Nucleotide-induced conformational changes of tetradecameric GroEL mapped by H/D exchange monitored by FT-ICR mass spectrometry

Zhang, Qian; Chen, Jin; Kuwajima, Kunihiro; Zhang, Huimin; Xian, Feng; Young, Nicolas L.; Marshall, Alan G.

doi:10.1038/srep01247

Cited by 29 publications

(28 citation statements)

References 54 publications

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“…The multi‐charge distribution of proteins was preserved, but the m/z value was centered on 1933.78, indicating an average exchange of 75 H atoms (±10). It is well known that the ESI study of protein–DNA interactions can be challenging: source parameters suitable to retain non‐covalent interactions should be selected, an adequate in‐solution protein/aptamer ratio should be evaluated to avoid non‐specific binding or ESI protein signal suppression. For this reason, different initial tests were performed on the protein/aptamer ratio to establish a suitable 1:5 ratio (data not shown).…”

Section: Resultsmentioning

confidence: 99%

Surface modification of chitosan films with a fibronectin fragment–DNA aptamer complex to enhance osteoblastic cell activity: A mass spectrometry approach probing evidence on protein behavior

Saccani

Parisi

Bergonzi

et al. 2019

Rapid Comm Mass Spectrometry

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Rationale Protein dynamics are fundamental for biological activity. Direct surface functionalization with these biomolecules often creates regions presenting non‐homogeneous and altered protein functionality. Aptamers have been shown to be an effective method to enhance the biocompatibility of biomaterials by acting as docking points for biomolecules capable of improving cell responses and the colonization/proliferation processes. Here mass spectrometry (MS) was successfully applied as an analytical approach to study the interactions between a fibronectin fragment (FN) and anti‐fibronectin aptamers in solution and on a chitosan film. Methods A 30 kDa N‐terminal fibronectin fragment, a ssDNA anti‐fibronectin 40 nucleotide long aptamer and chitosan 95/50 (degree of deacetylation 92.6–97.5%; molecular weight 80–220 kDa) were used. First, the dynamics of the FN in aqueous solution, in the presence or absence of anti‐FN‐specific DNA aptamers, were described. Thus, the same study was carried out on 2% (w/w) chitosan‐based films, functionalized with FN alone or through aptamers as selective spacers. Results Amide hydrogen/deuterium exchange mass spectrometry (HDX‐MS) analysis identified the SYRIGDTWSKKDNRGNL and YRVGDTYERPKDSMI, YVVGETWEKPYQGWMM and WERTYLGNAL fragments as presumably involved in the interaction of FN with aptamers. MS findings indicated the improved functionality of FN on chitosan when aptamers were used as selective spacer, and this may explain why cells preferentially attached to aptamer‐bound FN rather than on chitosan‐FN films. Conclusions Aptamers did not affect the amount of adsorbed FN, but influenced its conformation enhancing its biological activity toward adhesion and growth of osteoblasts. HDX‐MS data allowed the identification of the FN/aptamer interaction regions. Differences in FN flexibility on the chitosan film in the presence or absence of the aptamer were established providing insights at the molecular level to better understand the protein biological functionality on cell proliferation.

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Section: Resultsmentioning

confidence: 99%

Surface modification of chitosan films with a fibronectin fragment–DNA aptamer complex to enhance osteoblastic cell activity: A mass spectrometry approach probing evidence on protein behavior

Saccani

Parisi

Bergonzi

et al. 2019

Rapid Comm Mass Spectrometry

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“…For example, in HX MS analysis of large viral capsids such as brome mosaic virus (106) or the P22 capsid (107), or the chaperonins GroEL/ES (108), there are many identical copies of each subunit. Upon digestion, the amount of unique sequence is low because each identical monomer produces identical peptides (e.g.…”

Section: Analytical Tools Requiredmentioning

confidence: 99%

Analytical Aspects of Hydrogen Exchange Mass Spectrometry

Engen

Wales

2015

Annual Rev. Anal. Chem.

121

151

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The analytical aspects of measuring hydrogen exchange by mass spectrometry are reviewed. The nature of analytical selectivity in hydrogen exchange is described followed by review of the analytical tools required to accomplish fragmentation, separation, and the mass spectrometry measurements under restrictive exchange quench conditions. In contrast to analytical quantitation that relies on measurements of peak intensity or area, quantitation in hydrogen exchange mass spectrometry depends on measuring a mass change with respect to an undeuterated or deuterated control, resulting in a value between zero and the maximum amount of deuterium that could be incorporated. Reliable quantitation is a function of experimental fidelity and to achieve high measurement reproducibility, a large number of experimental variables must be controlled during sample preparation and analysis. The method also reports on important qualitative aspects of the sample, including conformational heterogeneity and population dynamics.

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“…Collisional and electron transfer dissociation offer efficient protein structural characterization, and UV photodissociation [34] is planned. We expect the first applications of the instrument to focus on top-down proteomics [35], mapping contact surfaces in large protein complexes by hydrogen-deuterium exchange [36], and petroleomics [37]. The instrument is part of the NSF High Field FT-ICR Mass Spectrometry User Facility, so research directions will ultimately be chosen by user interest.…”

Section: Resultsmentioning

confidence: 99%

21 Tesla Fourier Transform Ion Cyclotron Resonance Mass Spectrometer: A National Resource for Ultrahigh Resolution Mass Analysis

Hendrickson

Quinn

Kaiser

et al. 2015

J. Am. Soc. Mass Spectrom.

Self Cite

226

219

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Abstract. We describe the design and initial performance of the first 21 tesla Fourier transform ion cyclotron resonance (FT-ICR) mass spectrometer. The 21 tesla magnet is the highest field superconducting magnet ever used for FT-ICR and features high spatial homogeneity, high temporal stability, and negligible liquid helium consumption. The instrument includes a commercial dual linear quadrupole trap front end that features high sensitivity, precise control of trapped ion number, and collisional and electron transfer dissociation. A third linear quadrupole trap offers high ion capacity and ejection efficiency, and rf quadrupole ion injection optics deliver ions to a novel dynamically harmonized ICR cell. Mass resolving power of 150,000 (m/Δm 50% ) is achieved for bovine serum albumin (66 kDa) for a 0.38 s detection period, and greater than 2,000,000 resolving power is achieved for a 12 s detection period. Externally calibrated broadband mass measurement accuracy is typically less than 150 ppb rms, with resolving power greater than 300,000 at m/z 400 for a 0.76 s detection period. Combined analysis of electron transfer and collisional dissociation spectra results in 68% sequence coverage for carbonic anhydrase. The instrument is part of the NSF High-Field FT-ICR User Facility and is available free of charge to qualified users.

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Nucleotide-induced conformational changes of tetradecameric GroEL mapped by H/D exchange monitored by FT-ICR mass spectrometry

Cited by 29 publications

References 54 publications

Surface modification of chitosan films with a fibronectin fragment–DNA aptamer complex to enhance osteoblastic cell activity: A mass spectrometry approach probing evidence on protein behavior

Surface modification of chitosan films with a fibronectin fragment–DNA aptamer complex to enhance osteoblastic cell activity: A mass spectrometry approach probing evidence on protein behavior

Analytical Aspects of Hydrogen Exchange Mass Spectrometry

21 Tesla Fourier Transform Ion Cyclotron Resonance Mass Spectrometer: A National Resource for Ultrahigh Resolution Mass Analysis

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