Screening of the Binding of Small Molecules to Proteins by Desorption Electrospray Ionization Mass Spectrometry Combined with Protein Microarray

Yao, Chenxi; Wang, Tao; Zhang, Buqing; He, Dacheng; Na, Na; Ouyang, Jin

doi:10.1007/s13361-015-1221-z

Cited by 7 publications

(4 citation statements)

References 35 publications

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“…Another commonly used method for investigating enzymatic activity is protein microarray. 51,52 Protein microarrays are typically designed for high-throughput analysis of purified proteins 24 ; therefore, in situ protein purification systems using immobilized metal affinity surfaces have been developed for a streamlined workflow. 28 We first evaluated the enzymatic activity of 5 μM His-SAHN in clarified E. coli cell lysate after purification with the Ni-NTA and Cu-NTA plates.…”

Section: Desi-ms Analysis Of His-tagged Proteins Purified From Cell L...mentioning

confidence: 99%

“…22 Recently, online buffer exchange chromatography has been coupled with online immobilized metal affinity chromatography and size exclusion chromatography for a streamlined native ESI-MS analysis of tagged recombinant proteins from bacterial cell lysate. 23 Desorption ESI (DESI)-MS of tagged recombinant proteins immobilized on agarose slides has been shown to provide rapid online purification and has been used successfully to screen small-molecule binding and monitor the enzymatic reactions 24 ; however, characterization of the immobilized tagged recombinant proteins has remained elusive.…”

Section: Introductionmentioning

confidence: 99%

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Analysis of histidine‐tagged recombinant proteins from nickel and copper coated surfaces by direct electrospray ionization and desorption electrospray ionization mass spectrometry

Javanshad

Taylor

Delavari

et al. 2023

Rapid Comm Mass Spectrometry

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RationalePurification of recombinant proteins is a necessary step for functional or structural studies and other applications. Immobilized metal affinity chromatography is a common recombinant protein purification method. Mass spectrometry (MS) allows for confirmation of identity of expressed proteins and unambiguous detection of enzymatic substrates and reaction products. We demonstrate the detection of enzymes purified on immobilized metal affinity surfaces by direct or ambient ionization MS, and follow their enzymatic reactions by direct electrospray ionization (ESI) or desorption electrospray ionization (DESI).MethodsA protein standard, His‐Ubq, and two recombinant proteins, His‐SHAN and His‐CS, expressed in Escherichia coli were immobilized on two immobilized metal affinity systems, Cu–nitriloacetic acid (Cu‐NTA) and Ni‐NTA. The proteins were purified on surface, and released in the ESI spray solvent for direct infusion, when using the 96‐well plate form factor, or analyzed directly from immobilized metal affinity‐coated microscope slides by DESI‐MS. Enzyme activity was followed by incubating the substrates in wells or by depositing substrate on immobilized protein on coated slides for analysis.ResultsSmall proteins (His‐Ubq) and medium proteins (His‐SAHN) could readily be detected from 96‐well plates by direct infusion ESI, or from microscope slides by DESI‐MS after purification on surface from clarified E. coli cell lysate. Protein oxidation was observed for immobilized proteins on both Cu‐NTA and Ni‐NTA; however, this did not hamper the enzymatic reactions of these proteins. Both the nucleosidase reaction products for His‐SAHN and the methylation product of His‐CS (theobromine to caffeine) were detected.ConclusionsThe immobilization, purification, release and detection of His‐tagged recombinant proteins using immobilized metal affinity surfaces for direct infusion ESI‐MS or ambient DESI‐MS analyses were successfully demonstrated. Recombinant proteins were purified to allow identification directly out of clarified cell lysate. Biological activities of the recombinant proteins were preserved allowing the investigation of enzymatic activity via MS.

