrate the 54-plex assay in several ways including measuring inhibition of PKA activity in MCF7 cell lysates for a panel of 9 compounds.

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In-Spray Supercharging of Peptides and Proteins in Electrospray Ionization Mass Spectrometry

In-Spray Supercharging of Peptides and Proteins in Electrospray Ionization Mass Spectrometry | Mass Spectrometry Daily | Scoop.it
Sasa Miladinovic's insight:

Saša M. Miladinović, Luca Fornelli, Yu Lu, Krzysztof M. Piech, Hubert H. Girault, Yury O. Tsybin

 

Abstract

 

Enhanced charging, or supercharging, of analytes in electrospray ionization mass spectrometry (ESI MS) facilitates high resolution MS by reducing an ion mass-to-charge (m/z) ratio, increasing tandem mass spectrometry (MS/MS) efficiency. ESI MS supercharging is usually achieved by adding a supercharging reagent to the electrospray solution. Addition of these supercharging reagents to the mobile phase in liquid chromatography (LC)-MS/MS increases the average charge of enzymatically derived peptides and improves peptide and protein identification in large-scale bottom-up proteomics applications but disrupts chromatographic separation. Here, we demonstrate the average charge state of selected peptides and proteins increases by introducing the supercharging reagents directly into the ESI Taylor cone (in-spray supercharging) using a dual-sprayer ESI microchip. The results are comparable to those obtained by the addition of supercharging reagents directly into the analyte solution or LC mobile phase. Therefore, supercharging reaction can be accomplished on a time-scale of ion liberation from a droplet in the ESI ion source.

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Orbitrap Mass Spectrometry

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Roman A. Zubarev and Alexander Alekseevich Makarov

 

Abstract

 

Orbitrap is the newest addition to the family of high-resolution mass spectrometry analyzers. With its revolutionarily new, miniature design, Orbitrap combines high speed with excellent quantification properties, ranking favorably in many analytical applications.

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Top-Down Structural Analysis of an Intact Monoclonal Antibody by Electron Capture Dissociation-Fourier Transform Ion Cyclotron Resonance-Mass Spectrometry

Top-Down Structural Analysis of an Intact Monoclonal Antibody by Electron Capture Dissociation-Fourier Transform Ion Cyclotron Resonance-Mass Spectrometry | Mass Spectrometry Daily | Scoop.it
Sasa Miladinovic's insight:

Yuan Mao, Santosh G. Valeja, Jason C. Rouse, Christopher L. Hendrickson, and Alan G. Marshall

 

ABSTRACT

 

Top-down electron capture dissociation (ECD) Fourier transform ion cyclotron resonance (FTICR) mass spectrometry was performed for structural analysis of an intact monoclonal antibody (IgG1kappa (κ) isotype, 148 kDa). Simultaneous ECD for all charge states (42+ to 58+) generates more extensive cleavages than ECD for an isolated single charge state. The cleavages are mainly localized in the variable domains of both heavy and light chains, the respective regions between the variable and constant domains in both chains, the region between heavy-chain constant domains CH2 and CH3, and the disulfide bond (S–S)-linked heavy-chain constant domain CH3. The light chain yields mainly N-terminal fragment ions due to the protection of the interchain disulfide bond between light and heavy chain, and limited cleavage sites are observed in the variable domains for each chain, where the S–S spans the polypeptide backbone. Only a few cleavages in the S–S-linked light-chain constant domain, hinge region, and heavy-chain constant domains CH1 and CH2 are observed, leaving glycosylation uncharacterized. Top-down ECD with a custom-built 9.4 T FTICR mass spectrometer provides more extensive sequence coverage for structural characterization of IgG1κ than does top-down collision-induced dissociation (CID) and electron transfer dissociation (ETD) with hybrid quadrupole time-of-flight instruments and comparable sequence coverage for top-down ETD with orbitrap mass analyzers.

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Bruker Introduce the High-Performance LC Triple Quadrupole Mass Spectrometers: | Bruker Corporation

Bruker Introduce the High-Performance LC Triple Quadrupole Mass Spectrometers: | Bruker Corporation | Mass Spectrometry Daily | Scoop.it
EVOQ Qube and EVOQ Elite LC-TQ Systems at Pittsburgh Conference for the First Time

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Beyond the Genome

Beyond the Genome | Mass Spectrometry Daily | Scoop.it
... in mass spectrometry and separations science. Genomics benefited first. Now, spectrometry techniques are moving to other omics disciplines, contributing to broad understanding of the proteome through targeted measurements.
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Multi-omics Cloud-Computing Environment | AB SCIEX

Multi-omics Cloud-Computing Environment | AB SCIEX | Mass Spectrometry Daily | Scoop.it
The SWATH Proteomics Cloud Toolkit provides the ability to upload and process data, as well as visualize protein expression results, all securely in the cloud.
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Front-End Electron Transfer Dissociation: A New Ionization Source - Analytical Chemistry (ACS Publications)

