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Analysis of noncovalent and covalent protein-ligand complexes by electrospray ionisation mass spectrometry
KTH, School of Biotechnology (BIO), Glycoscience.
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 [en]
noncovalent complex, droplet fission, nano-electrospray ionisation, mass spectrometry
National Category
Analytical Chemistry
Identifiers
URN: urn:nbn:se:kth:diva-4728ISBN: 978-91-7178-976-1 (print)OAI: oai:DiVA.org:kth-4728DiVA: diva2:13650
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
List of papers
1. 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
Open this publication in new window or tab >>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
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.

Keyword
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:nbn:se:kth:diva-8357 (URN)10.1002/jms.685 (DOI)000223868000010 ()2-s2.0-4544221115 (Scopus ID)
Note
QC 20100705Available from: 2008-05-07 Created: 2008-05-07 Last updated: 2011-09-23Bibliographically approved
2. Investigation of multiple binding sites on ribonuclease A using nano-electrospray ionization mass spectrometry
Open this publication in new window or tab >>Investigation of multiple binding sites on ribonuclease A using nano-electrospray ionization mass spectrometry
2005 (English)In: Rapid Communications in Mass Spectrometry, ISSN 0951-4198, E-ISSN 1097-0231, Vol. 19, no 8, 1011-1016 p.Article in journal (Refereed) Published
Abstract [en]

Multiple non-active site interactions between ribonuclease A (RNAse) and selected target molecules were investigated using nano-electrospray ionization mass spectrometry (nano-ESI-MS). Among the building blocks of RNA, phosphate and ribose showed such multiple interactions. Multiple phosphate interactions survived a high cone voltage, while multiple interactions with D-ribose disappeared already at a low cone voltage. Using nano-ESI-MS, only cytosine among the individual bases appeared to interact with RNAse. Interestingly, guanosine binds to the RNAse surface at high cone voltage, probably as a result of cooperative binding of the sugar and the guanine base. Upon binding of deoxycytidine oligonucleotides with six (dC(6)), nine (dC(9)) and twelve (dC(12)) deoxycytidine nucleotide units to RNAse, the dC(12) Unit showed the strongest interaction. Upon collision-induced dissociation (CID) of the RNAse/dC(6) complex, this complex survived dissociation at an energy level where covalently bound cytosine from dC(6) was lost. This is in contrast to CID of RNAse complexed with mononucleotide cytidine 2'-monophosphate (CMP), which dissociates from the protein without breaking of covalent bonds.

Keyword
CYTIDINE 2', 3'-CYCLIC PHOSPHATE, PANCREATIC RIBONUCLEASE, SUBSITES STRUCTURE, ACTIVE-SITE, GAS-PHASE, RNASE-A, COMPLEXES, INHIBITORS, CLEAVAGE, ACIDS
National Category
Analytical Chemistry
Identifiers
urn:nbn:se:kth:diva-8349 (URN)10.1002/rcm.1880 (DOI)000228571700006 ()2-s2.0-17844386884 (Scopus ID)
Note
QC 20100913Available from: 2008-05-07 Created: 2008-05-07 Last updated: 2010-09-13Bibliographically approved
3. A general, robust method for the quality control of intact proteins using LC–ESI-MS
Open this publication in new window or tab >>A general, robust method for the quality control of intact proteins using LC–ESI-MS
Show others...
2007 (English)In: Journal of chromatography. B, ISSN 1570-0232, E-ISSN 1873-376X, Vol. 852, no 1-2, 188-194 p.Article in journal (Refereed) Published
Abstract [en]

A simple and robust method for the routine quality control of intact proteins based on liquid chromatography coupled to electrospray ionization mass spectrometry (LC-ESI-MS) is presented. A wide range of prokaryotic and eukaryotic proteins expressed recombinantly in Escherichia coli or Pichia pastoris has been analyzed with medium- to high-throughput with on-line desalting from multi-well sample plates. Particular advantages of the method include fast chromatography and short cycle times, the use of inexpensive trapping/desalting columns, low sample carryover, and the ability to analyze proteins with masses ranging from 5 to 100 kDa with greater than 50 ppm accuracy. Moreover, the method can be readily coupled with optimized chemical reduction and alkylation steps to facilitate the analysis of denatured or incorrectly folded proteins (e.g., recombinant proteins sequestered in E. coli inclusion bodies) bearing cysteine residues, which otherwise form intractable multimers and non-specific adducts by disulfide bond formation.

