Fine Structure in Isotopic Peak Distributions Measured Using a

Article pubs.acs.org/ac

Fine Structure in Isotopic Peak Distributions Measured Using a Dynamically Harmonized Fourier Transform Ion Cyclotron Resonance Cell at 7 T Eugene N. Nikolaev,*,†,‡,§ Roland Jertz,∥ Anton Grigoryev,†,⊥ and Gökhan Baykut*,∥ †

The Institute for Energy Problems of Chemical Physics, Russian Academy of Sciences, Leninskij pr. 38, k.2 Moscow Russia 119334 Institute of Biochemical Physics, Russian Academy of Sciences, Kosygina 4, Moscow Russia 119334 § The Institute of Biomedical Chemistry, Russian Academy of Medical Sciences, Pogodiskaja 10 Moscow Russia 119121 ∥ Bruker Daltonik GmbH, Fahrenheitstrasse 4, 28359 Bremen, Germany ⊥ The Institute for Information Transmission Problems, Russian Academy of Sciences, Bolshoy Karetny per. 19 Moscow Russia 127994 ‡

S Supporting Information *

ABSTRACT: The fine structure of isotopic peak clusters in mass spectra of reserpine and substance P are measured using Fourier transform ion cyclotron resonance mass spectrometry at a 7 T magnetic field. The resolved peaks in the fine structure consist of 13C, 15N, 17O, 18O, 2H, 33S, 34S, and combinations of them. A recently introduced high-resolution ion cyclotron resonance cell (Nikolaev, E. N.; Boldin, I. A.; Jertz, R.; Baykut, G. J. Am. Soc. Mass Spectrom. 2011, 22, 1125−1133) is used in these experiments. The positions of the experimentally obtained fine structure peaks on the mass scale agree with the isotopic distribution simulations with ≤200 ppb error. Some deviation from the theoretical isotopic distribution is observed, less abundant peaks in the fine structure patterns are a little suppressed compared to the larger ones. We present a method for atomic composition determination using accurate mass data and fine isotopic structure of the mass spectrum. Our method combines the traditional atomic composition determination from accurate mass data by enumeration of all possible formulas within constraints defined a priori with the estimation of element coefficients from resolved isotopic structures. These estimated values allow one to narrow the search space for the composition and therefore to reduce the number of candidate formulas.

M

only one 2H, or one 15N, or one 17O, or one 33S). The next one with the nominal mass mMP + 2 consists of a cluster of peaks representing molecules having either two 13C atoms or two 15N atoms or one 13C plus one 15N, or one 34S, etc. Thus, each of the isotopic peaks at the nominal masses mMP + n consists of a unique multiple-peak system, and depending on the size of the molecule and on the heteroatoms, they can be quite complex. Although each of the isotopic peak clusters at the nominal masses mMP + n consist of multiple-peak systems, in the organic mass spectrometry it became almost customary to refer to the nonmonoisotopic peaks as 13C peaks. This is mainly due to the insufficient resolving power of most of the mass spectrometers used for the analytical investigations so that each one of these isotopic clusters at the nominal masses mMP + n appear as one unresolved peak. Therefore, the information hidden in the fine structure of the isotopic peak clusters cannot be used. The

ass spectrometric studies always involve the consideration of atomic isotopes in the compounds studied. In the mass spectrometry of organic compounds, the atoms carbon, oxygen, nitrogen, sulfur, phosphorus, and hydrogen play the main role. Most of these elements (12C, 14N, 1H, 16O) have one most abundant isotope with around 99% or more abundance, 32S with around 95% abundance, while 31P is the only stable isotope of phosphorus. The remaining isotope(s) of these elements have very minor abundances (e.g., 13C, 1.070%; 2 H, 0.0115%; 15N, 0.368%; 18O, 0.205%; 34S, 4.290%; and 33S, 0.75%). However, with increasing the size of the molecule, i.e., with increasing the number of atoms, isotopic peaks even with minor abundances start appearing in the mass spectrum. In the mass spectrum of an organic compound measured in the narrow m/z region covering all detecting isotopic combinations for the molecular ion, the monoisotopic peak (MP) represents the molecule (the ion) composed of the main isotopes 12C, 1H, 14 N, 16O, etc. only. Other isotopic combinations appear next to the monoisotopic peak in approximately 1 Da distances (mMP + n with n = 1, 2, 3, ...). The first of them at the nominal mass mMP + 1 is a cluster of peaks of the molecules that contain only one of other than main isotopes of C, H, N, O, and S (13C, or © 2012 American Chemical Society

