Subscriber access provided by READING UNIV
Letter
Molecular Structure Characterization by Isotopic Splitting in Nonlinear Ion Mobility Spectra Julia L Kaszycki, Matthew A Baird, and Alexandre A. Shvartsburg Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.7b04610 • Publication Date (Web): 08 Dec 2017 Downloaded from http://pubs.acs.org on December 10, 2017
Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a free service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are accessible to all readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.
Analytical Chemistry is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.
Page 1 of 5 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
Analytical Chemistry
Molecular Structure Characterization by Isotopic Splitting in Nonlinear Ion Mobility Spectra Julia L. Kaszycki,† Matthew A. Baird, Alexandre A. Shvartsburg* Department of Chemistry, Wichita State University, 1845 Fairmount, Wichita, Kansas 67260, United States ABSTRACT: Nearly all compounds comprise numerous isotopologues ensuing from stable natural isotopes for constituent elements. The consequent isotopic envelopes in mass spectra can reveal the ion stoichiometry, but not geometry. We found those envelopes to split in differential ion mobility (FAIMS) spectra in a manner dependent on the ion geometry and buffer gas composition. The resulting multidimensional matrix of isotopic shifts is specific to isomers, providing a fundamentally new approach to the characterization of chemical structure. The physical origins of the effect remain to be clarified, but likely ensue from the transposition of center of mass of the ion within its geometry frame affecting the partition of energy in above-thermal collisions between the translational and rotational degrees of freedom. The additivity of shifts, holding with no exception so far, may be the key to unraveling the foundations of observed behavior.
Breakthroughs in chemistry were historically enabled by novel tools for molecular structure characterization, such as infrared or x-ray spectroscopy and nuclear magnetic resonance (NMR).1-3 Of humongous impact has been mass spectrometry (MS) that offers exceptional sensitivity, specificity, and speed across compound classes and sizes.4 Huge advances in the MS resolving power and accuracy now allow determination of exact stoichiometry from measured mass.5,6 However, isomers have same mass and are not distinguishable by single-stage MS. Multi-stage MS involving ion dissociation by various mechanisms has tremendously expanded our knowledge of molecular structures, but is limited by lack of unique fragments for important isomer types (e.g., modified peptide variants and lipid regioisomers).7-10 This is a real issue with isomeric mixtures, where one must connect the products to precursors. A growing complementary method is ion mobility spectrometry (IMS) that separates ions by properties of gasphase transport driven by electric fields.11 Linear IMS (normally at moderate field intensity, E) sorts species by mobility (K), which depends on the orientationally averaged ion-molecule collision cross section (Ω) according to the Mason-Schamp equation:12 𝛺=
(18π)1/2
𝑞
16
(𝑘𝐵 𝑇)1/2
1
1 1/2 1
𝑚
𝑀
( + )
𝑁
×
1 𝐾
(1)
where kB is the Boltzmann constant, m and M are the ion and gas molecule masses, q is the ion charge, and T and N are the gas temperature and number density. One can deduce the ion structures from Ω using molecular dynamics simulations. A major problem of linear IMS is modest orthogonality to MS, arising from inherent correlation between the ion mass and size.12,13
The mobilities of ions in any gas depend on the field. This underlies differential or field asymmetric waveform IMS (FAIMS)14 that captures ∆K - the difference between K at high and low E. In practice, ions are pulled by gas flow through a gap between electrodes carrying an asymmetric waveform of some amplitude (dispersion voltage, DV). The resulting strong oscillatory field deflects all ions toward the electrodes, but weak compensation field (EC) produced by compensation voltage (CV) superposed on the waveform can equilibrate species with given ∆K and permit it to pass the gap and be detected. Scanning EC yields a spectrum of ions entering the gap. With ∆K (and thus EC) correlated to ion mass much weaker than K itself, FAIMS can distinguish isomers and isobars substantially better8,10,15 than linear IMS at equal resolving power (R). Modeling this nonlinear effect is drastically harder than absolute K at low field, and no known quantitative first-principles procedure can extract the ion structures from FAIMS data. Most elements, including common H, C, N, O, S, feature multiple stable isotopes. Hence, all organic/biological and most other compounds are mixtures of isotopologues with fractions defined by the natural isotopic abundances for constituent elements. The ensuing ubiquitous mass spectral envelopes (with sufficient resolution) tell the stoichiometry,5,6 but contain no structural information and are identical for isomers. Isotopologues have miniscule geometry differences, except possibly for H/D substitution. Non-isobaric ions can materially differ in mobility because of unequal reduced mass in eq (1), revealing the masses of drifting ions (with complexed gas molecules) and furnishing the intrinsic mass scale.16,17 Such isotopic envelopes with 1 Da increments
ACS Paragon Plus Environment
Analytical Chemistry were recently seen in high-resolution linear IMS.18 However, they also hold no information about the ion structure. No nominally isobaric ions or isotopic isomers (isotopomers) have been resolved, in line with theory.16
Table 1. Computed intensities for H+MCA isotopologues
atoms
M
I*
m
I*
% at unit m
Conversely, FAIMS separations of isotopologues are not governed by the mass factor. Isobars and isotopomers with 13C, 15N, and/or D on specific sites were resolved for amino acids and peptides.19,20 As the peak order depends on gas composition, the effect ought to originate in the details of energetic ion-molecule scattering. Those studies addressed ions of same geometry with potential application to proteomics and metabolomics employing isotopic labeling, where the ability to distinguish differently labeled species and pinpoint the labeled site(s) may help.
