TY - JOUR A1 - Ruhe, L. A1 - Ickert, Stefanie A1 - Hochkirch, U. A1 - Hofmann, J. A1 - Beck, S. A1 - Thomale, J. A1 - Linscheid, M. W. T1 - Comprehensive molecular characterization of a cisplatin-specific monoclonal antibody N2 - Despite their immense and rapidly increasing importance as analytical tools or therapeutic drugs, the detailed structural features of particular monoclonal antibodies are widely unknown. Here, an antibody already in use for diagnostic purposes and for molecular dosimetry studies in cancer therapy with very high affinity and specificity for cisplatin-induced DNA modifications was studied extensively. The molecular structure and modifications as well as the antigen specificity were investigated mainly by mass spectrometry. Using nano electrospray ionization mass spectrometry, it was possible to characterize the antibody in its native state. Tandem-MS experiments not only revealed specific fragments but also gave information on the molecular structure. The detailed primary structure was further elucidated by proteolytic treatment with a selection of enzymes and high resolution tandem-MS. The data were validated by comparison with known antibody sequences. Then, the complex glycan structures bound to the antibody were characterized in all detail. The Fc-bound oligosaccharides were released enzymatically and studied by matrix-assisted laser desorption/ionization mass spectrometry. Overall 16 different major glycan structures were identified. The binding specificity of the antibody was investigated by applying synthetic single and double stranded DNA oligomers harboring distinct Pt adducts. The antibody− antigen complexes were analyzed by mass spectrometry under native conditions. The stability of the complex with double stranded DNA was also investigated. KW - Antibody KW - Native MS KW - Cisplatin KW - PTM PY - 2017 U6 - https://doi.org/10.1021/acs.molpharmaceut.7b00575 SN - 1543-8384 SN - 1543-8392 VL - 14 IS - 12 SP - 4454 EP - 4461 AN - OPUS4-43997 LA - eng AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER - TY - JOUR A1 - Ruhe, L. A1 - Ickert, Stefanie A1 - Beck, S. A1 - Linscheid, M. W. T1 - A new strategy for metal labeling of glycan structures in antibodies N2 - Quantitative analysis of complex proteins is a challenging task in modern bioanalytical chemistry. Commonly available isotope labels are still suffering from limitations and drawbacks, whereas new metal labels open numerous possibilities in mass spectrometric analyses. In this work, we have developed a newmetal labeling strategy to tag glycan structures of proteins, more particularly antibodies. The oligosaccharide glycans were selectively trimmed to the last N-acetylglucosamine to which an artificial azide containing galactose residue was bound. This azide can be used for subsequent cycloaddition of an alkyne. Therefore, we developed a lanthanide-containing macrocyclic reagent to selectively connect to this azido galactose. In summary, the glycan structures of an antibody can be labeled with a metal functionality using this approach. Furthermore, the functionality of the antibodies can be fully maintained by labeling the Fc glycans instead of using labeling reagents that target amino or thiol groups. This approach enables the possibility of using elemental, besides molecular mass spectrometry, for quantitative analyses or imaging experiments of antibodies in complex biological samples. KW - Antibody KW - Metal labeling KW - Glycans KW - DOTA KW - Lanthanide PY - 2018 U6 - https://doi.org/10.1007/s00216-017-0683-1 SN - 1618-2650 SN - 1618-2642 VL - 410 IS - 1 SP - 21 EP - 25 PB - Springer AN - OPUS4-44000 LA - eng AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER - TY - JOUR A1 - Ickert, Stefanie A1 - Hofmann, J. A1 - Riedel, Jens A1 - Beck, S. A1 - Pagel, K. A1 - Linscheid, M. W. T1 - Charge-induced geometrical reorganization of DNA oligonucleotides studied by tandem mass spectrometry and ion mobility N2 - Mass spectrometry is applied as a tool for the elucidation of molecular structures. This premises that gas-phase structures reflect the original geometry of the analytes, while it requires a thorough understanding and investigation of the forces controlling and affecting the gas-phase structures. However, only little is known about conformational changes of oligonucleotides in the gas phase. In this study, a series of multiply charged DNA oligonucleotides (n¼15–40) has been subjected to a comprehensive tandem mass spectrometric study to unravel transitions between different ionic gas-phase structures. The nucleobase sequence and the chain length were varied to gain insights into their influence on the geometrical oligonucleotide organization. Altogether, 23 oligonucleotides were analyzed using collision-induced fragmentation. All sequences showed comparable correlation regarding the characteristic collision energy. This value that is also a measure for stability, strongly correlates with the net charge density of the precursor ions. With decreasing charge of the oligonucleotides, an increase in the fragmentation energy was observed. At a distinct charge density, a deviation from linearity was observed for all studied species, indicating a structural reorganization. To corroborate the proposed geometrical change, collisional cross-sections of the oligonucleotides at different charge states were determined using ion mobility-mass spectrometry. The results clearly indicate that an increase in charge density and thus Coulomb repulsion results in the transition from a folded, compact form to elongated structures of the precursor ions. Our data show this structural transition to depend mainly on the charge density, whereas sequence and size do not have an influence. KW - Ion mobility KW - Collision-induced dissociation KW - Mass spectrometry KW - Oligonucleotide KW - Fragmentation