Separation of <i>β</i><sub>2</sub>‐microglobulin conformers by high‐field asymmetric waveform ion mobility spectrometry (FAIMS) coupled to electrospray ionisation mass spectrometry

Borysik, Antoni J.; Read, Paul A.; Little, David; Bateman, Robert; Radford, Sheena E.; Ashcroft, Alison E.

doi:10.1002/rcm.1613

Cited by 60 publications

(62 citation statements)

References 27 publications

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“…In DMA (17-19) ions traverse a space between 2 electrodes while being displaced laterally by gas flow. Only ions of specific K (determined by the voltage across the gap and flow speed) exit the gap, and mobility spectra are obtained by scanning the voltage.Recently, field asymmetric waveform ion mobility spectrometry (FAIMS) or differential mobility spectrometry has emerged as a major analytical tool (6,(20)(21)(22)(23)(24)(25)(26)(27)(28). FAIMS filters ions based on the difference between K at low and high electric field intensity (E).…”

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confidence: 99%

“…Hence, all peptide, protein, and other macromolecular ions were believed (6,(22)(23)(24)(25)(26)(27)(28) to belong to type C, meaning E C Ͻ 0 for negative E(t) polarity. These studies involved small proteins with m Ͻ16 kDa, such as ubiquitin (8.6 kDa) and cytochrome c (cyt c; 12.2 kDa).…”

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confidence: 99%

“…These studies involved small proteins with m Ͻ16 kDa, such as ubiquitin (8.6 kDa) and cytochrome c (cyt c; 12.2 kDa). Protein ions have many structural conformers for a particular charge state (z), and several are often resolved (6,(23)(24)(25)(26)(27)(28). However, all had E C Ͻ 0 within a limited range of Ϸ5 V/mm, limiting the peak capacity to Ϸ10 with the then-best-achievable FAIMS resolution (r) of Ϸ0.5 V/mm.…”

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Pendular proteins in gases and new avenues for characterization of macromolecules by ion mobility spectrometry

Shvartsburg

Noskov

Purves

et al. 2009

Proc. Natl. Acad. Sci. U.S.A.

110

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Polar molecules align in electric fields when the dipole energy (proportional to field intensity E ؋ dipole moment p) exceeds the thermal rotational energy. Small molecules have low p and align only at inordinately high E or upon extreme cooling. Many biomacromolecules and ions are strong permanent dipoles that align at E achievable in gases and room temperature. The collision cross-sections of aligned ions with gas molecules generally differ from orientationally averaged quantities, affecting ion mobilities measured in ion mobility spectrometry (IMS). Field asymmetric waveform IMS (FAIMS) separates ions by the difference between mobilities at high and low E and hence can resolve and identify macroion conformers based on the mobility difference between pendular and free rotor states. The exceptional sensitivity of that difference to ion geometry and charge distribution holds the potential for a powerful method for separation and characterization of macromolecular species. Theory predicts that the pendular alignment of ions in gases at any E requires a minimum p that depends on the ion mobility, gas pressure, and temperature. At ambient conditions used in current FAIMS systems, p for realistic ions must exceed Ϸ300 -400 Debye. The dipole moments of proteins statistically increase with increasing mass, and such values are typical above Ϸ30 kDa. As expected for the dipole-aligned regime, FAIMS analyses of protein ions and complexes of Ϸ30 -130 kDa show an order-of-magnitude expansion of separation space compared with smaller proteins and other ions. mass spectrometry ͉ protein structure S eparation and characterization of ions by using their drift in gases forced by electric field, called ion mobility spectrometry (IMS), is becoming common in analytical and structural chemistry (1-6). As with mass spectrometry (MS), initial applications were to small molecules (1, 5, 7) that could be readily ionized. Coupling to MS and to electrospray (ESI) and matrix-assisted laser desorption ionization (MALDI) ion sources in the 1990s (8, 9) brought IMS to biological analyses (2, 4, 6). In particular, peptide and protein folding (2, 4, 9-14) and amyloidogenic misfolding (15) were explored. Application of IMS and IMS/MS is expanding to ever-larger macromolecules, including noncovalent assemblies with mass (m) Ͼ 1 MDa (16-18). Such studies have used both drift tube (DT) IMS (1-5, 7-15) and differential mobility analyzers (DMA) (17, 18) to separate ions by absolute mobility (K). In DT IMS, ions are separated while being pulled through still gas by an electric field. In DMA (17-19) ions traverse a space between 2 electrodes while being displaced laterally by gas flow. Only ions of specific K (determined by the voltage across the gap and flow speed) exit the gap, and mobility spectra are obtained by scanning the voltage.Recently, field asymmetric waveform ion mobility spectrometry (FAIMS) or differential mobility spectrometry has emerged as a major analytical tool (6,(20)(21)(22)(23)(24)(25)(26)(27)(28). FAIMS filters ions based on th...