Received: November 3, 2011 Accepted: January 30, 2012 Published: January 30, 2012 2275

dx.doi.org/10.1021/ac202804f | Anal. Chem. 2012, 84, 2275−2283

Analytical Chemistry

Article

Figure 1. The dynamically harmonized ICR cell1,2 used for the measurements of highly resolved isotopic fine structures at 7 T.

drying gas, heated up to 200 °C. The sample concentration was chosen very high in order to be able to select all desired 13C isotope cluster groups at proper intensities using the same sample preparation; the intensity ratio of the highest mass peak in the fourth 13C isotope cluster peak of reserpine to the monoisotopic 12C mass peak is about 0.12%. Reserpine ions were generated only as singly protonated (m/ z 609) molecules, substance P ions as singly (m/z 1347), doubly (m/z 674), and triply (m/z 452) protonated molecules. In the case of substance P, only the doubly protonated molecular ions were used in the experiments. In the first step, a narrow band mass spectrum of the pseudomolecular ion containing the monoisotopic peak and several isotopic peak clusters was acquired. Therefore, the ion cluster group in a mass range of about 4 Da was isolated in the quadrupole mass selector, subsequently accumulated in the collision cell for typically several hundreds of milliseconds, and finally transferred through the hexapole ion guide into the ICR cell. Ions captured in the ICR cell were excited by a dipolar broadband excitation chirp (frequency sweep from 150 to 3500 Da) applied for about 16 ms, with each single frequency step applied for 15 μs. The detection was performed in the heterodyne mode using a detection time duration of 7 s resulting in a moderately high resolving power so that at least the two most abundant isotope peaks in each of the first four nonmonoistopic clusters could be resolved. Typically 50 scans were accumulated. The ion population in the ICR cell was kept high enough to determine the fourth isotopic cluster with a reasonable intensity but low enough to avoid peak coalescence and minimize ion− ion interaction effects in the abundant peak clusters. In order to distinguish low-intensity isotope peaks from mathematical FFT sidebands of adjacent high-intensity peaks, a sine square window function was applied for the apodization of the transient. However, this reduced the initial resolving power by almost 40%. The time domain signal was then transformed using the FFT magnitude calculation, including a zero filling to 4 times the initial time domain size. These narrow band spectra acquired by mass selecting and isolating the ions of (practically) the complete isotopic pattern will be called in the following the “complete pattern spectra”. In the second step, each of the first four 13C isotope cluster groups were measured separately. For this, one individual isotope cluster (e.g., the cluster group containing one 13C isotope) was isolated in the quadrupole mass selector. After accumulation in the collision cell for typically several hundreds of milliseconds it was transferred into the ICR cell. In order to be able to measure all cluster groups (of different abundances) with roughly equal peak intensities, the ion population in the

isotopic fine structure only becomes visible when ultra highresolution mass spectrometry is used. Fourier transform ion cyclotron resonance mass spectrometry delivers the highest resolution of all mass spectrometric techniques. In our recent paper,1 we demonstrated the ultra high-resolving power with a new ICR cell in just a moderate magnetic field of 7 T. With this ultra high resolving power, the fine structures of the isotopic peak clusters can be resolved. A very important task of the mass spectrometry is to determine the atomic composition of a compound. One of the options is a very accurate determination of the molecular mass. The number of possible compositions to be assigned to the investigated compound significantly decreases with increasing mass accuracy. Another option is to get help from fragmentation experiments (MSn) while discussing possible and impossible compositions and structures for the compound. In the present work, we show the utility of isotopic peaks intensity distribution analysis for narrowing the formula search space.

