JOURNAL OF MASS SPECTROMETRY J. Mass Spectrom. 2002; 37: 792–802 Published online 20 June 2002 in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002/jms.336 Isomer separation of hyperbranched polyesteramides with gas-phase H/D exchange and a novel MS n approach: DoDIP Sander Koster, 1† Marc C. Duursma, 1 Xinghua Guo, 1 Rolf. A. T. M. van Benthem, 2 Chris G. de Koster, 2,3 Jaap J. Boon 1 and Ron M. A. Heeren 1* 1 Unit for Macromolecular Mass Spectrometry, FOM-Institute for Atomic and Molecular Physics, Kruislaan 407, 1098 SJ Amsterdam, The Netherlands 2 DSM Research, P.O. Box 18, 6160 MD Geleen, The Netherlands 3 University of Amsterdam, Swammerdam Institute for Life Sciences, Nieuwe Achtergracht 166, 1018 WV Amsterdam, The Netherlands Received 11 December 2001; Accepted 15 April 2002 Two approaches are introduced that provide information about the isomeric composition of hyperbranched polyesteramides. The first approach is based on a novel tandem mass spectrometric (MS n ) approach that allows the study of different types of isomeric structures by a separation based on their difference in appearance energy. The method is called DoDIP: dissociation of depleted ion populations. A first MS/MS step is used to fragment isomers with relatively low appearance energy. The isomers with higher appearance energy are fragmented in a second MS/MS step of higher energy. The second approach is based on gas-phase H/D exchange experiments that result in a bimodal isotopic distribution for oligomers X n D n+1 of which one distribution corresponds to a type of isomeric structure that exhibits H/D exchange behaviour and the other to an isomeric structure that does not exhibit H/D exchange behaviour. X is a difunctional anhydride of phthalic acid (P), 1,2-cyclohexanedicarboxylic acid (C), succinic acid (S) or glutaric acid (G). D in X n D n+1 is a trifunctional diisopropanolamine and n the degree of polymerization. The type of isomeric structure that does not exhibit H/D exchange behaviour has a non-alternating monomer sequence that contains an amine bond with a relatively high proton affinity. The other isomeric structure that does exhibit H/D exchange behaviour has an alternating monomer sequence containing only amide and ester bonds with relatively low proton affinity. Oligomer structures were confirmed with additional MS 2 experiments after H/D exchange. H/D exchange experiments on the fragments obtained after MS 2 of the parent ion show that next to previously postulated mechanisms for the cleavage of the ester and amide bond another reaction pathway must be operational. A new mechanism is introduced to explain the H/D exchange behaviour of the fragments that requires a cleavage of the amide bonds only. Two types of fragments are formed by this mechanism. One type is protonated due to the cleavage of the amide bond whereas the other type has an oxazolonium ion structure due to the loss of an additional H 2 O. Copyright  2002 John Wiley & Sons, Ltd. KEYWORDS: isomers; hyperbranched polymers; electrospray ionization Fourier transform ion cyclotron resonance mass spectrometry; H/D exchange; DoDIP; dissociation of depleted ion populations INTRODUCTION Ion–molecule reactions have long been used to obtain detailed structural information on organic compounds. The first studies involved small molecules such as amino acids. 1–4 Studies of large molecules have become popular in the last L Correspondence to: Ron M. A. Heeren, Unit for Macromolecular Mass Spectrometry, FOM-Institute for Atomic and Molecular Physics, Kruislaan 407, 1098 SJ Amsterdam, The Netherlands. E-mail: heeren@amolf.nl † Present address: Institute of Microtechnology, University of Neuchˆ atel, Rue Jacquet-Droz 1, P.O. Box 3, CH-2007 Neuchˆ atel, Switzerland. Contract/grant sponsor: FOM. Contract/grant sponsor: DSM Research. Contract/grant sponsor: Akzo Nobel Chemicals Research Arnhem. decade after the soft ionization techniques electrospray ion- ization (ESI) and matrix-assisted laser desorption/ionization (MALDI) were introduced. These ionization techniques allow the transfer of large molecules intact into the gas phase to study their chemical structure. Most ion–molecule reac- tions reported on large molecules concern biomolecules. 5–15 Isomer identification poses a special problem for mass spectrometry as isomers have the same m/z value. Iso- meric structures of small molecules, e.g. xylenes, can be distinguished with hydrogen/deuterium (H/D) exchange experiments owing to a difference in the number of exchange- able hydrogens and exchange rate. 16,17 For larger molecules, mainly biomolecules, different rates of deuterium incorpora- tion have also been observed. This is not a result of differences in isomeric structures, however, but is caused by differences Copyright  2002 John Wiley & Sons, Ltd.