High-Throughput Mass Spectrometry Applied to  Structural Genomics

Chalk, R.; Berridge, G.; Shrestha, L.; Strain-Damerell, Claire; Mahajan, P.; Yue, Wyatt W.; Gileadi, O.; Burgess-Brown, N.

doi:10.3390/chromatography1040159

Cited by 5 publications

(4 citation statements)

References 31 publications

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“…Samples were introduced via an Agilent 1290 HPLC under denaturing conditions or via a syringe pump under native conditions. Denaturing mass spectra and native spectra were acquired as described previously [7] [31]. LC-MSMS spectra were acquired on a Bruker HCT ion trap coupled to a Thermo U3000 nano HPLC using previously described protocols [7],…”

Section: Methodsmentioning

confidence: 99%

“…All proteins generate multiple m/z values in electrospray ionisation under all conditions. Generalizations of this kind are seldom possible in biology, but we have analysed over 10,000 proteins in our laboratory and have always observed this [7]. Indeed, the process of deconvolution which transforms m/z data to neutral spectra both assumes and requires that multiple charge states must exist.…”

Section: Introductionmentioning

confidence: 99%

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Electrospray Surface Charge Describes Protein Molecular Motion

Chalk

Borkowska

Azeez

et al. 2019

Preprint

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The electrospray mass/charge distribution for a protein is an instantaneous measurement of surface areas during the transition from liquid to gas phase. Protonation is dependent upon surface area and surface area is related to protein folding under all conditions. M/z distributions for proteins and protein analogues all with different degrees of entropy were compared. Rigid dipeptide self-assemblies of any size possess a single m/z, whereas protein always displays multiple m/z distributions. Native proteins have narrow, defined m/z distributions, while denatured proteins and synthetic homopolypetides possess the widest possible m/z distributions. These observations are consistent with dynamic changes in surface area resulting from molecular motion.

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Section: Methodsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Electrospray Surface Charge Describes Protein Molecular Motion

Chalk

Borkowska

Azeez

et al. 2019

Preprint

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“…12. Where the targets express well, it is possible to verify the mutation by determining the intact mass on a mass spectrometer [11]. However if the target does not express or is low expressing, then DNA sequencing is required to verify the mutation.…”

Section: Note 11)mentioning

confidence: 99%

High-Throughput Site-Directed Mutagenesis

Strain-Damerell

Burgess-Brown

2019

Methods in Molecular Biology

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Protein engineering has an array of uses; whether you are studying a disease mutation, removing undesirable sequences, adding stabilizing mutations for structural purposes, or simply dissecting protein function. Protein engineering is almost exclusively performed using site-directed mutagenesis (SDM) as this provides targeted modification of specific amino acids, as well as the option of re-writing the native sequence to include or exclude certain regions. Despite its widespread use, SDM has often proved to be a bottleneck, requiring precision manipulation on a sample-by-sample basis to make it work. When dealing with large volumes of samples it is not possible to use such a low-throughput approach. Here we describe a high-throughput (HTP) method for SDM, optimized and used by the Structural Genomics Consortium (SGC) to complement structural studies.

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“…Native (intact) mass spectrometry was performed as outlined 52 . Denaturing mass spectrometry and phosphorylation mapping were performed as described 53 .…”

Section: Mass Spectrometrymentioning

confidence: 99%

Structural basis of human 5,10-methylenetetrahydrofolate reductase (MTHFR) regulation by phosphorylation and S-adenosylmethionine inhibition

Froese

Kopec

Rembeza

et al. 2018

Preprint

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The folate and methionine cycles are crucial to the biosynthesis of lipids, nucleotides and proteins, and production of the global methyl donor S-adenosylmethionine (SAM). 5,10-methylenetetrahydrofolate reductase (MTHFR) represents a key regulatory connection between these cycles, generating 5-methyltetrahydrofolate for initiation of the methionine cycle, and undergoing allosteric inhibition by its end product SAM. Our 2.5 Å resolution crystal structure of human MTHFR reveals a unique architecture, appending the well-conserved catalytic TIM-barrel to a eukaryote-only SAM-binding domain. The latter domain of novel fold provides the predominant interface for MTHFR homodimerization, positioning the N-terminal serine-rich phosphorylation region into proximity with the Cterminal SAM-binding domain. This explains how MTHFR phosphorylation, identified on 11 N-terminal residues (16-total), increases sensitivity to SAM binding and inhibition. Finally, we demonstrate the 25-amino-acid inter-domain linker enables conformational plasticity and propose it to be a key mediator of SAM regulation.

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High-Throughput Mass Spectrometry Applied to Structural Genomics

Cited by 5 publications

References 31 publications

Electrospray Surface Charge Describes Protein Molecular Motion

Electrospray Surface Charge Describes Protein Molecular Motion

High-Throughput Site-Directed Mutagenesis

Structural basis of human 5,10-methylenetetrahydrofolate reductase (MTHFR) regulation by phosphorylation and S-adenosylmethionine inhibition

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