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EXPERIMENTAL SECTION Experiments were performed using the ESI-Qh-FTICR mass spectrometer which is described in detail elsewhere.1 The mass spectrometer was equipped with an electrospray ion source (Bruker Daltonics, Apollo II), a quadrupole mass selector, a hexapole collision cell, and a hexapole ion guide for transferring ions to the ICR cell, which is placed in the center of an actively shielded 7 T superconducting magnet (Bruker Biospin, Wissembourg, France). The new type of ICR cell is also described in detail elsewhere.1 The specific design and electrode geometry were chosen based on digital simulations2,3 using an ion motion simulation program.4 The length L of the cell is 150 mm, and the inner diameter is 56 mm. The end-cap electrodes have orifices of 6 mm diameter in their centers (Figure 1). An additional electrode at the entrance of the cell is added here to provide focusing of incoming ions. On both end-cap electrodes as well as the entrance lens, a typical dc voltage of 1.5 V is directly applied during the detection sequence, and the same dc voltage is applied to the convex electrodes of the cylindrical surface, while the concave electrodes remain grounded. During the injection of ions, the entrance lens and the front end-cap electrode are pulsed down to typically −10 V. Two different samples were used, reserpine (no. R-0875 by Sigma Aldrich, Munich, Germany) and substance P (no. S-6883 by Sigma Aldrich, Munich, Germany). Both samples were sprayed by direct infusion at a concentration of 1 pmol/μL in water/methanol (1:1 in vol) into which 0.1% formic acid was added, with a flow rate of 120 μL/h. Nitrogen was used as the 2276



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ICR cell was controlled by adjusting the ion transfer parameters of the electrospray source and the accumulation time in the collision cell. This allowed us to use the same sample concentration for all experiments. The ion population in the ICR cell was kept low enough to avoid peak coalescence and ion−ion interactions. The detection was again performed in heterodyne mode, now choosing a detection time duration of about 45 s. Here also typically 50 scans were accumulated. This procedure turned out to be a good compromise between ideally high resolving power (3 900 000) and good long-term resonance frequency stability during the overall detection time of about 40 min. The time domain signal again was transformed into a mass spectrum as described above. These spectra acquired by isolating individual 13C isotope cluster groups and measuring them separately will be called in the following the “isolated isotope spectra”.

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RESULTS Figure 2 shows the complete pattern spectra containing the monoisotopic pure 12C peak and the first four isotopic peak clusters of protonated reserpine (a) at a resolving power of 1 310 000 and of doubly protonated substance P (b) at a resolving power of 1 390 000 measured at the monoisotopic peak. The inserts in each spectrum show a close view at each individual cluster group. For evaluation of the acquired mass spectrum, the “Data Analysis” software (Bruker Daltonik, Bremen, Germany) was used. For comparison, simulated isotopic distributions were generated at approximately the same resolving power conditions as measured at the monoisotopic (12C) peak in both cases. The measured mass spectrum was single point mass calibrated using the monoisotopic mass peak position as reference. All m/z values and relative intensities of the clearly resolved isotope peaks are listed in Tables 1 and 2. Figure 3a−d shows for singly protonated reserpine the isolated isotope spectra, i.e., the fine structure spectra of each individual 13C isotopic peak cluster in detail from the first 13C cluster (a) to the third 13C cluster (d), respectively. Using the Data Analysis software, isotopic distributions for each 13Cn fine structure clusters were simulated for approximately the same resolving power conditions. The peak intensity of each isotope was calculated in reference to the 13Cn isotope peak. Each individual measured fine structure spectrum was then mass calibrated using the most abundant peak in the cluster as a reference mass for single point calibration. All mass values and relative intensities are listed in Table 1. The spectra of the doubly protonated substance P are shown in Figure 4a−d. The results listed in the Table 2 were evaluated the same way as described for the reserpine experiments. After single-point calibration of each fine structure spectrum in reference to the 13Cn peak, the maximum mass deviation of all clearly identified isotopes is 160 ppb. The average value of the absolute deviations is 69 ppb which corresponds to 45 μu.

Figure 2. Complete pattern spectra of protonated reserpine (a) and doubly protonated substance P (b) acquired in narrowband (heterodyne) mode. Ions corresponding to the monoisotopic peak and first four 13C isotope clusters were isolated in the quadrupole and injected into the ICR cell for measurement. The inserted spectra show a closer view at the individual 13C clusters, in which the peaks containing only 13C isotopes are labeled, mixed isotope cluster peaks are not labeled. Mass spectra were single-point calibrated using the monoisotopic peak. Gray colored spectra marked with “S” are simulated fine structure pattern, the blue ones marked with “M” are the measured spectra. The reserpine spectrum (a) is acquired with a resolving power of 1 310 000 measured at the monoisotopic peak, spectrum (b) with a resolving power of 1 390 000, again measured at the monoisotopic peak. Note that the isotopic peak clusters show with increasing order an increasing shift to higher masses compared to the simulated spectra when single point calibration is applied to the monoisotopic peak.

