Extension of the Kendrick Mass Defect Analysis of Homopolymers to

Letter pubs.acs.org/ac

Extension of the Kendrick Mass Defect Analysis of Homopolymers to Low Resolution and High Mass Range Mass Spectra Using Fractional Base Units Thierry Fouquet* and Hiroaki Sato* National Institute of Advanced Industrial Science and Technology (AIST), 1-1-1 Higashi, Tsukuba, Ibaraki 305-8565, Japan S Supporting Information *

ABSTRACT: Beyond the high resolution/low mass range data traditionally used, a Kendrick mass defect analysis (KMD) using the new concept of fractional base units has been successfully conducted on low resolution/low mass range and high resolution/high mass range data for the first time. Relying on a mathematical framework to rationalize the effect of the fractional base units, the electrospray ionization single stage and multistage mass spectra of a poly(vinylpyrrolidone) recorded from a low resolution ion trap analyzer were turned into information-rich KMD plots using vinylpyrrolidone/112 and pyrrolidone/86 as base units. The distributions detected in the matrix assisted laser desorption ionization spiralTOF mass spectra of high molecular weight poly(ethylene oxide) and poly(caprolactone) were conveniently discriminated in KMD plots using (ethylene oxide)/45 and caprolactone/113 as base units with an unprecedented resolution at such a mass range. The high resolution KMD analysis using fractional base units opens new perspectives for the acquisition, visualization, and presentation of mass spectra of polymers with less restrictions in terms of required resolution and molecular weights.

M

unit chemically senseless but mathematically acceptable. Depending on the value of the divisor X, the computed KMD plots discriminate with an unprecedented resolution the isotopes of a given distribution (one horizontal line per 12 C/13Cx isotope) and/or the components of a blend or cooligomers without any modification of the raw data.16 If the main drawback of a regular KMD analysis remains its need for high resolution mass spectral data (i.e., low mass range and high resolution analyzers such as Fourier transform ion cyclotron resonance, orbitrap, or spiralTOF17), the introduction of the fractional base units and the associated high resolution KMD plots open new perspectives. As a founding postulate, improving the resolution of the KMD analysis would allow a decrease of the required resolution for the MS analysis with no loss of information. This Letter validates the postulate and reports on the successful KMD analysis of data recorded with (a) a low resolution ion trap device in a low mass range (average resolution at m/z 1000: ∼4000) and (b) a high resolution spiralTOF analyzer in high mass range (average resolution at m/z 15 000: ∼20 000) using a fractional base unit with a rationally chosen divisor.

ass spectrometry (MS) has emerged as a major analytical technique for the characterization of polymers1 thanks to soft ionization sources such as electrospray ionization (ESI)2,3 and matrix assisted laser desorption ionization (MALDI)4,5 and a variety of mass analyzers and experiments.6,7 However, technical polymers are often blended samples containing several homopolymers/copolymers and additives. Consequently, mass spectra of polymers are notoriously complex, and the data mining is extremely timeconsuming. To circumvent this issue, the Kendrick mass defect analysis8 (KMD) has been recently adapted from petroleomics9 to the field of polymer chemistry.10 In lieu of the traditional CH2 base unit11 (at 14.0157 in the IUPAC scale, set at 14.0000 in the Kendrick scale), Sato et al.10 have used the repeat unit of a polymer backbone as the new base unit for the calculation of the Kendrick masses (KM). Thus, all the congeners of a given distribution possess the same mass defect and line up horizontally in the associated KMD plot (KMD vs nominal Kendrick mass NKM) while other distributions with different end-groups or a different repeat unit line up horizontally at different KMD values or obliquely, respectively. Since its first use for polymers, several authors have refined the KMD analysis by using higher order mass defects12 or other base units such as a comonomer for a copolymeric sample10,13,14 or the expelled neutral for tandem mass spectra (MS/MS).15 The latest improvement consists of the use of a f raction of the repeat unit ((repeat unit)/X with X being a positive integer) as a base © 2017 American Chemical Society

Received: December 27, 2016 Accepted: February 9, 2017 Published: February 9, 2017 2682

DOI: 10.1021/acs.analchem.6b05136 Anal. Chem. 2017, 89, 2682−2686

Letter

Analytical Chemistry

Figure 1. (A) ESI-MS spectrum of PVP (inset: peak shapes recorded with ion trap and spiralTOF analyzers). (B) Full scale VP-based KMD plot. (C) Zoom from (B) (KMD range: −0.14 to +0.04). (D) Full scale VP/112-based KMD plot.