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Section: Desi-ms Analysis Of His-tagged Proteins Purified From Cell L...mentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Analysis of histidine‐tagged recombinant proteins from nickel and copper coated surfaces by direct electrospray ionization and desorption electrospray ionization mass spectrometry

Javanshad

Taylor

Delavari

et al. 2023

Rapid Comm Mass Spectrometry

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“…The noncovalent molecular ions could not be detected in some cases such as complexes formed by polyhydroxy compounds; even many fragment ions generated also appeared in the spectrum. The free drugs were quantified to determine the binding ability between proteins and drugs by desorption electrospray ionization and venturi easy ambient sonic‐spray ionization mass spectrometry which cannot provide the stoichiometries . CSI‐MS was milder than ESI‐MS, provided a stable solvation‐ionization at low temperature, preserved labile noncovalent complexes, and has become one of the most promising and useful tools to study noncovalent drug‐protein complexes …”

Section: Introductionmentioning

confidence: 99%

Study of the noncovalent interactions of ginsenosides and amyloid‐β‐peptide by CSI‐MS and molecular docking

Zhou

Qiao

et al. 2019

J Mass Spectrom

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Noncovalent interactions between drugs and proteins play significant roles for drug metabolisms and drug discoveries. Mass spectrometry has been a commonly used method for studying noncovalent interactions. However, the harsh ionization process in electrospray ionization mass spectrometry (ESI-MS) is not conducive to the preservation of noncovalent and unstable biomolecular complexes compared with the cold spray ionization mass spectrometry (CSI-MS). A cold spray ionization providing a stable solvation-ionization at low temperature is milder than ESI, which was more suitable for studying noncovalent drug-protein complexes with exact stoichiometries.In this paper, we apply CSI-MS to explore the interactions of ginsenosides toward amyloid-β-peptide (Aβ) and clarify the therapeutic effect of ginsenosides on Alzheimer's disease (AD) at the molecular level for the first time. The interactions of ginsenosides with Aβ were performed by CSI-MS and ESI-MS, respectively. The ginsenosides Rg1 bounded to Aβ at the stoichiometries of 1:1 to 5:1 could be characterized by CSI-MS, while dehydration products are more readily available by ESI-MS. The binding force depends on the number of glycosyls and the type of ginsenosides. The relative binding affinities were sorted in order as follows: Rg1 ≈ Re > Rd ≈ Rg2 > Rh2, protopanaxatriol by competition experiments, which were supported by molecular docking experiment. CSI-MS is expected to be a more appropriate approach to determine the weak but specific interactions of proteins with other natural products especially polyhydroxy compounds.

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“…Importantly, MS is generally so rapid and sensitive such that feeding the instrument quickly with low amounts of the sample becomes the bottleneck for high-speed analysis . Various ESI − or ESI-derived − microfluidic autosamplers were developed to tackle this issue. However, most of these autosamplers were only used to analyze small molecules.…”

mentioning

confidence: 99%

Bioaffinity Screening with a Rapid and Sample-Efficient Autosampler for Native Electrospray Ionization Mass Spectrometry

et al. 2021

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Fast and efficient handling of ligands and biological targets are required in bioaffinity screening based on native electrospray ionization mass spectrometry (ESI-MS). We use a prototype microfluidic autosampler, called the "gap sampler", to sequentially mix and electrospray individual small molecule ligands together with a target protein and compare the screening results with data from thermal shift assay and surface plasmon resonance. In a first round, all three techniques were used for a screening of 110 ligands against bovine carbonic anhydrase II, which resulted in five mutual hits and some false positives with ESI-MS presumably due to the high ligand concentration or interferences from dimethyl sulfoxide. In a second round, 33 compounds were screened in lower concentrations and in a less complex matrix, resulting in only true positives with ESI-MS. Within a cycle time of 30 s, dissociation constants were determined within an order of magnitude accuracy consuming only 5 pmol of ligand and less than 15 pmol of protein per screened compound. In a third round, dissociation constants of five compounds were accurately determined in a titration experiment. Thus, the gap sampler can rapidly and efficiently be used for high-throughput screening.

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Screening of the Binding of Small Molecules to Proteins by Desorption Electrospray Ionization Mass Spectrometry Combined with Protein Microarray

Cited by 7 publications

References 35 publications

Analysis of histidine‐tagged recombinant proteins from nickel and copper coated surfaces by direct electrospray ionization and desorption electrospray ionization mass spectrometry

Analysis of histidine‐tagged recombinant proteins from nickel and copper coated surfaces by direct electrospray ionization and desorption electrospray ionization mass spectrometry

Study of the noncovalent interactions of ginsenosides and amyloid‐β‐peptide by CSI‐MS and molecular docking

Bioaffinity Screening with a Rapid and Sample-Efficient Autosampler for Native Electrospray Ionization Mass Spectrometry

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