measurement accuracy of the instrument. To narrow the search space, additional constraints on coefficient values are typically introduced. We risk obtaining a seemingly unambiguous identification yet missing the true composition due to its falling outside the search space if we set the constraints too narrow. By consideration of the isotopic distribution pattern, we can derive a set of rather narrow constraints for intensity ratios of peaks corresponding to different isotopic compositions.6,7 Because we know the natural distribution of isotopes for all elements in the compound, we could determine coefficient values for these elements in the compound formula. Since the

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DETERMINATION OF MOLECULAR ION ATOMIC COMPOSITION FROM THE FINE ISOTOPIC STRUCTURE In the traditional approach to determination of the atomic composition of molecules from accurate mass data, the result set of candidate formulas is formed by enumerating all molecular formulas and selecting those with masses equal to that of the monoisotopic peak within the limits of the mass 2277



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Table 1. Mass and Peak Intensity Values of the Measured and Simulated Isotopic Fine Structure Spectra of Reserpine and Substance P with Resolving Powers of 1 310 000 and 1 390 000, Respectivelya isotope group 12

C

13

C

13

C2

13

C3

13

C4

12

C

isotope 12

C 15 N 13 C 2 H 13 15 C N 18 O 13 C2 2 H 13C 13 C2 15N 13 18 C O 13 C3 13 C2 18O 13 C4

609.280 657 610.277 692 610.284 012 610.286 934 611.281 047 611.284 904 611.287 367 611.290 289 612.284 402 612.288 259 612.290 722 613.291 613 613.294 101

12

674.371 350 674.871 043 674.873 030 675.369 251 675.371 548 675.374 711 675.867 767 675.870 065 675.870 932 675.872 721 675.873 227 675.875 153 675.876 393 675.877 849 676.369 447 676.371 476 676.372 612 676.373 757 676.374 907 676.376 076 676.376 821 676.378 077

C S 13 C 34 S 13 15 C N 13 C2 15 N 34S 13 15 C N2 13 34 C S 15 N 18O 13 C2 15N 13 18 C O 13 C3 2 H 13C2 13 15 C N 34S 34 18 S O 13 C2 34S 13 15 C N 18O 13 C3 15N 13 C3 33S 13 C2 18O 13 C4 33

13

C

13

C2

13

C3

13

C4

theoretical mass value [u]

measured mass value [u]

mass deviation [mu]

mass deviation [ppb]

Reserpine + H+ (C33H41O9N2+) 609.280 657 0.0 0 610.277 927 0.23 385 610.284 181 0.17 277 610.286 952 0.02 29 611.281 264 0.22 355 611.285 145 0.24 394 611.287 575 0.21 340 611.290 469 0.18 294 612.284 543 0.14 230 612.288 480 0.22 361 612.290 938 0.22 353 613.291 853 0.24 391 613.294 228 0.13 207 Substance P + 2H+ (C63H100O13N18S12+) 674.371 49 0.0 0 674.870 427 −0.62 −913 674.873 107 0.08 114 675.369 844 0.59 878 675.372 137 0.59 872 675.374 959 0.25 367 675.868 417 0.65 962 675.870 514 0.45 664 675.871 509 0.58 854 675.872 078 −0.64 −951 675.873 840 0.61 907 675.875 848 0.70 1028 675.876 908 0.51 762 675.878 466 0.62 913 676.370 081 0.63 937 676.371 901 0.42 628 676.373 202 0.59 872 676.374 489 0.73 1082 676.375 470 0.56 832 676.376 969 0.89 1320 676.377 514 0.69 1025 676.378 619 0.54 801

theoretical peak intensity [%]

measured peak intensity [%]

peak intensity difference [%]

100.0 0.7 35.7 0.5 0.3 1.8 6.2 0.2 0.05 0.7 0.7 0.1 0.1

100.0 0.4 36.7 0.2 0.1 1.4 5.5 0.1 0.02 0.4 0.5 0.1 0.02

0.0 −44.1 2.7 −64.1 −47.3 −25.1 −10.7 −46.3 −47.8 −34.0 −33.8 −33.9 −64.0

100.0 6.6 68.6 4.5 4.5 23.2 0.3 0.1 3.1 0.2 1.5 1.8 5.2 0.3 0.2 0.2 1.1 0.2 0.4 0.1 0.6 0.9

