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Identification of Isomers by Multi-Dimensional Isotopic Shifts in High-Field Ion Mobility Spectra Pratima Pathak, Matthew A. Baird, and Alexandre A. Shvartsburg Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.8b02057 • Publication Date (Web): 03 Jul 2018 Downloaded from http://pubs.acs.org on July 4, 2018
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Analytical Chemistry
Identification of Isomers by Multi-Dimensional Isotopic Shifts in High-Field Ion Mobility Spectra Pratima Pathak, Matthew A. Baird, Alexandre A. Shvartsburg* Department of Chemistry, Wichita State University, 1845 Fairmount, Wichita, Kansas 67260, United States *
[email protected] ABSTRACT: Nearly all molecules incorporate elements with stable isotopes. The resulting isotopologue envelopes in mass spectra tell the exact stoichiometry, but nothing about the geometry. Chromatography and electrophoresis at high resolution also can distinguish isotopologues, again without revealing structural information. In high-definition differential ion mobility (FAIMS) spectra, these envelopes universally split in structure-specific manner, providing a new general approach to isomer delineation. Here we show that the peak shifts from instances of same isotope are equal and can be averaged into characteristic elemental shifts, namely 13C and 37Cl for dichloroanilines (DCA). Matrices of these shifts, including the gas composition dimension, are unique to the structure. Hence, all six DCA isomers (with four making two unresolved pairs) are readily delineated in the 13C/37Cl maps with He/CO2 buffer gases. Mixtures of co-eluting isomers are also distinguished from pure components.
Most elements (including the common H, C, N, O, S, Cl, Br) have multiple stable isotopes. Thus all organic/biological and nearly all other compounds comprise numerous isotopologues (molecules with different isotopes for one or more atoms). The resulting mass distribution is unique to each stoichiometry, which in principle allows deducing it from the isotopic envelopes measured by mass spectrometry (MS).1-3 The needed resolving power (R) and mass accuracy are high and rapidly rise for heavier ions with increasing numbers of both potential molecular formulas and isotopologues for each per unit mass. Spectacular advances in resolution of Fourier Transform (FT) MS now make stoichiometry assignments relying on “exact” mass realistic up to ~1 kDa (especially within constraints of a compound class).1-4 This capability expands as new Orbitrap and FT Ion Cyclotron Resonance (FTICR) platforms using yet stronger electric or magnetic fields deliver3-5 ever greater R values approaching and exceeding 106. Hence, the frontline is moving to structural characterization of species and differentiation of isomers ubiquitous in proteomics, lipidomics, and petroleomics. As all molecular geometries share the isotopic envelopes, isotopic analyses are unrelated to structural MS. Modern MS analyses of complex samples typically involve prior separation step(s), traditionally by liquid chromatography (LC) or capillary electrophoresis (CE). Isotopologues necessarily have unequal geometries, with the differences sensitive to underlying morphology. Therefore, their separation properties theoretically differ in ways that (unlike in MS) depend on the structure. The LC6-9 and CE10-15 elution times for atomic and molecular species, including pure and substituted anilines similar to the chloroanilines studied here,10,13 shift due to various heavy isotopes (D, 11B, 15N, 17O, 18O, 37Cl, 81Br). With long gradients (up to 20 hr.), some non-isobaric isotopologues were fully resolved. These shifts appear mostly mass-dependent -
none between nominally isobaric isotopologues or isotopic isomers (isotopomers) were reported, and conveyed no structural information. A growing replacement or complement to condensed-phase separations is ion mobility spectrometry (IMS) employing ion transport through gases driven by electric fields.16,17 In the original linear IMS mode,16 ions are sorted by drift velocity (v) at moderate field intensity (E), where v(E) is a linear function. Its slope called mobility (K) is proportional to the inverse orientationally averaged ion-molecule collision cross section (Ω) per the Mason-Schamp equation:18 𝐾=
(18π)1/2
𝑧𝑒
16𝑁𝛺
(𝑘𝐵 𝑇)1/2
1
1 1/2
𝑚
𝑀
( + )
(1)
where kB is the Boltzmann constant, m and M are the ion and gas molecule masses, e is elementary charge, z is the charge state, and T and N are the gas temperature and number density. Isotopologues differ in mobility because of unequal reduced mass in eq (1). The resolved peaks for isotopic envelopes with 1 Da increment furnish intrinsic mass scale, revealing the masses of drifting ions (with adsorbed gas molecules).19,20 The structural information in theory resides in shifts of Ω, but isotopologues have negligible geometry differences except possibly for H/D exchange. Indeed, no mobility deviations (leave alone peak resolution) were observed for nominal isobars or isotopic isomers (isotopomers) in linear IMS, and structural elucidation based on the isotopic splitting21 remains hypothetical. A newer form of IMS is differential or field asymmetric waveform IMS (FAIMS).17,22-32 The mobility of any ion in a gas depends on E above certain threshold.17,18 That is exploited in FAIMS, where a periodic asymmetric field separates species by ∆K - the difference between K values at low and high E. In practice, a gas flow pulls ions through a gap between two electrodes carrying a waveform of some amplitude (dispersion voltage, DV). The consequent oscillatory field deflects all ions toward