None
128.027
100
128
100
100
15N
129.024
0.361
129
6.963
5.19
13C
129.030
6.489
93.20
D
129.033
0.112
1.61
37Cl
130.024
32.40
13C15N
130.027
0.023
0.07
13C13C
130.033
0.175
0.54
13CD
130.036
0.007
Here we explore unlabeled structural isomers - three monochloroanilines (MCA, C6H6NCl) with Cl in ortho (2), meta (3), and para (4) positions (Fig. 1), and distinguish them by spectral shifts between natural isotopologues.
37Cl15N
131.021
0.117
37Cl13C
131.027
2.102
93.05
37ClD
131.030
0.0363
1.61
Our FAIMS/MS system with electrospray ionization (ESI) source has been described.8,20 Briefly, a custom FAIMS device with 1.88-mm planar gap is coupled to the Thermo LTQ XL ion trap via a slit aperture/electrodynamic funnel interface. The high-definition bisinusoidal waveform has 1 MHz frequency and DV = 3.8 - 4.0 kV (peak field of 20 - 21 kV/cm). The CV scan speed is 1 V/min. The carrier gas (He/CO2 mixtures) is supplied at 3 L/min, meaning the filtering time of 0.1 s. The maximum He fraction (v/v) to avoid electrical breakdown under these conditions is ~65%, similar to the threshold with He/N2 buffers.8,10 The MCA standards (Sigma Aldrich, monoisotopic mass 127 Da) were dissolved to 0.1 mM in 99:1 methanol/formic acid and infused to the ESI emitter at ~0.5 μL/min. The CV scale was anchored using an internal calibrant (2-monobromoaniline, 172 Da) in similar concentration by linear dilation of the axis as established in peptide and lipid analyses.8,10 In particular, this corrects for variations of the ambient pressure and temperature.
13
All three MCAs yield 1+ ions, with H+ presumably attaching to N atom. The observed MS spectra (Fig. 1) match the isotopic distribution computed (Table 1) with unit resolution: disentangling isobars that differ by at most 0.01 Da necessitates R > 104 that is beyond the capability of ion trap even in the highest-resolution (ultrazoom) mode. First, we acquired FAIMS spectra for each isomer at the base peak (m/z = 128) across He percentages (Fig. 2). As expected in this mass range, all MCA ions belong to type A,14,20 which means a positive K(E) slope and negative EC. Upon He addition up to the maximum 63%, the absolute EC increase 100
2
3
80 60 40 20
4
0 127
128
129
130
131
m/z
Figure 1. MCA isomers and measured mass spectrum
132
Heavy
13
Accurate mass
13
Unit mass
130
32.61
99.37
0.02 131
2.259
5.18
C C C
131.037
0.0025
37Cl13C15N
132.024
0.0076
37Cl13C 2
132.030
0.0568
85
37Cl13CD
132.033
0.0024
4
0.11 132
0.067
11
* Intensity relative to the base peak (100%) for all species with I* ≥ 0.002%. -50
4
Signal
-150
2 220
120
130
230
240
250
-EC,V/cm
4
2
3 110
210
He 30%
Mix
4
He 63%
3 200
-200
Mix
Signal
-100
EC,V/cm
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
Page 2 of 5
60%
2
3
140
-EC,V/cm
-250 0
10
20
30
40
50
60
He fraction, %
Figure 2. Separations of MCA isomers at DV = 3.8 kV: EC depending on He % and spectra at 30% and 63% He. while peaks narrow with growing K values.22 As the result, the resolving power improves from ~10 in CO2 to ~100 at 63% He. These trends and R metrics mirror those for various ions in He/N2 mixtures.8,10,21,23 The │EC│ value for 2 greatly exceeds those for 3 and 4 at any He concentration, and 2 is resolved baseline at ≥20% He. The peaks for 3 and 4 lie much closer, but 3 edges to higher│EC│ as the He fraction goes up, allowing baseline resolution by 60% He (Fig. 2). Separations were verified using the isomolar 2/3/4 mixture (different peak intensities reflect unequal ionization efficiency and/or lower losses in the FAIMS gap14,22 for species with larger Ω). While the resolution of these isomers is satisfying, it was anticipated [for example, from the separations of analogous phthalic (o), isophthalic (m), and terephthalic (p)