(eight values tested per data set, from round(R) − 4 to round(R) + 4). The low resolution ESI-MS spectrum of PVP is depicted in Figure 1A and displays a main distribution of sodium adducts ([PVP + Na]+, red squares) and two minor proton adducts ([PVP + H]+, black triangles) and sodiated dimers ([2PVP + Na]+, black circles). Two representative peak shapes from the low resolution ESI-MS (ion trap) and high resolution MALDI-MS data (spiralTOF, Figure S2) are depicted in the inset for the sake of comparison of the resolving power (2-digit m/z value with the ion trap, 4-digit value with the spiralTOF). The data set recorded from the ion trap device is normally not suited to a KMD analysis owing to the lack of accuracy in the measurement of m/z. The VP-based full scale KMD plot is depicted in Figure 1B, and its magnification (KMD: −0.14 to +0.04) is depicted in Figure 1C. The KMD analysis clearly fails as points are roughly aligned along an oblique direction with no clear separation of either the three distributions or the isotopes. On the contrary, a KMD analysis using VP/112 as a newly introduced fractional base unit produces a sufficiently resolved KMD plot (Figure 1D) with three distinct point alignments readily assigned to the three series of sodiated PVP (red dots), protonated PVP, and sodiated PVP dimers (R = 111.0684, round(R) = 111, optimal X = 112, cf. Supporting Information). It should be pointed out that the average mass difference between two consecutive oligomer peaks in the ESI-MS spectrum is not 111.0684 (the exact mass of a VP unit) but 111.0860 (δ ∼ 159 ppm) owing to the low resolution. Consequently, data points do not line up along the horizontal axis but slightly obliquely while the KMD plots computed with 111.0860 and 111.0860/112 as base units display horizontally aligned series (Figure S3). Such deviation is nevertheless not detrimental to the interpretation of the KMD plot, and the exact masses of the repeat units will be used for sake of simplicity. The selection of points from the KMD plot to highlight the associated peaks in the MS spectrum is easily achievable (Figure S4) since the discrimination of the series is clear enough.

Experimental protocols are detailed in an extended experimental part in the Supporting Information, and the structures of the polymers are depicted in Scheme S1. Concisely, the ESI-MS and MSn (n = 3, 4) spectra of a polyvinylpyrrolidone (PVP) have been recorded using an amaZon SL-STT2 ion trap (Bruker, Bremen, Germany). The MALDI-MS spectra of a polyethylene glycol (PEO) and polycaprolactone (PCL) were recorded using a JMS-S3000 SpiralTOF mass spectrometer (JEOL, Tokyo, Japan).17 For every experiment, mMass 5.5.0.018 was used for data processing and artworks. The mass-to-charge ratios of ions (m/z) were converted to KM, NKM, and KMD according to KM(ion) = m /z(ion)·

round(m(baseunit)) m(baseunit)

with “round(x)” being

the rounded value of x to the nearest integer, NKM(ion) = round (KM(ion)), and KMD(ion) = NKM(ion) − KM(ion). The KMD plot displays the KMD of the detected oligomeric adducts as a function of their corrected NKM (CNKM) using a bubble chart (data triplet: CNKM, KMD, abundance), CNKM(ion) = NKM(ion) − ceiling(NKM(ion) − m/z(ion)) with “ceiling(x)” being the smallest integer greater than or equal to x.16 “Full scale” plot refers to KMD plots with KMD ranging from −0.5 to 0.5. MSRepeatFinder (JEOL) was used to extract points of interest from the KMD plots and highlight the corresponding peaks in the mass spectra (so-called grouping mode). As the missing link between the founding article16 and the present Letter, the rigorous mathematical framework accounting for the expansion of the KMD dimension using a fractional base unit (repeat unit)/X is reported in Figure S1 and the associated comment. In particular, it provides a rationalized method to choose the divisor X, avoiding a time-consuming screening of possible values. Briefly, the best values of X to improve the resolution of the KMD plot in a controlled extent are close to round(R) and round(2R). Relying on this finding, the divisors used throughout the text have been voluntarily chosen near round(R) following a rapid trial-and-error step 2683


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Figure 2. (A) ESI-MS3 spectrum of a dehydrated PVP 7-mer at m/z 820 (red squares: eliminations of pyrrolidone P). (B) Full scale P-based KMD plot. (C) Zoom from (B) (KMD range: −0.08 to +0.10). (D) Full scale P/86-based KMD plot.

Figure 3. (A) MALDI-spiralTOF-MS spectrum of a 10 000 g mol−1 PEO. (B) Full scale EO-based KMD plot. (C) Zoom from (B) (KMD range: −0.03 to +0.09). (D) Full scale EO/45-based KMD plot.