100.0 2.2 70.1 1.8 1.8 16.3 0.1 0.1 1.5 0.1 0.6 0.8 2.7 0.1 0.1 0.1 0.4 0.03 0.1 0.1 0.2 0.3

0.0 −66.5 2.2 −60.8 −59.1 −29.7 −52.3 1.3 −51.2 −60.7 −58.3 −55.2 −48.1 −62.4 −41.9 −66.5 −63.3 −78.5 −73.1 −6.9 −64.2 −70.4

a

These values refer to the spectra where the monoisotopic peak and the first four 13C isotope clusters were isolated and measured (complete pattern spectra).

isolated isotope spectra. We chose the second option because intensity ratios are less accurate if peaks belong to different isolated isotope spectra (in isotope clusters of different 13C isotope numbers). Also in the second option we need to measure only a few isolated isotope spectra, (say, one or two), and this is decreasing the measurement time. It is possible to derive a set of equations that allows one to estimate coefficients in compound’s composition from intensity ratios without assembling the complete pattern spectrum. Suppose we have a compound consisting of C, H, O, N, S, and P atoms. In the fine structure, any observable isotopic substitution for these elements result in either the shift from the monoisotopic peak to higher masses by approximately 1 u (e.g., 1H to 2H, 32S to 33S; case “A”) or the shift to higher masses by approximately 2 u (e.g., 16O to 18O or 32S to 34S; case “B”). Let A and B be fictitious elements with isotopes M(A)A,

mass spectrum approximates the isotopic distribution by intensities (I) of the peak with a particular m/z, instead of probabilities (P), we can use intensities I of the corresponding isotopic compositions. We need to consider though the potential errors in measurements of intensities caused by nonlinearity of signal dependence on the number of ions in FTICR MS. For this reason, we cannot calculate the exact composition but only estimate the range of coefficient values (in many cases, it is enough for unambiguous writing of the formula) . Previously reported methods6,7 operate on the whole mass spectrum of the isotope cluster (complete pattern in our definitions), whereas the resolution and dynamic range are better in a narrow band spectra of isolated isotope spectra. There are two options: either assemble a single mass list from isolated isotope spectra or devise a method that can operate on 2278



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Table 2. Mass and Peak Intensity Values of the Measured and Simulated Isotopic Fine Structure Spectra of Reserpine and Substance Pa isotope group

isotope 15

N C 17 O 2 H 13 15 C N 18 O 13 C2 13 17 C O 2 H 13C 15 N 18O 13 C2 15N 13 18 C O 13 C3 13 C2 17O 2 H 13C2 13 15 C N 18O 13 C3 15N 18 O2 13 C2 18O 13 17 C O 18O 13 C4 2 H 13C 18O 13 C3 17O 2 H 13C3

610.277 692 610.284 012 610.284 874 610.286 934 611.281 047 611.284 903 611.287 367 611.288 229 611.290 289 612.281 939 612.284 402 612.288 259 612.290 722 612.291 584 612.293 644 613.285 293 613.287 757 613.289 150 613.291 613 613.292 476 613.294 077 613.294 535 613.294 939 613.296 999

15

674.869 867 674.871 043 674.873 027 674.873 458 675.368 384 675.369 248 675.371 544 675.372 721 675.373 472 675.374 704 675.376 165 675.867 765 675.870 062 675.870 925 675.871 990 675.873 221 675.874 398 675.875 150 675.876 382 675.877 843 676.368 854 676.369 442 676.371 371 676.371 738 676.372 602 676.372 994 676.373 668 676.374 063 676.374 897

13

13

C

13

C2

13

C3

13

C4

N S 13 C 17 O 15 N2 34 S 13 15 C N 13 33 C S 18 O 13 C2 2 H 13C 15 N 34S 13 15 C N2 13 34 C S 15 N 18O 13 C2 15N 13 C2 33S 13 18 C O 13 C3 2 H 13C2 15 N3 17O 13 15 C N 34S 34 18 S O 2 H 15N2 33S 13 C2 34S 2 H 13C 15N2 13 15 C N 18O 15 N 17O 18O 13 C3 15N 33

13

C

13

C2

13

C3

13

C4

theoretical mass value [u]


mass deviation [mu]