Keyword
Alkylation; Column regeneration; Electrospray ionization; Glycoprotein; Liquid chromatography; Mass spectrometry; Protein analysis; Quality control; Reduction; Xyloglucan endo-transglycosylase (XET)
National Category
Biological Sciences
Identifiers
urn:nbn:se:kth:diva-8350 (URN)10.1016/j.jchromb.2007.01.011 (DOI)000247286700026 ()2-s2.0-34249689690 (Scopus ID)
Note
QC 20100818Available from: 2008-05-07 Created: 2008-05-07 Last updated: 2010-08-18Bibliographically approved
4. Mechanism-based labeling defines the free energy change for formation of the covalent glycosyl-enzyme intermediate in a xyloglucan endo-transglycosylase
Open this publication in new window or tab >>Mechanism-based labeling defines the free energy change for formation of the covalent glycosyl-enzyme intermediate in a xyloglucan endo-transglycosylase
Show others...
2008 (English)In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 283, no 32, 21864-21872 p.Article in journal (Refereed) Published
Abstract [en]

Xyloglucan endo-transglycosylases (XETs) are key enzymes involved in the restructuring of plant cell walls during morphogenesis. As members of glycoside hydrolase family 16 (GH16), XETs are predicted to employ the canonical retaining mechanism of glycosyl transfer involving a covalent glycosyl-enzyme intermediate. Here, we report the accumulation and direct observation of such intermediates of PttXET16-34 from hybrid aspen by electrospray mass spectrometry in combination with synthetic "blocked" substrates, which function as glycosyl donors but are incapable of acting as glycosyl acceptors. Thus, GalGXXXGGG and GalGXXXGXXXG react with the wild-type enzyme to yield relatively stable, kinetically competent, covalent GalG-enzyme and GalGXXXG-enzyme complexes, respectively (Gal = Gal beta(1 -> 4), G = Glc beta(1 -> 4), and X = Xyl alpha(1 -> 6) Glc beta(1 -> 4)). Quantitation of ratios of protein and saccharide species at pseudo-equilibrium allowed us to estimate the free energy change (Delta G(0)) for the formation of the covalent GalGXXXG-enzyme as 6.3-8.5 kJ/mol (1.5-2.0 kcal/mol). The data indicate that the free energy of the beta(1 -> 4) glucosidic bond in xyloglucans is preserved in the glycosyl-enzyme intermediate and harnessed for religation of the polysaccharide in vivo.

Keyword
Biomaterials, Free energy, Gene transfer, High performance liquid chromatography, Mass spectrometry, Plant cell culture, Plants (botany), Polysaccharides, Sugars, Direct observations, Electrospray mass spectrometries, Enzyme complexes, Free energy changes, Glucosidic bonds, Glycoside hydrolase family 16, Glycosyl, Glycosyl acceptors, Glycosyl donors, In-vivo, Key enzymes, Plant cell walls, Transglycosylases, Xyloglucan, Xyloglucans
National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:kth:diva-8351 (URN)10.1074/jbc.M803057200 (DOI)000258114700005 ()2-s2.0-52049094160 (Scopus ID)
Note
QC 20100913. Uppdaterad från manuskript till artikel (20100913).Available from: 2008-05-07 Created: 2008-05-07 Last updated: 2010-09-13Bibliographically approved

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