ACS Paragon Plus Environment
Page 3 of 5
acid ions]24 and is not our present concern. Rather, we focus on the relative EC of isotopologues within an isomer at nominal m/z = 128, 129, 130, 131 (peak positions were imprecise at the relative signal of 93%, >99%, and >93% of intensity at m/z = 129, 130, and 131 respectively (Table 1). As with most labeled amino acids and peptides, heavier isotopologues have lower │EC│ values (Fig. 3a). This may produce a shoulder on the low-EC side of peaks averaged over the isotopic envelope, resembling those typical in IMS due to partly merged structural isomers (Fig. 3b). Of the inter-isotopologue EC shifts
Signal
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
Analytical Chemistry
20
30
40
50
60
0
10
20
30
40
50
60
He fraction, %
Figure 4. Measured ∆EC for isotopologic shifts in MCA isomers (DV = 4 kV and 0 - 63 % He). Points are the averages of ~12 replicates, with bars showing the std. error of mean. The results at DV = 3.8 kV were similar. Smooth trends as a function of He % prove the data accuracy further. identification. As a consequence, the differences in ∆EC(129 - 128) shift overall invert in ∆EC(130 - 129), Fig. 4d. The latter still resolves 3 and 4 significantly better and well enough at ≥40% He. Present shifts are so tightly reproduced (usually to 0.1 V/cm) since they are between the isotopologues that (i) elute within 30 s (less for smaller ∆EC), during which FAIMS conditions fluctuate negligibly, and (ii) have virtually identical geometries and close EC values that respond to any change in concert. Still, random errors can be compressed by multiplexing - adding concurrent measurements of same quantity. Indeed, summing the assumedequal ΔEC(129 - 128) and ΔEC(131 - 130) into cumulative 13C shift smoothens the trends and provides ready isomer differentiation (Fig. 5a). While the orders of 13C shifts and │EC│ for isomers match (Fig. 2), the differences for ΔEC are much larger and expressing the shifts in relative ΔEC/EC terms conserves their order (Fig. 5b). Those relative shifts somewhat decrease at higher He fractions. The capability to distinguish two peaks is assessed by resolution r - difference of position divided by mean peak width at half maximum (w). As w halves between 0 and 60 % He (Fig. 5c), the resolution is maximized at ~50% He stronger than the EC shifts (Fig. 5d). In contrast, structural isomers are nearly always best resolved at the maximum He fraction permitted by the electrical breakdown or sensitivity considerations.8,10,23,25 This follows the scenario with labeled amino acids19 and peptides20 and may be related to the impact momentum (speculated to control these isotopic effects, below) being smaller for light molecules. The maximum r is ~0.3, whereas good mixture separation requires r > 1.0. Then characterizing the mixtures of isomers by ∆EC would be challenging if their isotopologue envelopes overlap in FAIMS spectra. This is not the case here at ~60% He, where ∆EC patterns identify features fully resolved for any m/z (Fig. 2). Other systems may exhibit
ACS Paragon Plus Environment
Comb. EC shift/EC, %
3.0 2.5
(a) (129 - 128) + (131 - 130)
worse isomer separation but greater isotopologic shifts, where isomers would actually be resolved based on the latter.
