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Influence of droplet size, capillary-cone distance and selected instrumental parameters for the analysis of noncovalent protein-ligand complexes by nano-electrospray ionization mass spectrometry
KTH, Superseded Departments, Chemistry.
KTH, Superseded Departments, Chemistry.
Biovitrum AB, Dept Analyt Sci.
KTH, Superseded Departments, Chemistry.
2004 (English)In: Journal of Mass Spectrometry, ISSN 1076-5174, E-ISSN 1096-9888, Vol. 39, no 9, 1059-1067 p.Article in journal (Refereed) Published
Abstract [en]

It has been suggested in the literature that nano-electrospray ionization (nano-ESI) mass spectrometry better reflects the equilibrium between complex and free protein in solution than pneumatically assisted electrospray ionization (ESI) in noncovalent interaction studies. However, no systematic studies of the effects of ionization conditions have been performed to support this statement. In the present work, different instrumental and sample-derived parameters affecting the stability of noncovalent complexes during analysis by nano-ESI were investigated. In general, increased values of parameters such as drying gas flow-rate, ion-source temperature, capillary tip voltage and buffer concentration lead to a dissociation of ribonuclease A (RNAse)-cytidine 2'-monophosphate (CMP) and cytidine 5'-triphosphate (CTP) complexes. The size of the electrosprayed droplets was shown to be an important issue. Increasing the capillary to cone distance yielded an increased complex to free protein ratio when a hydrophilic ligand was present and the reverse effect was obtained with a hydrophobic ligand. Important in this regard is the degree of sampling of ions originating from late-generation residue droplets, that is, ions present in the droplet bulk. Sampling of these ions increases with longer capillary-cone distance (flight time). Furthermore, when the sample flow-rate was increased by increasing the capillary internal tip i.d. from 4 to 30 mum, a decreased complex to free protein ratio for the RNAse-CTP system was observed. This behavior was consistent with the change in surface to volume ratio for flow-rates between 2 and 100 nl min(-1). Finally, polarity switching between positive and negative ion modes gave a higher complex to free protein ratio when the ligand and the protein had the same polarity.

Place, publisher, year, edition, pages
2004. Vol. 39, no 9, 1059-1067 p.
Keyword [en]
nano-electrospray ionization; noncovalent complexes; droplet size; capillary-cone distance; H-FABP; RNAse; BINDING INTERACTIONS; QUANTITATIVE-DETERMINATION; COMPETITIVE-BINDING; RESOLUTION; RECEPTOR; CHARGE; DNA
National Category
Analytical Chemistry
Identifiers
URN: urn:nbn:se:kth:diva-8357DOI: 10.1002/jms.685ISI: 000223868000010Scopus ID: 2-s2.0-4544221115OAI: oai:DiVA.org:kth-8357DiVA: diva2:13658
Note
QC 20100705Available from: 2008-05-07 Created: 2008-05-07 Last updated: 2011-09-23Bibliographically approved
In thesis
1. Electrospray Ionization Mass Spectrometry for Determination of Noncovalent Interactions in Drug Discovery
Open this publication in new window or tab >>Electrospray Ionization Mass Spectrometry for Determination of Noncovalent Interactions in Drug Discovery
2008 (English)Doctoral thesis, comprehensive summary (Other scientific)
Abstract [en]

Noncovalent interactions are involved in many biological processes in which biomolecules bind specifically and reversibly to a partner. Often, proteins do not have a biological activity without the presence of a partner, a ligand. Biological signals are produced when proteins interact with other proteins, peptides, oligonucleotides, nucleic acids, lipids, metal ions, polysaccharides or small organic molecules. Some key steps in the drug discovery process are based on noncovalent interactions. We have focused our research on the steps involving ligand screening, competitive binding and ‘off-target’ binding. The first paper in this thesis investigated the complicated electrospray ionization process with regards to noncovalent complexes. We have proposed a model that may explain how the equilibrium between a protein and ligand changes during the droplet evaporation/ionization process.

The second paper describes an evaluation of an automated chip-based nano-ESI platform for ligand screening. The technique was compared with a previously reported method based on nuclear magnetic resonance (NMR), and excellent correlation was obtained between the results obtained with the two methods. As a general conclusion we believe that the automated nano-ESI/MS should have a great potential to serve as a complementary screening method to conventional HTS. Alternatively, it could be used as a first screening method in an early phase of drug development programs when only small amounts of purified targets are available.

In the third article, the advantage of using on-line microdialysis as a tool for enhanced resolution and sensitivity during detection of noncovalent interactions and competitive binding studies by ESI-MS was demonstrated. The microdialysis device was improved and a new approach for competitive binding studies was developed.

The last article in the thesis reports studies of noncovalent interactions by means of nanoelectrospray ionization mass spectrometry (nanoESI-MS) for determination of the specific binding of selected drug candidates to HSA. Two drug candidates and two known binders to HSA were analyzed using a competitive approach. The drugs were incubated with the target protein followed by addition of site-specific probes, one at a time. The drug candidates showed predominant affinity to site I (warfarin site). Naproxen and glyburide showed affinity to both sites I and II.