PVP 7-mer (formed upon the release of water from the protonated precursor ion19) is depicted in Figure 2A. The main product ion series arising from the consecutive expulsions of the pyrrolidone pendant group (abbreviated as P, 85.0528 Da) is marked with red squares. Several minor product ion series are slightly seen and formed upon the disruption of the polymer backbone and the loss of the end-groups. The full scale and magnified P-based KMD plots are depicted in Figure 2B,C, respectively. As for the MS stage, a rough point alignment is reached but the KMD analysis fails at distinguishing the product ion series.15 On the contrary, a KMD plot computed

Using an ion trap for the mass analysis of polymers is obviously within the scope of recording MSn mass spectra. The fragmentation pathways of polymer ions is indeed rich of information in terms of end-groups and architectures.6 So far, the KMD analysis has been successfully extended to the MS/ MS stage using data recorded from a high resolution orthogonal-acceleration TOF.15 The previous results suggest a KMD analysis would be satisfactorily conducted on the MSn data recorded with a low resolution ion trap considering the high resolution of the KMD analysis achieved with the fractional base units. The ESI-MS3 spectrum of a dehydrated 2684


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Figure 4. MALDI-spiralTOF-MS spectrum of a SEC-fractionated PCL. (A) Mp ∼ 7000 g mol−1 and (B) Mp ∼ 15 000 g mol−1 (inset: representative peaks from the two PCL (H, OH) and PCL (H, C3H7O) series adducted with sodium and potassium). (C, D) Full scale CL-based KMD. (E, F) Full scale CL/113-based KMD plot.

with P/86 as the base unit (Figure 2D) displays five wellresolved lines (R = 85.0528, round(R) = 85, optimal X = 86, cf. Supporting Information) with the main series plotted in red. It constitutes (a) an alternative visualization of the MSn data with intuitive alignments and (b) an easy way to extract data points using any appropriate software. Similar results are found at the MS4 step (Figure S5, using X = 89) and confirm the possibility to conduct a KMD analysis for low resolution mass spectral data at any stage (MS or MSn) with a well-chosen f ractional base unit. The KMD analyses for polymer ions conducted with high resolution mass analyzers have been reported for samples of low molecular weights only owing to the degradation of the resolution with the increase of the mass range. It is worth noting that low resolution/low mass range (10 000 Da) mass spectra exhibit a similar peak shape. As such, the high resolution KMD analysis proposed above for low resolution data in the low mass range should be extendable to the case of high resolution data in the high mass range, as a first KMD analysis for high molecular weight polymers. The MALDI-MS spectrum of a 10 000 g mol−1 PEO is depicted in Figure 3A. A high laser fluence has been voluntarily used to form ions in the low mass range in addition to the main distribution in the high mass range as this situation is commonly faced when analyzing high molecular weight samples. The mass spectrum displays four

main peak series: the main distribution centered around 9000 Da ([PEO + Na]+), a doubly charged series ([PEO + 2Na]+), and in source decay (ISD) product ions [ln (linear product ions, same elemental composition as the intact chains) and cn (cyclic product ions, no end-groups)].6 A EO-based KMD analysis provides the KMD plots depicted in Figure 3B (full scale) and Figure 3C (magnification, KMD: −0.03 to +0.09). If the points are satisfactorily aligned along the horizontal axis, no discrimination is achieved between singly charged, doubly charged, and product ions, but an unresolved cloud is plotted instead. Using a fractional EO/45 as the base unit (R = 44.0262, round(R) = 44, optimal X = 45, cf. Supporting Information), the full scale KMD plot depicted in Figure 3D offers a strikingly enhanced visualization of the data. The doubly charged congeners (green dots), the cyclic product ion cn (blue dots), and the singly charged oligomers (+ the ln product ions, same composition/KMD but in low mass range) are now distinctly grouped at different KMD values. Signals can then be readily extracted from the KMD plots for a convenient interpretation of the mass spectra (Figure S6). Last but not least, the EO/45-based KMD plot is isotopically resolved in the high mass range with several horizontal lines assigned to the 13 Cx isotopes (e.g., the 13C5 series plotted in red, Figure 3D). Exemplifying another situation for which several distributions are found in the high mass range, the MALDI mass spectra of an 8000 g mol−1 PCL sample fractionated by size exclusion 2685


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(FY2015) and a Grant-in-Aid “JSPS KAKENHI” (Grant number: JP 15F15344).