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an electrode, but weak compensation field (EC) produced by compensation voltage (CV) superposed on the waveform equilibrates the species with given ∆K and permits it to pass the gap to a detector. Scanning EC yields a spectrum of ions entering the gap.17 The ∆K and thus EC correlate with ion mass weaker than K does (usually by 3 - 4 fold for biomolecules), rendering FAIMS more orthogonal to MS than linear IMS is.31,32 This enables FAIMS to discriminate isomers (e.g., peptide isoforms31 and lipid regioisomers32) better than linear IMS can. A striking example is the FAIMS resolution of amino acid and peptide isotopomers labeled on specific sites. 33,34 Unlike with linear IMS, separations of isotopologues in FAIMS are not governed by the mass even qualitatively: heavy atom substitution may push EC in either direction, depending on the ion species and gas.33 Then the effect must originate in the details of energetic ion-molecule scattering. We recently discovered that EC shifts between non-isobaric isotopologues reflecting the natural isotopic abundances differ between the isomers.35 The protonated monochloroanilines, MCA (base peak at m/z = 128) with Cl in alternate positions on the benzene ring (o-, p-, m-) were distinguished by the EC shifts between species with no and one 13C atom, i.e., ∆EC(129 − 128) and ∆EC(131 − 130), where ∆EC(m1 − m2) = EC(m1) − EC(m2) (2) The shifts between isotopologues with 35Cl and 37Cl [∆EC(130 − 128)] exceeded those (sensible for 2 Da increment), but lied much closer for all isomers - surprising as those differed in the location of Cl (not C) atoms. The features at m/z = 129 and 131 were slightly broader than the other two (of equal width), presumably because of the convolution over four or six unresolved isotopomers with inequivalent 13C sites. As the shifts for artificially labeled species,33,34 those for 13C and 37Cl were additive. Therefore, ∆EC(129 − 128) and ∆EC(131 − 130) were identical within the experimental uncertainty and could be summed into the cumulative 13C shift to compress the random data scatter for superior isomer differentiation.35 Project Definition and Experimental Methods The above work35 has opened a fundamentally new path to isomer identification via the peak shifts between natural isotopologues. Some major ensuing questions are: (a) How broad is the approach, are there molecular symmetry constraints? (b) What are the typical shift magnitudes and how they depend on the instrumental parameters? (c) Up to what mass are such analyses feasible? (d) Are shifts not involving C atoms additive as well? (e) Are more than two concurrent shifts additive? (f) Can other (e.g., 37Cl) shifts be structurally informative? (g) How specific are these shifts in terms of the number of distinguishable isomers? (h) Can shifts characterize unresolved isomer mixtures? To start answering these, here we examine dichloroaniline C6H5NCl2 (DCA) that has six isomers with variant Cl positions on the ring (Figure 1). All DCAs also make protonated 1+ ions in electrospray ionization (ESI) source, with H+ presumably on the N atom. The observed mass spectra (Figure 1) match the isotopic distribution computed (Table 1) with unit resolution: disentangling isobars spaced by 104 that is beyond the capability of ion trap even in the ultrazoom mode.
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Figure 1. Geometries of DCA isomers and MS spectrum over the relevant FAIMS range. The peaks at m/z = 167 and 168 are scaled as labeled. The ion flux depends on the isomer and is lowest for 2,6, presumably as two Cl adjacent to amine reduce its proton affinity. The highest-mass MCA peak intense enough to precisely measure EC (at least ~1% relative intensity) was the monoisotopic mass +3 Da, mostly due to 13C37Cl. Here, the two Cl atoms and high 37Cl fraction yield abundance of ~1% or more for species comprising 37Cl2 at the base monoisotopic peak (m/z = 162) plus 4 and 5 Da (Table 1). As with MCAs, >93% of the signal at any of those masses arises from the dominant stoichiometry, here with one or two 37Cl and/or one 13C (but no 15N or D). Hence we can again ascribe the observed peaks to specific isotopologues. This expands the shift matrix from three to five entries, creating a more informative set. In particular, we can address (d, e) using the 37Cl2 and 13C37Cl2 isotopologues. Higher ion counts herein (below) have further allowed measuring EC for some isomers at the base peak +6 Da (m/z = 168) with 0.02% intensity, albeit less precisely. Table 1. Intensities computed for H+DCA isotopologues (using the calculator on www.sisweb.com). Heavy Atoms
M
Accurate mass I*
Unit mass M
I*
% at unit m
None
161.988
100
162
100
100
15
N
162.985
0.361
163
6.947
5.20
13
C
162.991
6.489
93.41
D
162.994
0.096
1.4
37
Cl
163.985
64.80
13
C15N
163.988
0.023
0.035
13
C2
163.994
0.175
0.269
13
CD
163.997
0.006
37
Cl15N
164.982
0.234
37
13
Cl C
164.988
4.205
93.34
37
ClD
164.991
0.062
1.4
13
C3
164.998
0.003
37
Cl2
165.982
10.50
37
Cl13C15N
165.985
0.015
0.14
37
Cl13C2
165.991
0.114
1.07
37
13
Cl CD
165.994
0.004
37
Cl215N
166.979
0.038
37
Cl213C
166.985
0.681
37
Cl2D
166.988
0.010
37
Cl213C15N
167.982
0.003
37
Cl213C2
167.989
0.018
164
65.00
99.69
0.009 165
4.505
5.19
0.07 166
10.63
98.78
0.04 167
0.731
5.2 93.2 1.4
168
0.022
12 86
* Intensity relative to the base peak (100%) for all species with I* ≥ 0.002%. The I* values ≥ 0.02% are bolded.