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confidence: 99%

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Pendular proteins in gases and new avenues for characterization of macromolecules by ion mobility spectrometry

Shvartsburg

Noskov

Purves

et al. 2009

Proc. Natl. Acad. Sci. U.S.A.

110

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“…The gentle nature of the ESI process allows the transfer of intact protein-ligand and proteinprotein complexes into the gas phase [6,[27][28][29]; thus, insights into conformations and interactions are obtained in a single experiment [17, 30 -36]. A further dimension can be added to these studies by incorporating ion mobility spectrometry [37,38].…”

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confidence: 99%

Irreversible thermal denaturation of cytochrome c studied by electrospray mass spectrometry

Liu

Konermann

2009

J. Am. Soc. Mass Spectrom.

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This work uses electrospray ionization mass spectrometry (ESI-MS) in conjunction with hydrogen/deuterium exchange (HDX) and optical spectroscopy for characterizing the solutionphase properties of cytochrome c (cyt c) after heat exposure. Previous work demonstrated that heating results in irreversible denaturation for a subpopulation of proteins in the sample. However, that study did not investigate the physical reasons underlying this interesting effect. Here we report that the formation of oxidative modifications at elevated temperature plays a key role for the observed behavior. Tryptic digestion followed by tandem mass spectrometry is used to identify individual oxidation sites. Trp59 and Met80 are among the modified amino acids. In native cyt c both of these residues are buried deep within the protein structure, such that covalent modifications would be expected to be particularly disruptive. ESI-MS analysis after heat exposure results in a bimodal charge-state distribution. Oxidized protein appears predominantly in charge states around 11ϩ, whereas a considerably lower degree of oxidation is observed for the 7ϩ and 8ϩ peaks. This finding confirms that different oxidation levels are associated with different solution-phase conformations. HDX measurements for different charge states are complicated by peak distortions arising from oxygen adduction. Nonetheless, comparison with simulated peak shapes clearly shows that the HDX properties are different for high-and low-charge states, confirming that interconversion between unfolded and folded conformers is blocked in solution. In addition to oxidation, partial aggregation upon heat exposure likely contributes to the formation of irreversibly denatured protein. A number of well-established tools are available for experiments of this kind, including X-ray crystallography, nuclear magnetic resonance, calorimetry, and various spectroscopic techniques. Electrospray ionization mass spectrometry (ESI-MS) has become another widely used approach for exploring the properties of proteins in solution, providing information complementary to that obtained by traditional methods [1]. The ESI process generates intact gas-phase ions from proteins in solution. In the commonly used positive-ion mode these [M ϩ nH] nϩ species are multiply charged because of proton attachment.ESI of natively folded proteins results in protonation states n similar to those predicted for water droplets of the same size that are charged to the Rayleigh limit. This finding supports the idea that ionization follows the charged-residue model [2-6], although alternative scenarios have been proposed as well [7][8][9]. Much higher protonation states and wider charge-state distributions are generally seen for proteins that are unfolded in solution. Based on this empirical relationship, ESI-MS is now routinely being used for monitoring structural transitions in response to changes in pH, temperature, organic co-solvents, or covalent modifications [1, 10 -16]. The physical basis for the striking dependence of...