Reserpine + H+ (C33H41O9N2+) 610.277 745 0.05 87 610.284 012 0.00 0 610.284 961 0.09 143 610.286 975 0.04 67 611.281 089 0.04 69 611.284 934 0.03 51 611.287 367 0.00 0 611.288 280 0.05 83 611.290 336 0.05 77 612.281 986 0.05 77 612.284 449 0.05 77 612.288 259 0.00 0 612.290 722 0.00 0 612.291 639 0.06 90 612.293 690 0.05 75 613.285 322 0.03 47 613.287 786 0.03 47 613.289 168 0.02 29 613.291 596 −0.02 −28 613.292 513 0.04 60 613.294 077 0.00 0 613.294 520 −0.01 −24 613.294 949 0.01 16 613.297 029 0.03 49 Substance P + 2H+ (C63H100O13N18S12+) 674.869 968 0.10 150 674.871 114 0.07 105 674.873 027 0.00 0 674.873 469 0.01 16 675.368 466 0.08 121 675.369 318 0.07 104 675.371 613 0.07 102 675.372 841 0.12 178 675.373 541 0.07 102 675.374 704 0.00 0 675.376 218 0.05 78 675.867 840 0.08 111 675.870 134 0.07 107 675.870 952 0.03 40 675.872 078 0.09 130 675.873 269 0.05 71 675.874 504 0.11 157 675.875 186 0.04 53 675.876 382 0.00 0 675.877 889 0.05 68 676.368 812 −0.04 −62 676.369 468 0.03 38 676.371 387 0.02 24 676.371 764 0.03 38 676.372 600 0.00 −3 676.372 910 −0.08 −124 676.373 704 0.04 53 676.374 114 0.05 75 676.374 911 0.01 21 2279




2.1 100.0 1.0 1.3 4.3 29.9 100.0 2.0 2.7 2.0 6.5 95.6 100.0 3.1 4.2 4.2 4.4 13.3 100.0 1.8 49.0 2.7 2.1 2.8

1.3 100.0 0.5 0.7 2.8 23.5 100.0 1.3 2.1 1.6 4.2 100.0 98.4 1.3 3.4 2.4 3.4 9.9 100.0 1.0 43.5 1.1 1.2 1.9

−38.1 0.0 −50.0 −46.2 −34.9 −21.4 0.0 −35.0 −22.2 −20.0 −35.4 4.6 −1.6 −58.1 −19.0 −42.9 −22.7 −25.6 0.0 −44.4 −11.2 −59.3 −42.9 −32.1

9.8 1.2 100.0 0.7 0.9 19.8 19.8 2.4 11.7 100.0 3.4 5.9 2.8 60.7 3.5 30.3 3.6 36.2 100.0 5.2 1.0 19.6 11.7 4.7 100.0 2.6 11.7 3.4 33.7

4.5 0.6 100.0 0.6 0.6 10.9 12.5 1.5 8.7 100.0 1.7 4.7 3.1 50.6 2.1 23.1 2.2 29.3 100.0 3.2 1.4 16.4 11.3 4.4 100.0 1.9 7.8 2.9 24.0

−54.1 −50.0 0.0 −14.3 −33.3 −44.9 −36.9 −37.5 −25.6 0.0 −50.0 −20.3 10.7 −16.6 −40.0 −23.8 −38.9 −19.1 0.0 −38.5 40.0 −16.3 −3.4 −6.4 0.0 −26.9 −33.3 −14.7 −28.8



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Table 2. continued isotope group

isotope 18

O2 C3 33S 13 C2 18O 13 C4 2 H 13C3 13

theoretical mass value [u] 676.375 596 676.376 076 676.376 828 676.378 059 676.379 520


mass deviation [mu]

Substance 676.375 630 676.376 093 676.376 833 676.378 059 676.379 544


P + 2H+ (C63H100O13N18S12+) 0.03 50 0.02 25 0.00 7 0.00 0 0.02 35




3.2 3.9 59.7 79.7 5.7

2.9 3.9 51.9 73.5 6.0

−9.4 0.0 −13.1 −7.8 5.3

a

The data is obtained from individually isolated 13C isotopic clusters (isolated isotope spectra). The relative peak intensities are normalized to the most abundant peak in each 13C cluster (pure 13C in all but the last cluster).