2
2.0
3
1.5 1.0
4
0.5 1.6 1.4 1.2 1.0 0.8 0.6 0.4 0.2 0.0 6
2
(b)
w, V/cm
The six C and seven H atoms in H+MCA 3 lead to numerous isotopomers that 4 could split in IMS spectra.17 With one (c) 13C, there are six for 2 5 and 3 and four for 4 4 where the positions 3 2 and 6 and 3 and 5 0.25 (d) 2-4 are equivalent. (The 0.20 five or three isoto0.15 2-3 pomers due to one 0.10 3 - 4 low-abundant D are 0.05 much less perti0.00 nent). Isotopomers 0 10 20 30 40 50 60 should have unequal He fraction, % 13 ∆EC, in principle Figure 5. Cumulative C shifts for splitting the features MCA isomers in absolute (a) and for m/z = 129 and 131 relative (b) terms, mean peak width (c), and resolution between the into peaks of equal peaks at m/z = 129 and 128 (d). height for 2 and 3 Dashed lines are cubic regressions. and 2:2:1:1 ratio for 4. We did not encounter that in any regime, so the splitting must be within the peak width. Indeed, the peaks at m/z = 129 are slightly broader than the (equally wide) peaks at 128 and 130 for 2, 3, and perhaps 4 (Table 2). As one may expect from the trends of EC shifts and their differences, this broadening increases at ≥40% He. The minor isotopologues far more abundant at m/z = 129 than 130 (Table 1) may also contribute to this broadening. Anyhow, the narrower peaks for 4 at m/z = 129 indicate that ion geometries can in principle also be differentiated by peak widths in FAIMS spectra.
r
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
Combined EC shift, V/cm
Analytical Chemistry
All differentiation of MCA isomers comes from the 13C rather than 37Cl shifts, even though the structures differ only Table 2. Peak widths at m/z = 129 and 130 relative to m/z = 128 (from ~60 - 180 replicates, with 95% confidence interval) Isomers
2
3
4
Mean of 2/3/4
129 He 0-63%
1.068± 0.019
1.068± 0.020
1.017± 0.014
1.046±0.010
130 He 0-63%
1.009± 0.014
1.009± 0.009
0.994± 0.o10
1.003±0.007
Peaks
129 He 40-63% 130 He 40-63%
1.070±0.015 Insufficient data statistics 0.992±0.010
in the Cl location and 37Cl substitution increments the mass by 2 Da (vs 1 Da for 13C). The reason for and generality of this situation remain to be grasped. Concluding, we have demonstrated that natural isotopologues split in FAIMS spectra with distinct peak shifts and/or widths for each isomer. This phenomenon not previously encountered in any separation enables a new approach to molecular geometry elucidation. Of note is operational similarity to NMR (particularly 13C NMR), where chemical shifts for active nuclei carry structural information,3 though the underlying physics is obviously unrelated. However, present method can have sensitivity approaching that of MS (though inevitably below that of the used MS platform in view of ion losses in the FAIMS stage) and can probe individual species in complex mixtures with wide dynamic range. While resolving isotopomers would help, here we distinguished all isomers without that. Hence the described approach (identifying isomers based on FAIMS spectral shifts between isotopologues) qualitatively differs from the hypothetical method of Valentine and Clemmer,17 where one would distinguish isomers based on the ratios of isotopomer peak heights. Most stoichiometries allow multiple alternative and simultaneous isotopic substitutions that yield many isotopologue masses and thus a set of spectral shifts. That set depends on the buffer gas composition, creating a multidimensional array of fingerprint specificity. The ion species can then be assigned by matching the measured array to tabulated data. In principle, one needs those for all conceivable isomers to certainly exclude all but one. Whether said arrays are specific enough to make that redundant remains to be determined in a larger study. This approach may obviate the need for accurate EC scale calibration presently required to assign the resolved isomers, or deliver complementary confirmation. The observed behavior likely comes from the transposition of ion center of mass within its frame influencing the partition of energy in above-thermal ion-molecule collisions between rotational and translational degrees of freedom.19,20 Better understanding and modeling of these effects should lead to a capability to interpret isotopologic shifts in structural terms a priori, without reference to past experiments. This effort will be facilitated by empiric trends derived from analyses of diverse compounds with representative morphologies. One appears to be the additivity of shifts for multiple isotopes,19,20 holding across compound classes and atom types. The FAIMS and MS resolving powers of current platform are major limitations. For instance, no isotopomers or nominally isobaric isotopologues were distinguished here although both had been (by FAIMS) for labeled synthetic standards.19,20 Their separation would greatly augment the content and thus specificity of characteristic shift set. We are integrating high-definition FAIMS to Orbitrap MS (R > 105) to resolve nearly all isobars with m/z < 300 (including present chloroanilines incorporating 15N or D vs. 13C with respective −6 and 3 mDa differences).26 We are also raising
ACS Paragon Plus Environment
Page 4 of 5
Page 5 of 5 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
Analytical Chemistry FAIMS resolving power to improve the separation of all features but especially isotopomers not distinguishable by MS. Those advances will elevate the power of present approach and extend it to larger molecules with greater number of isotopologues and isotopomers in total and per mass unit. Corresponding Author * E-mail:
[email protected] † Presently at Excellims (Acton, MA)
ACKNOWLEDGMENT We thank Prof. David E. Clemmer for insightful discussions of isotopic effects in IMS, and Gordon A. Anderson (GAACE) and Andrew P. Bowman (WSU) for experimental help. This work was supported by NSF FIRST (EPS-0903806) and CAREER (CHE-1552640). A.S. has interest in Heartland MS that makes FAIMS and ion funnel systems utilized in this research.
