Mass spectrometric studies of dissociation constants of noncovalent complexes

Erba, Elisabetta; Zenobi, Renato

doi:10.1039/c1pc90006d

Cited by 46 publications

(45 citation statements)

References 162 publications

(172 reference statements)

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“…30 Extensive reviews indicate that MS has an increasing role in gauging the composition, conformation, binding strength, and dissociation constants of noncovalent interactions. 1,31–33 …”

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confidence: 99%

Rapid Assessment of Human Amylin Aggregation and Its Inhibition by Copper(II) Ions by Laser Ablation Electrospray Ionization Mass Spectrometry with Ion Mobility Separation

Donaldson

et al. 2015

Anal. Chem.

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Native electrospray ionization (ESI) mass spectrometry (MS) is often used to monitor noncovalent complex formation between peptides and ligands. The relatively low throughput of this technique, however, is not compatible with extensive screening. Laser ablation electrospray ionization (LAESI) MS combined with ion mobility separation (IMS) can analyze complex formation and provide conformation information within a matter of seconds. Islet amyloid polypeptide (IAPP) or amylin, a 37-amino acid residue peptide, is produced in pancreatic beta-cells through proteolytic cleavage of its prohormone. Both amylin and its precursor can aggregate and produce toxic oligomers and fibrils leading to cell death in the pancreas that can eventually contribute to the development of type 2 diabetes mellitus. The inhibitory effect of the copper(II) ion on amylin aggregation has been recently discovered, but details of the interaction remain unknown. Finding other more physiologically tolerated approaches requires large scale screening of potential inhibitors. Here, we demonstrate that LAESI-IMS-MS can reveal the binding stoichiometry, copper oxidation state, and the dissociation constant of human amylin–copper(II) complex. The conformations of hIAPP in the presence of copper(II) ions were also analyzed by IMS, and preferential association between the β-hairpin amylin monomer and the metal ion was found. The copper(II) ion exhibited strong association with the –HSSNN– residues of the amylin. In the absence of copper(II), amylin dimers were detected with collision cross sections consistent with monomers of β-hairpin conformation. When copper(II) was present in the solution, no dimers were detected. Thus, the copper(II) ions disrupt the association pathway to the formation of β-sheet rich amylin fibrils. Using LAESI-IMS-MS for the assessment of amylin–copper(II) interactions demonstrates the utility of this technique for the high-throughput screening of potential inhibitors of amylin oligomerization and fibril formation. More generally, this rapid technique opens the door for high-throughput screening of potential inhibitors of amyloid protein aggregation.

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“…30 Extensive reviews indicate that MS has an increasing role in gauging the composition, conformation, binding strength, and dissociation constants of noncovalent interactions. 1,31–33 …”

mentioning

confidence: 99%

Rapid Assessment of Human Amylin Aggregation and Its Inhibition by Copper(II) Ions by Laser Ablation Electrospray Ionization Mass Spectrometry with Ion Mobility Separation

Donaldson

et al. 2015

Anal. Chem.

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“…1d,e, α- M Glu forms two hydrogen bonds with each ICA, resulting in a more stable complex than that of β- M Glu, which interacts with each ICA through a single hydrogen bond. We have observed 1:1 and 2:1 complexes formed between ICA and these two anomers by analysing aqueous solutions of ICA mixed with the analytes in a 2:1 ratio using electrospray ionization mass spectrometry (ESI-MS), a method that provides a snapshot of solution equilibria in the gas phase16 (Supplementary Tables 2–4 and Supplementary Methods; MS data for the other monosaccharides and disaccharides used in the present work are also included there). The RT measurements were carried out using a scanning tunnelling microscope (STM), following a process of functionalizing palladium (Pd) STM probes and Pd-substrates with ICA in ethanolic solutions (see Methods for details of functionalization and characterization of probes and substrates), mounting them in a PicoSPM instrument (Agilent), recording a RT spectrum of the phosphate buffer solution, pH 7.4 to assure that a clean baseline is achieved (see Methods, Supplementary Fig.…”

Section: Resultsmentioning

confidence: 99%

Electronic single-molecule identification of carbohydrate isomers by recognition tunnelling

Im¹,

Biswas²,

Líu³

et al. 2016

Nat Commun

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Carbohydrates are one of the four main building blocks of life, and are categorized as monosaccharides (sugars), oligosaccharides and polysaccharides. Each sugar can exist in two alternative anomers (in which a hydroxy group at C-1 takes different orientations) and each pair of sugars can form different epimers (isomers around the stereocentres connecting the sugars). This leads to a vast combinatorial complexity, intractable to mass spectrometry and requiring large amounts of sample for NMR characterization. Combining measurements of collision cross section with mass spectrometry (IM–MS) helps, but many isomers are still difficult to separate. Here, we show that recognition tunnelling (RT) can classify many anomers and epimers via the current fluctuations they produce when captured in a tunnel junction functionalized with recognition molecules. Most importantly, RT is a nanoscale technique utilizing sub-picomole quantities of analyte. If integrated into a nanopore, RT would provide a unique approach to sequencing linear polysaccharides.

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“…To obtain relative binding affinities of the HIV-PIs, we determined their solution phase dissociation constants with established mass spectrometry methods 3,26 . We varied the concentration of lopinavir and ritonavir in intervals of 2.5 μM from 4 μM to 20 μM (and used a fixed concertation of ZMPSTE24, 15 μM).…”

Section: Resultsmentioning

confidence: 99%

Mass spectrometry captures off-target drug binding and provides mechanistic insights into the human metalloprotease ZMPSTE24

et al. 2016

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Off-target binding of hydrophobic drugs can lead to unwanted side effects, either through specific or nonspecific binding to unintended membrane protein targets; however, distinguishing the binding of drugs to membrane proteins from that of detergents, lipids and cofactors is challenging. Here we use high-resolution mass spectrometry to study the effects of HIV protease inhibitors on the human zinc metalloprotease ZMPSTE24. This intramembrane protease plays a major role in converting prelamin A to mature lamin A. We monitored proteolysis of farnesylated prelamin A peptide by ZMPSTE24 and unexpectedly found retention of the C-terminal peptide product with the enzyme. We also resolved binding of zinc, lipids, and HIV protease inhibitors and showed that drug binding blocked prelamin A peptide cleavage and conferred stability to ZMPSTE24. Our results not only have relevance for the progeria-like side effects of certain HIV protease inhibitor drugs but also highlight new approaches for documenting off-target drug binding.

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Mass spectrometric studies of dissociation constants of noncovalent complexes

Cited by 46 publications

References 162 publications

Rapid Assessment of Human Amylin Aggregation and Its Inhibition by Copper(II) Ions by Laser Ablation Electrospray Ionization Mass Spectrometry with Ion Mobility Separation

Rapid Assessment of Human Amylin Aggregation and Its Inhibition by Copper(II) Ions by Laser Ablation Electrospray Ionization Mass Spectrometry with Ion Mobility Separation

Electronic single-molecule identification of carbohydrate isomers by recognition tunnelling

Mass spectrometry captures off-target drug binding and provides mechanistic insights into the human metalloprotease ZMPSTE24

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