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MS noted for lipids 167 166 165 164 163 2,5 and peptides.29-31 162 Nonetheless, 2,3 and 168 2,4 and 3,4 and 3,5 always coincide (Figure 2). The 2,5 can be largely resolved from 2,3 and 2,4 at 43% He, better at lower Q (below). 214 216 218 220 222 224 226 Single FAIMS peaks 164 165 167 168 3,5 persist at heavier m/z, 163 166 reflecting the domi162 nant isotope set: 13C, 37 Cl, 13C37Cl, 37Cl2, 13 37 C Cl2, and 13C237Cl2 at m/z = 163, 164, 165, 166, 167, and 168 (Table 1). These generally are not coincident, 178 180 182 184 186 188 190 EC, V/cm with isomer-specific patterns. For example, augFigure 3. FAIMS spectra for menting the mass uniformly 2,5 and 3,5 at unit masses. moves peaks to lower EC for 2,5, while for 3,5 the presence of 37Cl decreases EC but that of 13 C increases it (Figure 3). Hence DCAs also exhibit structurally informative isotopologic shifts in FAIMS spectra. To survey them, we grouped the independent ∆EC by isotope: 13 C shifts [∆EC(163 − 162), ∆EC(165 − 164), ∆EC(167 − 166)] and 37Cl shifts [∆EC(164 − 162), ∆EC(166 − 164)]. The 13C shift for ∆EC(168 − 167) was not quantified because of (at best) miniscule signal at m/z = 168. The three 13C and two 37Cl shifts are close for any given isomer and He fraction, showing the asserted33-35 additivity (Figure 4). Hence we can again reduce the scatter by averaging the associated ∆EC. Then a larger number of individual ∆EC for DCAs than MCAs does not expand the ∆EC set but improves its precision. The absolute ∆EC and their inter-isomer differences increase upon He addition, as with35 MCAs up to 40% He. So we focus on the shifts at 40% He. While present ∆EC make 4; 2,5 > 3,4 > 3,5; 2,5 > 2,6 > 2,4 (at 40% He). That the insertion of one atom in same position on different isomers can invert their relative EC shifts (within a conserved peak order) further illustrates their structural specificity. The consistency of 13C and 37Cl shifts within each isomer is remarkable, especially considering that most dominant isotopologus comprise multiple (up to 15) isotopomers (Table 2) with a priori unequal EC, producing heterogeneity for all shifts but 37 Cl in 2,6 and 3,5. However, the peak widths for all isomers at m/z = 163 - 167 are 100 - 110% of those at m/z = 162. Hence all isotopomers have close EC values, extending that finding for MCAs35 to a greater number of isotopomers and 37Cl shifts.