REFERENCES 1. Larkin, P. Infrared and Raman Spectroscopy. Elsevier, 2011. 2. van der Heide, P. X-Ray Photoelectron Spectroscopy: an Introduction to Principles and Practices. Wiley: NY, 2011. 3. Keeler, J. Understanding NMR Spectroscopy. Wiley: NY, 2011. 4. Gross, J. H. Mass Spectrometry: a Textbook. Springer: 2017. 5. Marshall, A. G.; Rodgers, R. P. Proc. Natl. Acad. Sci. USA 2008, 105, 18090. 6. Klitzke, C. F.; Corilo, Y. E.; Siek, K.; Binkley, J.; Patrick, J.; Eberlin, M. N. Energy Fuels 2012, 26, 5787. 7. Xuan, Y.; Creese, A. J.; Horner, J. A.; Cooper, H. J. Rapid Commun. Mass Spectrom. 2009, 23, 1963. 8. Shliaha, P.; Baird, M. A.; Nielsen, M. M.; Gorshkov, V.; Bowman, A. P.; Kaszycki, J. L.; Jensen, O. N.; Shvartsburg, A. A. Anal. Chem. 2017, 89, 5461. 9. Han, X. Lipidomics: Comprehensive Mass Spectrometry of Lipids. Wiley: NY, 2016.
10. Bowman, A. P.; Abzalimov, R. R.; Shvartsburg, A. A. J. Am. Soc. Mass Spectrom. 2017, 28, 1552. 11. Eiceman, G. A.; Karpas, Z.; Hill, H. H. Ion Mobility Spectrometry. CRC Press: Boca Raton, 2013. 12. Mason, E. A.; McDaniel, E. W. Transport Properties of Ions in Gases. Wiley: NY, 1988. 13. May, J. C.; Goodwin, C. R.; Lareau, N. M.; Leaptrot, K.; Morris, C. B.; Kurulugama, R. T.; Mordehai, A.; Klein, C.; Barry, W.; Darland, E.; Overney, G.; Imatani, K.; Stafford, G. C.; Fjeldsted, J. C.; McLean, J. A. Anal. Chem. 2014, 86, 2107. 14. Shvartsburg, A. A. Differential Ion Mobility Spectrometry. CRC Press: Boca Raton, 2009. 15. Shvartsburg, A. A.; Mashkevich, S. V.; Smith, R. D. J. Phys. Chem. A 2006, 110, 2663. 16. Valentine, S. J.; Clemmer, D. E. Anal. Chem. 2009, 81, 5876. 17. Valentine, S. J.; Clemmer, D. E. Apparatus for Determining Masses at High Pressure. PCT Application. US 2010/044367. 18. Kirk, A. T.; Raddatz, C. R.; Zimmermann, S. Anal. Chem. 2017, 89, 1509. 19. Shvartsburg, A. A.; Clemmer, D. E.; Smith, R. D. Anal. Chem. 2010, 82, 8047. 20. J. L. Kaszycki, Bowman, A. P.; Shvartsburg, A. A. J. Am. Soc. Mass Spectrom. 2016, 27, 795. 21. Shvartsburg, A. A.; Tang, K.; Smith, R. D. Anal. Chem. 2004, 76, 7366. 22. Shvartsburg, A. A.; Tang, K.; Smith, R. D. J. Am. Soc. Mass Spectrom. 2004, 15, 1487. 23. Shvartsburg, A. A.; Danielson, W. F.; Smith, R. D. Anal. Chem. 2010, 82, 2456. 24. Barnett, D. A.; Purves, R. W.; Ells, B.; Guevremont, R. J. Mass Spectrom. 2000, 35, 976. 25. Shvartsburg, A. A.; Prior, D.; Tang, K.; Smith, R. D. Anal. Chem. 2010, 82, 7649. 26. Zhurov, K.O.; Kozhinov, A. N.; Tsybin, Y. O. Energy Fuels 2013, 27, 2974.
TOC Graphic
ACS Paragon Plus Environment