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“…The introduction of commercial DMS instruments and particularly their integration with mass spectrometry (MS) andI or liquid or gas chromatography since 2003 has enabled rapid growth of the number and diversity of applications that include environmental analyses [6,7], food and water quality assurance [8][9][10], bacterial typing [11,12], forensic investigations [13], proteomics and metabolomics [14][15][16][17], pharmaceutical studies [18][19][20], and protein folding research [21][22][23][24][25]. Since its earliest days, DMS has been employed to detect explosives, drugs, and chemical warfare agents, and its role in defense, security, and law enforcement settings continues expanding [26][27][28][29][30][31].…”

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Optimum waveforms for differential ion mobility spectrometry (FAIMS)

Shvartsburg

Smith

2008

J. Am. Soc. Mass Spectrom.

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Differential mobility spectrometry or field asymmetric waveform ion mobility spectrometry (FAIMS) is a new tool for separation and identification of gas-phase ions, particularly in conjunction with mass spectrometry. In FAIMS, ions are filtered by the difference between mobilities in gases (K) at high and low electric field intensity (E) using asymmetric waveforms. An infinite number of possible waveform profiles make maximizing the performance within engineering constraints a major issue for FAIMS technology refinement. Earlier optimizations assumed the non-constant component of mobility to scale as E 2 , producing the same result for all ions. Here we show that the optimum profiles are defined bl the full series expansion of K(E) that includes terms beyond the first that is proportional to E . For many ionlgas pairs, the first two terms have different signs, and the optimum profiles at sufficiently high E in FAIMS may differ substantially from those previously reported, improving the resolving power by up to 2.2 times. This situation arises for some ions in all FAIMS systems, but becomes more common in recent miniaturized devices that employ higher E. With realistic K(E) dependences, the maximum waveform amplitude is not necessarily optimum, and reducing it by up to~20% to 30% is beneficial in some cases. In DMS, a(EIN) is elicited directly using a periodic time-dependent electric field E(t) that comprises short segments E+(t) with high E and longer segments L(t) with lower E of opposite polarity such that mean E over the period t c is null (the zero-offset condition) [33, 39-43]:D ifferential ion mobility spectrometry (DMS) is becoming a powerful method of broad utility for analysis of gas-phase ions and separation of their mixtures [1][2][3][4][5]. The introduction of commercial DMS instruments and particularly their integration with mass spectrometry (MS) andI or liquid or gas chromatography since 2003 has enabled rapid growth of the number and diversity of applications that include environmental analyses [6,7], food and water quality assurance [8][9][10], bacterial typing [11,12], forensic investigations [13], proteomics and metabolomics [14][15][16][17], pharmaceutical studies [18][19][20], and protein folding research [21][22][23][24][25]. Since its earliest days, DMS has been employed to detect explosives, drugs, and chemical warfare agents, and its role in defense, security, and law enforcement settings continues expanding [26][27][28][29][30][31].As captured by the name, DMS separates ions based on the difference between their mobilities (K) at high and relatively low electric field intensity (E) [1,5]. The mobility of any ion depends on E and the gas number density (N), and we can expand K(EIN) in a series [7,30,32,33]:

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Separation of β₂‐microglobulin conformers by high‐field asymmetric waveform ion mobility spectrometry (FAIMS) coupled to electrospray ionisation mass spectrometry

Cited by 60 publications

References 27 publications

Pendular proteins in gases and new avenues for characterization of macromolecules by ion mobility spectrometry

Pendular proteins in gases and new avenues for characterization of macromolecules by ion mobility spectrometry

Irreversible thermal denaturation of cytochrome c studied by electrospray mass spectrometry

Optimum waveforms for differential ion mobility spectrometry (FAIMS)

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Separation of β2‐microglobulin conformers by high‐field asymmetric waveform ion mobility spectrometry (FAIMS) coupled to electrospray ionisation mass spectrometry

Cited by 60 publications

References 27 publications

Pendular proteins in gases and new avenues for characterization of macromolecules by ion mobility spectrometry

Pendular proteins in gases and new avenues for characterization of macromolecules by ion mobility spectrometry

Irreversible thermal denaturation of cytochrome c studied by electrospray mass spectrometry

Optimum waveforms for differential ion mobility spectrometry (FAIMS)

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Separation of β₂‐microglobulin conformers by high‐field asymmetric waveform ion mobility spectrometry (FAIMS) coupled to electrospray ionisation mass spectrometry