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Table 2. Number of isotopomers for dominant isotopologues at unit masses, per isomer Heavy atoms
13
37
13
37
13
13
2,3; 2,4; 2,5; 3,4 2,6; 3,5
6
3
12
1
6
15
4
1
12
1
4
9
C
Cl
C37Cl
Cl2
C37Cl2
C237Cl2
Conclusions The isomer-specific shifts for natural isotopologues in FAIMS spectra are not singular to monochloroanilines, but enable a general novel approach to molecular structure identification. Present results answer the posed questions: (a) The approach supposes no particular ion symmetry. While the 4-MCA and DCAs 2,6 and 3,5 have a mirror plane, other MCAs and DCAs hold no symmetry. (b) The EC shifts can have either sign, but “+” (lower │EC│ for higher mass) appears more common - as observed previously.3335 The magnitudes are up to 2 V/cm, apparently decreasing for heavier ions. Stronger dispersion field at lower He fractions in He/CO2 yields qualitatively similar, but uniformly greater shifts than lower ED at greater fractions. This tracks the situation for peptide isotopomers, but is unusual for general isomer separations. Perhaps isotopic separations are favored by heavier gases that raise the resolution of non-isobaric isotopologues in linear IMS via increasing the difference of reduced ion-molecule masses.19 Then yet heavier buffers such as pure CO2 or SF6 may be of benefit. The shifts may also be simply more pronounced at greater │EC│, wherein one should choose the gas to maximize those. Pertinent investigations are ongoing. (c) The approach extends at least to ~170 Da, and the mass scaling of ∆EC suggests feasibility in the >200 Da range. For larger ions, the decrease of absolute individual shifts is at least partly offset by their increasing number and diversity across isomers. Thus the approach may work for much larger ions - in parallel to peptides with variant PTM localization, where the decrease of resolution due to smaller fractional mass and size of PTM for longer sequences is mitigated by greater number of charge states and accessible low-energy conformations.29 We are moving to larger species to explore these trends. (d) The shifts involving two 37Cl or 37Cl and 13C are also remarkably additive. Whether small discrepancies (noticeable in Figure 4) are real deserves exhaustive research. (e) The shifts with three atoms (13C and two 37Cl) are as additive as those with two. This highlights additivity as the overarching property of isotopologic shifts in FAIMS,33-35 which enables their instant verification and superior determination by comparing and averaging ∆EC for multiple instances of same isotope.35 The shift comprising two 13C and two 37Cl for 3,5 seems to deviate from additivity (Figure 3), but poor precision of measured EC near the detection limit precluded clarification. (f) Most importantly, the shifts for isotopes beyond 13C (namely 37 Cl) are structurally informative. This allows constructing multidimensional matrices of excellent specificity. (g) Present robust delineation of all six DCA isomers using 2D shift maps proves the power of approach. Yet higher specificity would be readily achieved by (i) use of diverse buffers beyond He/CO2 to add another separation dimension(s) and (ii) combining with high-resolution MS platforms (proximately Orbitrap with resolving power >105) to improve the accuracy of
major shifts and establish new ones by isolating minor nominally isobaric isotopologues - here comprising D or 15N with 3 or 6 mDa mass difference.35 (Whereas low-resolution curvedgap FAIMS units were integrated with FTICR30 and Orbitrap27 MS, the effort for high-resolution FAIMS is in progress). The ability to measure EC across ~104 dynamic range demonstrated here portends well for the expansion of approach to minor isotopologues currently hidden under dominant isobars. For example, DCAs have 14 isotopologues more abundant than the total at presently measured m/z = 168 (in bold in Table 1) versus six captured here. The extra ∆EC points (four for 15N, two for D, and two for 13C2) would, assuming additivity, increase the precision of 13C shift and provide the mean 15N and D shifts. Improved ESI/FAIMS and FAIMS/MS interfaces46-49 ensuring greater initial ion flux and better transmission and thus wider dynamic range ought to permit measuring yet less abundant (isobaric or not) isotopologues, with nearly all stoichiometries in the relevant mass range resolved by FTMS1-5 and many by multi-pass time-of-flight mass spectrometers50 with R > 105. Those advances will elevate the power of approach and extend it to larger molecules with greater number of constituent elements/isotopes and isotopologues in total and per mass unit. (h) The method can distinguish unresolved isomer mixtures from pure components and possibly gauge their ratio (to be assessed). It also helps a lot to identify the resolved isomers. For example, distinguishing 2,5 from 2,4 by EC (differing by 2%, Figure 6) is a challenge that requires exceptional control of all conditions (DV, waveform shape, and gas composition, pressure and temperature), confirmed by thorough multipoint calibration. In contrast, the 13C shift for 2,5 dramatically differs from the near-zero for 2,4 across the ED values and He fractions (Figures 4, 7), allowing trivial calibration-free differentiation. Although the physics is obviously unrelated, identification of compounds based on isotopologic shifts in FAIMS operationally resembles35 NMR where chemical shifts for active nuclei contain structural information.51 Then the 2-D maps (Figure 5) would be analogous to 2-D NMR, specifically heteronuclear correlation methods such as HSQC and HMBC.51-53
ACKNOWLEDGMENT We thank Gordon A. Anderson (GAACE), Dr. Keqi Tang (PNNL), and Andrew P. Bowman and Julia L. Kaszycki (WSU) for experimental help, and Prof. David E. Clemmer for insightful advice. This work was supported by NSF CAREER Award (CHE1552640). A.S. also holds a faculty appointment at the Moscow Engineering Physics Institute (MEPhI), Russia.
Supporting Information Available Examples of replicate FAIMS spectra and 2-D shift maps at other He fractions.
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