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Lee Earley, Lissa C. Anderson, Dina L. Bai, Christopher Mullen, John E. P. Syka, A. Michelle English, Jean-Jacques Dunyach, George C. Stafford , Jr, Jeffrey Shabanowitz, Donald F. Hunt, and Philip D. Compton

 

Abstract

 

Electron transfer dissociation (ETD), a technique that provides efficient fragmentation while depositing little energy into vibrational modes, has been widely integrated into proteomics workflows. Current implementations of this technique, as well as other ion–ion reactions like proton transfer, involve sophisticated hardware, lack robustness, and place severe design limitations on the instruments to which they are attached. Described herein is a novel, electrical discharge-based reagent ion source that is located in the first differentially pumped region of the mass spectrometer. The reagent source was found to produce intense reagent ion signals over extended periods of time while having no measurable impact on precursor ion signal. Further, the source is simple to construct and enables implementation of ETD on any instrument without modification to footprint. Finally, in the context of hybrid mass spectrometers, relocation of the reagent ion source to the front of the mass spectrometer enables new approaches to gas phase interrogation of intact proteins.

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Elemental Composition Validation from Stored Waveform Inverse Fourier Transform (SWIFT) Isolation FT-ICR MS Isotopic Fine Structure

Journal: Journal of The American Society for Mass Spectrometry ArticleTitle: Elemental Composition Validation from Stored Waveform Inverse Fourier Transform (SWIFT) Isolation FT-ICR MS Isotopic Fine Structure...
Sasa Miladinovic's insight:

Brian M. Ruddy, Gregory T. Blakney, Ryan P. Rodgers, Christopher L. Hendrickson, Alan G. Marshall

 

Abstract

 

Elemental composition assignment confidence in mass spectrometry is typically assessed by monoisotopic mass accuracy. For a given mass accuracy, resolution and detection of other isotopologues can further narrow the number of possible elemental compositions. However, such measurements require ultrahigh resolving power and high dynamic range, particularly for compounds containing low numbers of nitrogen and oxygen (both 15N and 18O occur at less than 0.4 % natural abundance). Here, we demonstrate validation of molecular formula assignment from isotopic fine structure, based on ultrahigh resolution broadband Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR MS). Dynamic range is enhanced by external quadrupole and internal stored waveform inverse Fourier transform (SWIFT) isolation to facilitate detection of low abundance heavy atom isotopologues.

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Absorption Mode Fourier Transform Electrostatic Linear Ion Trap Mass Spectrometry

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Ryan T. Hilger , Phillip Wyss , Robert E. Santini , and Scott A. McLuckey

 

Abstract

 

In Fourier transform mass spectrometry, it is well known that plotting the spectrum in absorption mode rather than magnitude mode has several advantages. However, magnitude spectra remain commonplace due to difficulties associated with determining the phase of each frequency at the onset of data acquisition, which is required for generating absorption spectra. The phasing problem for electrostatic traps is much simpler than for FT-ICR instruments, which greatly simplifies the generation of absorption spectra. Here we present a simple method for generating absorption spectra from a Fourier transform electrostatic linear ion trap mass spectrometer. The method involves time shifting the data prior to Fourier transformation in order to synchronize the onset of data acquisition with the moment of ion acceleration into the electrostatic trap. Under these conditions, the initial phase of each frequency at the onset of data acquisition is zero. We demonstrate that absorption mode provides a 1.7 fold increase in resolution (FWHM) as well as reduced peak tailing. We also discuss methodology that may be applied to unsynchronized data in order to determine the time shift required to generate an absorption spectrum.

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High Definition Multiple Reaction Monitoring: The Application of Quadrupole Ion Mobility Time-of-Flight Mass Spectrometry of Targeted Proteomic Studies

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Sasa Miladinovic's insight:

Recently the specificity of SRM/MRM peptide quantification has been questioned, prompting the use of high-resolution accurate mass instruments for peptide quantification. This application note demonstrates the benefits of the SYNAPT G2-Si System, in a new High Definition MRM mode, for the quantification of proteins and peptides.

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Target-Decoy Approach and False Discovery Rate: When Things May Go Wrong

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Nitin Gupta, Nuno Bandeira, Uri Keich, Pavel A. Pevzner


Abstract


The target-decoy approach (TDA) has done the field of proteomics a great service by filling in the need to estimate the false discovery rates (FDR) of peptide identifications. While TDA is often viewed as a universal solution to the problem of FDR evaluation, we argue that the time has come to critically re-examine TDA and to acknowledge not only its merits but also its demerits. We demonstrate that some popular MS/MS search tools are not TDA-compliant and that it is easy to develop a non-TDA compliant tool that outperforms all TDA-compliant tools. Since the distinction between TDA-compliant and non-TDA compliant tools remains elusive, we are concerned about a possible proliferation of non-TDA-compliant tools in the future (developed with the best intentions). We are also concerned that estimation of the FDR by TDA awkwardly depends on a virtual coin toss and argue that it is important to take the coin toss factor out of our estimation of the FDR. Since computing FDR via TDA suffers from various restrictions, we argue that TDA is not needed when accurate p-values of individual Peptide-Spectrum Matches are available.