chromatography (Figure S7) are depicted in Figure 4A (Mp ∼ 7000 g mol−1) and Figure 4B (Mp ∼ 15 000 g mol−1). Representative peaks are depicted in the insets to highlight the resolving power of a spiralTOF in this mass range and shows four series of peaks assigned to two PCL distributions carrying (H, OH) and (H, C3H7O) as end-groups10 (Scheme S1) adducted with sodium and potassium. The full scale CL-based KMD plots are depicted in Figure 4C (Mp ∼ 7000 Da) and Figure 4D (Mp ∼ 15 000 Da) and displayed two unresolved clouds from which no information can be extracted. With CL/ 113 as the base unit (R = 114.0681, round(R) = 114, optimal X = 113, cf. Supporting Information), the full scale KMD plots of the two fractions now display four well-discriminated groups readily assigned to the sodium and potassium adducts of the two PCL (H, OH) (red and green dots, respectively) and (H, C3H7O)-ended distributions. The two KMD plots with the fractional CL/113 as the base units provide an indubitably enhanced visualization of the mass spectral data as compared to the mass spectrum itself, and the regular CL-based KMD plot and grouping of points would be readily done with no additional data treatment using the appropriate software. In conclusion, the KMD analyses of (a) a low resolution mass spectrum in low mass range at the MS and MSn stages and (b) a high resolution mass spectrum in high mass range are thus successfully conducted. The use of a fractional base unit improves the resolution of the KMD analysis itself and counterbalances the lower resolution of the MS analysis. On the basis of these findings and relying on a rational choice of the divisor, the concept of fractional base units is expected to widely extend the scope of the KMD analysis in the field of polymer chemistry by allowing the use of more affordable low resolution mass analyzers and making high molecular weight samples suited to the mass defect analysis with all its advantages (fast data mining, easy visualization, and presentation to wide audiences) as long as the mass spectra are isotopically resolved.

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REFERENCES

(1) Montaudo, G.; Lattimer, R. P. Mass spectrometry of polymers; CRC Press, Taylor and Francis group: Oxford, 2001. (2) Hakkarainen, M. Mass spectrometry of polymers − new techniques; Springer-Verlag: Berlin, Heidelberg, New York, 2012. (3) Gruendling, T.; Weidner, S.; Falkenhagen, J.; Barner-Kowollik, C. Polym. Chem. 2010, 1, 599−617. (4) Pasch, H.; Schrep, W. MALDI-TOF mass spectrometry of synthetic polymers; Springer-Verlag: Berlin, Heidelberg, New York, 2003. (5) Charles, L. Mass Spectrom. Rev. 2014, 33, 523−543. (6) Wesdemiotis, C.; Solak, N.; Polce, M. J.; Dabney, D. E.; Chaicharoen, K.; Katzenmeyer, B. C. Mass Spectrom. Rev. 2011, 30, 523−559. (7) Wesdemiotis, C. Angew. Chem., Int. Ed. 2017, 56, 1452−1464. (8) Sleno, L. J. Mass Spectrom. 2012, 47, 226−236. (9) Marshall, A. G.; Rodgers, R. P. Acc. Chem. Res. 2004, 37, 53−59. (10) Sato, H.; Nakamura, S.; Teramoto, K.; Sato, T. J. Am. Soc. Mass Spectrom. 2014, 25, 1346−1355. (11) Kendrick, E. Anal. Chem. 1963, 35, 2146−2154. (12) Dimzon, I. K.; Trier, X.; Frömel, T.; Helmus, R.; Knepper, T. P.; de Voogt, P. J. Am. Soc. Mass Spectrom. 2016, 27, 309−318. (13) Fouquet, T.; Nakamura, S.; Sato, H. Rapid Commun. Mass Spectrom. 2016, 30, 973−981. (14) Fouquet, T.; Aizawa, H.; Sato, H. Rapid Commun. Mass Spectrom. 2016, 30, 1818−1822. (15) Fouquet, T.; Sato, H. Rapid Commun. Mass Spectrom. 2016, 30, 1361−1364. (16) Fouquet, T.; Sato, H. Mass Spec. (Tokyo), submitted for publication. (17) Satoh, T.; Tsuno, H.; Iwanaga, M.; Kammei, Y. J. Am. Soc. Mass Spectrom. 2005, 16, 1969−1975. (18) Strohalm, M.; Kavan, D.; Novák, P.; Volný, M.; Havlícek, V. Anal. Chem. 2010, 82, 4648−4651. (19) Fouquet, T.; Torimura, M.; Sato, H. Mass Spectrom. 2016, 5, A0050.

ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.analchem.6b05136. Extended experimental section; PVP, PEO, and PCL structures; discussion of the fractional base units; MALDI-spiralTOF MS spectrum and KMD plot of PVP; KMD plots from the ESI-MS and ESI-MS4 spectra of PVP; mass spectrum and KMD plot of PEO; SEC chromatogram of PCL (PDF)

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AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected]. *E-mail: [email protected]. ORCID

Thierry Fouquet: 0000-0002-9473-9425 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS T.F. and H.S. acknowledge the ongoing financial support by the Japan Society for the Promotion of Science (JSPS) under the postdoctoral fellowship for overseas researchers program 2686


Extension of the Kendrick Mass Defect Analysis of Homopolymers to

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