Figure 3. Isolated isotope spectra of singly protonated reserpine acquired in narrowband (heterodyne) mode. Ions corresponding to each isotopic cluster (first 13C, second 13C, third 13C, and fourth 13C) are individually isolated in the quadrupole and injected into the ICR cell for measurement. All fine structure spectra are individually single-point calibrated using the pure 13C signal in the cluster. The fine structure spectrum in part a shows the first 13C isotopic cluster in comparison to the simulated spectrum. Similarly, the spectra in parts b, c, and d show the second, the third, and the fourth 13C isotopic clusters, respectively, in comparison to the corresponding simulated spectra.

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Figure 4. Isolated isotope spectra of doubly protonated substance P acquired in narrowband (heterodyne) mode. Ions corresponding to each isotopic cluster (first 13C, second 13C, third 13C, and fourth 13C) are individually isolated in the quadrupole and injected into the ICR cell for measurement. All fine structure spectra are individually single-point calibrated using the pure 13C signal in the cluster. The fine structure spectrum in part a shows the first 13C isotopic cluster in comparison to the simulated spectrum. Similarly, the spectra in parts b, c, and d show the second, the third, and the fourth 13C isotopic clusters, respectively, in comparison to the corresponding simulated spectra. M(A)+1

A, M(B)B, M(B)+2B (and maybe others), and C assumes no special meaning and denotes carbon. Expanding the multinomial distribution that describes the isotopic pattern of a compound AXBYCZ consisting of elements A, B, and C, we obtain expressions for intensities of the first several peaks given in S-Table 1 in the Supporting Information. Here A and B denote elements with isotopes shifted by 1 and 2 u correspondingly, and C is the carbon. Equations that allow estimating the coefficients X, Y, and Z corresponding to elements A, B and C are given in S-Table 2 in the Supporting Information. They are derived using one by one division of the expressions for peak intensity presented in S-Table 1 in the Supporting Information and then solving the equations for X, Y,

and Z. The coefficient Z for carbon is estimated from the complete pattern spectrum, which does not need the fine structure resolution and thus is readily observable in experiments.

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DISCUSSION The complete pattern spectra of protonated reserpine and doubly protonated substance P (resolving power ∼1.3 million) displayed in Figure 2a,b and the isolated isotope spectra of these substances (resolving power ∼4 million) displayed in Figures 3a−d and 4a−d clearly show a significant difference not only in resolving powers but also in intensity ratios of particular peaks. The reason for this is not the mass range of the isolation 2281



Article

number of ions in FTICR MS is clearly the limiting factor of the composition estimation procedure. Further work is required to find ways to measure and minimize these errors. Also a robust criterion is needed to determine the “safe” intensity ratio tolerance for the estimate to be applicable in identification of unknown compounds. Though further research is needed and we cannot propose such criterion right now, we can estimate the order of improvements in composition assessment. S-Figure 1 in the Supporting Information shows the reduction in the number of candidate formulas with narrowing the confidence interval for estimated coefficients. Suppose we are to identify a peak with m/z = 674.371 35 (Substance P + 2H+) with a m/z tolerance of 0.2 ppm. Without sophisticated chemical feasibility filters, we would get 65 candidate formulas (S-Table 4 in the Supporting Information). As S-Figure1 in the Supporting Information shows, if we have 10% accurate coefficient estimates, the composition becomes unique, with 20% accuracy we would get only 5 candidates to disambiguate, and even 100% tolerance (i.e., for each coefficient we know that it is less than its doubled value) allows us to cut the number of candidate formulas in half. This result shows the utility of the fine structure-based composition estimation, even if the estimates are very rough. The method of atomic composition determination we are proposing should be considered as an addition to (MSn) experiments. It should be especially helpful in cases when MS/MS does not work properly.