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New Perfinity iDP: Automated HPLC Trypsin Digestion Platform | Shimadzu

New Perfinity iDP: Automated HPLC Trypsin Digestion Platform | Shimadzu | Mass Spectrometry Daily | Scoop.it
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The Perfinity iDP (Integrated Digestion Platform) automates the protein analysis workflow from protein digestion to HPLC separation and MS detection, significantly reducing sample preparation times and enhancing reproducibility. 
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Multipurpose Dissociation Cell for Enhanced ETD of Intact Protein Species

Multipurpose Dissociation Cell for Enhanced ETD of Intact Protein Species | Mass Spectrometry Daily | Scoop.it
Sasa Miladinovic's insight:

Christopher M. Rose, Jason D. Russell, Aaron R. Ledvina, Graeme C. McAlister, Michael S. Westphall, Jens Griep-Raming, Jae C. Schwartz, Joshua J. Coon, John E. P. Syka

 

Abstract

 

We describe and characterize an improved implementation of ETD on a modified hybrid linear ion trap-Orbitrap instrument. Instead of performing ETD in the mass-analyzing quadrupole linear ion trap (A-QLT), the instrument collision cell was modified to enable ETD. We partitioned the collision cell into a multi-section rf ion storage and transfer device to enable injection and simultaneous separate storage of precursor and reagent ions. Application of a secondary (axial) confinement voltage to the cell end lens electrodes enables charge-sign independent trapping for ion–ion reactions. The approximately 2-fold higher quadrupole field frequency of this cell relative to that of the A-QLT enables higher reagent ion densities and correspondingly faster ETD reactions, and, with the collision cell’s longer axial dimensions, larger populations of precursor ions may be reacted. The higher ion capacity of the collision cell permits the accumulation and reaction of multiple full loads of precursor ions from the A-QLT followed by FT Orbitrap m/z analysis of the ETD product ions. This extends the intra-scan dynamic range by increasing the maximum number of product ions in a single MS/MS event. For analyses of large peptide/small protein precursor cations, this reduces or eliminates the need for spectral averaging to achieve acceptable ETD product ion signal-to-noise levels. Using larger ion populations, we demonstrate improvements in protein sequence coverage and aggregate protein identifications in LC-MS/MS analysis of intact protein species as compared to the standard ETD implementation.

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Laserspray and Matrix-Assisted Ionization Inlet Coupled to High-Field FT-ICR Mass Spectrometry for Peptide and Protein Analysis

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Leonard Nyadong, Ellen D. Inutan, Xu Wang, Christopher L. Hendrickson, Sarah Trimpin and Alan G. Marshall

 

Abstract

 

We present the first coupling of laser spray ionization inlet (LSII) and matrix assisted ionization inlet (MAII) to high-field Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR MS) for generation of electrospray-like ions to take advantage of increased sensitivity, mass range, and mass resolving power afforded by multiple charging. We apply the technique to top-down protein analysis and characterization of metalloproteins. We also present a novel method for generation of multiply-charged copper–peptide complexes with varying degrees of copper adduction by LSII. We show an application of the generated copper–peptide complexes for protein charge state and molecular weight determination, particularly useful for an instrument such as a linear ion trap mass analyzer.

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Charge Site Mass Spectra: Conformation-Sensitive Components of the Electron Capture Dissociation Spectrum of a Protein

Charge Site Mass Spectra: Conformation-Sensitive Components of the Electron Capture Dissociation Spectrum of a Protein | Mass Spectrometry Daily | Scoop.it
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Owen S. Skinner, Kathrin Breuker, Fred W. McLafferty

 

Abstract


A conventional electron capture dissociation (ECD) spectrum of a protein is uniquely characteristic of the first dimension of its linear structure. This sequence information is indicated by summing the primary c m+ and z m+• products of cleavage at each of its molecular ion’s inter-residue bonds. For example, the ECD spectra of ubiquitin (M + nH)n+ ions, n = 7–13, provide sequence characterization of 72 of its 75 cleavage sites from 1843 ions in seven c (1–7)+ and eight z (1–8)+• spectra and their respective complements. Now we find that each of these c/z spectra is itself composed of “charge site (CS)” spectra, the c m+ or z m+• products of electron capture at a specific protonated basic residue. This charge site has been H-bonded to multiple other residues, producing multiple precursor ion forms; ECD at these residues yields the multiple products of that CS spectrum. Closely similar CS spectra are often formed from a range of charge states of ubiquitin and KIX ions; this indicates a common secondary conformation, but not the conventional α-helicity postulated previously. CS spectra should provide new capabilities for comparing regional conformations of gaseous protein ions and delineating ECD fragmentation pathways.

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Longitudinal thinking

Increasing public and private sector cooperation to support long-term, longitudinal population studies is critical to advance translational research on complex diseases.
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Protein Target Quantification Decision Tree

Protein Target Quantification Decision Tree | Mass Spectrometry Daily | Scoop.it
The utility of mass spectrometry-(MS-) based proteomic platforms and their clinical applications have become an emerging field in proteomics in recent years. Owing to its selectivity ....
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