Place, publisher, year, edition, pages
Stockholm: KTH, 2008. viii, 60 p.
Series
Trita-CHE-Report, ISSN 1654-1081 ; 2008:33
Keyword
Mass spectrometry, electrospray ionization, drug discovery, noncovalent interaction, complexes, human serum albumin (HSA), fatty acid binding protein (FABP), ribonuclease, ligand screening
National Category
Analytical Chemistry
Identifiers
urn:nbn:se:kth:diva-4730 (URN)978-91-7178-947-1 (ISBN)
Public defence
2008-05-23, E2, KTH, Lindstedtsvägen 3, Stockholm, 00:00
Opponent
Supervisors
Note
QC 20100705Available from: 2008-05-07 Created: 2008-05-07 Last updated: 2010-07-05Bibliographically approved
2. Analysis of noncovalent and covalent protein-ligand complexes by electrospray ionisation mass spectrometry
Open this publication in new window or tab >>Analysis of noncovalent and covalent protein-ligand complexes by electrospray ionisation mass spectrometry
2008 (English)Doctoral thesis, comprehensive summary (Other scientific)
Abstract [en]

In this thesis, the application of electrospray ionisation mass spectrometry (ESI-MS) to the analysis of intact proteins is demonstrated. In papers I and II, the use of ESI-MS for the analysis of noncovalent protein-ligand complexes were discussed. In addition, the interfacing of liquid chromatography (LC) with ESI-MS and the development of an LC-ESI-MS method were demonstrated in paper III for the quality control of recombinant proteins. Furthermore, this method was applied in paper IV for the analysis of covalent glycosyl-enzyme intermediates.

The monitoring of noncovalent complexes by ESI-MS is well established. However, the varying characteristic of ESI-MS data, especially in the analysis of noncovalent complexes can make the quantification of such complexes troublesome. In paper I, it was demonstrated how the variation in the position of the ESI-emitter and the initial droplet size of the electrosprayed droplets, together with different partitioning of a protein and its ligand in these droplets, can be the cause of such varying characteristics. Furthermore, it was shown that the partitioning can be of electrostatic and/or hydrophobic/hydrophilic origin. Thus it was demonstrated that if the ligand is more hydrophobic and thereby more surface active relative to the protein, decreasing the droplet size or increasing the distance between the electrospray emitter and the sampling orifice will lead to more efficient sampling of the droplet bulk where the ligand concentration is low. This results in a favoured sampling of free protein relative to the protein ligand complex. The opposite was shown to occur if the ligand is more hydrophilic than the protein.

In paper II, Ribonuclease A (RNAse) was used as a model for enzymes acting on polymeric substrates with different chain lengths. Nano-ESI-MS was applied to monitor the noncovalent interactions between RNAse and different target ligands. Among the single building blocks of RNA, including ribose, the bases adenine, guanine, cytosine and uracil, and phosphate, only phosphate was observed to interact at multiple RNAse sites at a higher cone voltage. Furthermore, monobasic singlestranded deoxycytidylic acid oligomers (dCx) of different lengths (X=6, 9 and 12), and RNAse were analysed with nano-ESI-MS. The deoxycytidylic acid with 12 nucleotides was observed with the highest complex to free protein ratio, hence indicating the strongest interaction. Finally, collision induced dissociation of the noncovalent RNAseA-dC6 complex resulted in dissociation of covalently bound cytosine from the nucleotide backbone rather than break up of the noncovalent complex, illustrating the cooperative effect of multiple noncovalent interactions.

In paper III an LC-ESI-MS method was presented capable of analysing proteins 10-100 kDa in size, from salt-containing liquid samples. The proteins included human protein fragments for the largescale production of antibodies and human protein targets for structural determination, expressed in E. coli. Also, glycosylated proteins expressed in Pichia pastoris were analysed. The method provides fast chromatography, is robust and makes use of cheap desalting/trap columns. In addition it was used with optimised reduction and alkylation protocols in order to minimize protein aggregation of denatured and incorrectly folded proteins containing cysteins, which otherwise form adducts by disulfide bond formation. Furthermore, the method was used in paper IV for the quantification of covalent proteinligand intermediates formed enzymatically between PttXET16-34, a xyloglucan endo-transglycosylase (XET) from hybrid aspen, and the synthetic substrates GalGXXXGGG and GalXXXGXXXG designed in order to function as donor substrates only. Thus covalent GalG-enzyme and GalGXXXG-enzyme complexes were detected. Moreover, establishing of a pseudo equilibrium for the formation of the covalent GalGXXXG-enzyme complex enabled quantification of the saccharide and enzyme constituents of this equilibrium and determination of the free energy of formation (∆G0). The high mass resolution of the TOF-MS allowed unambiguous assessment of the covalent nature of the glycosyl-enzyme complexes. Morover, the formation of noncovalent complexes between excess substrate and protein, which can deteriorate MS-signal and increase spectrum complexity, was efficiently avoided by the chromatographic step, which separated the saccharide content from the protein content.

Place, publisher, year, edition, pages
Stockholm: KTH, 2008. iv, 51 p.
Keyword
noncovalent complex, droplet fission, nano-electrospray ionisation, mass spectrometry
National Category
Analytical Chemistry
Identifiers
urn:nbn:se:kth:diva-4728 (URN)978-91-7178-976-1 (ISBN)
Public defence
2008-05-26, FD5, Albanova Universitetscentrum, Roslagstullsbacken 21, Stockholm, 13:00
Opponent
Supervisors
Note
QC 20100913Available from: 2008-05-07 Created: 2008-05-07 Last updated: 2010-09-13Bibliographically approved

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