procedure but rather the total number of ions used in the measuring procedure. The complete pattern spectra include all isotopic peaks, with the monoisotopic peak being the most abundant peak in mass in the spectra. For this reason in the case of the complete pattern spectra, ion−ion interaction effects as well as the general space charge effects can be much stronger than in the isolated isotope spectra. The single point calibration in the complete pattern spectra is made for the monoisotopic peak. The peaks in the 13C clusters shift in the direction to higher masses (compared to the simulated position on the m/z scale). This is to be expected at least due to the space charge effects which reduces the cyclotron frequency to lower values. Table 1 shows peak positions and peak intensities in the complete pattern fine structure spectra. Table 2 shows the isolated isotope pattern spectra. The pure 13C peak is calibrated on the mass scale in each fine structure spectrum, and the distances of this peak to the other peaks are compared. As we know from a realistic modeling of the ion cloud motion using the particle-in-cell approach,5 during the detection period small ion clouds in an ICR cell strongly interact with the large clouds of ions of very close m/z because they are coming through each other with a frequency equal to the difference of their cyclotron frequencies. This interaction can lead to a peak coalescence phenomenon, and it can also lead to a complete or partial destruction of the small ion cloud, which either reduces the peak intensity or totally annihilates the small peak. Very accurate tuning of the total number of ions in the cell is needed to prevent a small peak elimination. Therefore, in an isotopic fine structure spectrum, due to the ion cloud interactions, the intensities of smaller peaks are generally suppressed by the nearest abundant peak which is in the first numbers of isotopic groups mostly the “pure” 13Cn. In Tables 1 and 2, the peak intensity deviation column is calculated in reference to the largest peak in the fine structure spectrum, (the pure 13C peak in all but the last isotopic cluster); therefore, it heavily contains this general suppression effect. If we could estimate the peak intensity deviations of the small peaks in the cluster, this would give us more valuable information. It seems to be more reasonable to compare intensities of small peaks in the spectra with the corresponding small peaks in the simulated isotopic distribution spectrum and suggesting that the effect of this peak suppression by the largest peak in the fine structure spectrum has a similar magnitude. In mass spectra of the larger order clusters (the number of 13 C atoms), an isotopic peak cluster consists of more than one abundant peak. The larger the number of abundant peaks, i.e., the closer the abundances of the peaks in an isotopic cluster, the less significant the suppression becomes in terms of relative intensities. As an example, two equally abundant peaks experience equal suppression effects by each other, and although their absolute intensities may suffer, their relative intensities will practically not be affected. The isotopic fine structure allows estimation of the elemental composition to facilitate compound identification using the accurate mass-based approach. We described a novel method for composition estimation that operates on isolated isotope spectra without the need for construction of the complete pattern spectrum. The results of such an estimation for reserpine and substance P are shown in S-Table 3 in the Supporting Information. Calculated coefficient values match reference ones with errors not exceeding 70%. The intensity deviations caused by nonlinearity in signal dependence on the

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ASSOCIATED CONTENT

S Supporting Information *

Additional information as noted in text. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected] (E.N.N.); [email protected] (G.B.). Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We would like to thank Matthias Witt, Jens Fuchser, Christian Berg, Christopher Thompson, and Claudia Kriete for helpful discussions. E.N. and A.G. acknowledge the support from the Russian Foundation of Basic Research (grants 09-04-00725, 1004-13306), from the Russian Federal program (state contracts 14.740.11.0755, 16.740.11.0369), and from the Fundamental Sciences for Medicine program of the Russian Academy of Sciences.

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REFERENCES

(1) Nikolaev, E. N.; Boldin, I. A.; Jertz, R.; Baykut, G. J. Am. Soc. Mass Spectrom. 2011, 22, 1125−1133. (2) Nikolaev, E. N.; Boldin, I. A.; Jertz, R.; Fuchser, J.; Baykut, G. Proceedings of the 59th ASMS Conference on Mass Spectrometry and Allied Topics, Denver, CO, June 5−9, 2011; WOB. (3) Boldin, I. A.; Nikolaev, E. N. Proceedings of the 58th ASMS Conference on Mass Spectrometry and Allied Topics, Salt Lake City, UT, May 23−27, 2010; WOB 2:50 (4) Boldin, I. A.; Nikolaev, E. N. Rapid Commun. Mass Spectrom. 2011, 25, 122−126. (5) Nikolaev, E. N.; Heeren, R. M. A.; Popov, A. M.; Pozdneev, A. V.; Chingin, K. S. Rapid Commun. Mass Spectrom. 2007, 21, 3527−3546. (6) Shi, S. D.-H.; Hendrickson, C. L.; Marshall, A. G. Proc. Natl. Acad. Sci. U.S.A. 1998, 95, 11532−11537.

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(7) Miura, D.; Tsuji, Y.; Takahashi, K.; Wariishi, H.; Saito, K. Anal. Chem. 2010, 82, 5887−5891.

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Fine Structure in Isotopic Peak Distributions Measured Using a

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