KW - Tandem-MS PY - 2018 U6 - https://doi.org/10.1177/1469066717746896 SN - 1469-0667 SN - 1751-6838 VL - 24 IS - 2 SP - 225 EP - 230 PB - Sage AN - OPUS4-44429 LA - eng AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER - TY - JOUR A1 - Ickert, Stefanie A1 - Riedel, Jens A1 - Beck, S. A1 - Linscheid, M. W. T1 - Negative nucleotide ions as sensitive probes for energy specificity in collision‐induced fragmentation in mass spectrometry N2 - Rationale: The most commonly used fragmentation methods in tandem mass spectrometry (MS/MS) are collision‐induced dissociation (CID) and higher energy collisional dissociation (HCD). While in CID the preselected ions in the trap are resonantly (and m/z exclusively) excited, in HCD the entire m/z range experiences the dissociative acceleration. The different excitation is reflected in different fragment distributions. Methods: As a test‐bed for particularly pronounced fragmentation specificity, here MS/MS experiments on several 4‐mer oligonucleotides were conducted employing both collision methods and the results were thoroughly compared. Oligonucleotides are shown to be sensitive probes to subtle changes, especially in the negative ion mode. A detailed analysis of these differences reveals insight into the dissociation mechanics. Results: Thedifferencesarerepresentedinheat‐maps,whichallowforadirectvisualinspection oflargeamountsofdata.Inthesefalsecolourrepresentationsthe,sometimessubtle,changesinthe individual dissociation product distributions become distinct. Another advantage of these graphic plots can be found in the formation of systematic patterns. These patterns reflect trends in dissociation specificity which allow for the formulation of general rules in fragmentation behavior. Conclusions: Instruments equipped with two different excitation schemes for MS/MS are today widely available. Nonetheless, direct comparisons between the individual results are scarcely made. Such comparative studies bear a powerful analytical potential to elucidate fragmentation reaction mechanism. KW - DNA KW - Tandem MS KW - HCD KW - CID PY - 2018 U6 - https://doi.org/10.1002/rcm.8062 SN - 0951-4198 SN - 1097-0231 VL - 32 IS - 7 SP - 597 EP - 603 PB - Wiley & Sons, Ltd. AN - OPUS4-44430 LA - eng AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER - TY - CONF A1 - Ickert, Stefanie A1 - Riedel, Jens A1 - Beck, S. A1 - Linscheid, M. W. T1 - Vacuum ultraviolet light as a new Tandem MS method N2 - Tandem MS techniques are widely used for both, structure and sequence elucidation of biopolymers. Thereby, fragmentation activation is realized by various methods, for example with lasers or collisions with neutral gases. In this study, we present a new Tandem MS system using a commercially available vacuum ultraviolet lamp. On the one hand, this approach provides efficient fragmentation in both ionization modes, positive as well as negative. On the other hand, it enables an additional previously not achieved post ionization of the fragments. While the first results in atypical fragment patterns and, thus provides orthogonal information, the second is crucial especially to identify low abundant ions. T2 - European Mass Spectrometry Conference CY - Saarbrücken, Germany DA - 10.03.2018 KW - Tandem MS KW - Vacuum Ultraviolet PY - 2018 AN - OPUS4-44484 LA - eng AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER - TY - JOUR A1 - Ickert, Stefanie A1 - Beck, S. A1 - Linscheid, M. A1 - Riedel, Jens T1 - VUV Photodissociation Induced by a Deuterium Lamp in an Ion Trap N2 - Tandem mass spectrometry represents an important analytical tool to unravel molecular structures and to study the gas-phase behavior of organic molecules. Besides commonly used methods like collision-induced dissociation and electron capture or transfer dissociation, new ultraviolet light–based techniques have the potential to synergistically add to the activation methods. Here, we present a new simple, yet robust, experimental design for polychromatic activation of trapped ions using the 115–160 nm output of a commercially available deuterium lamp. The resulting continuous dissociative excitation with photons of a wide energy range from 7.7 to 10.8 eV is studied for a comprehensive set of analyte classes in both positive and negative ion modes. While being simple, affordable, compact, and of low maintenance, the new setup initiates fragmentation of most precursor ions via their known dissociation pathways. Additionally, some new fragmentation patterns were discovered. Especially, electron loss and electron capture reactions with subsequent fragmentations were observed. For oligonucleotides, peptides, carbohydrates, and organic dyes, in comparison to collision-induced dissociation, a significantly wider fragment distribution was obtained, resulting in an information increase. Since the individual photons carry enough energy to post-ionize the nascent fragments, a permanent vacuum ultraviolet light exposure inside the ion trap potentially goes along with a general increase in detection capability. KW - Fragmentation activation KW - Vacuum ultraviolet (VUV) light KW - Mass spectrometry KW - Tandem MS PY - 2019 U6 - https://doi.org/10.1007/s13361-019-02282-8 SN - 1044-0305 VL - 30 IS - 10 SP - 2114 EP - 2122 PB - Springer Nature CY - Heidelberg AN - OPUS4-48756 LA - eng AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER - TY - CONF A1 - Ickert, Stefanie A1 - Riedel, Jens A1 - Beck, S. A1 - Linscheid, M. W. T1 - Vacuum ultraviolet light as a new Tandem MS method N2 - Tandem MS techniques are widely used for both, structure and sequence elucidation of biopolymers. Thereby, fragmentation activation is realized by various methods, for example with lasers or collisions with neutral gases. In this study, we present a new Tandem MS System using a commercially available vacuum ultraviolet lamp. On the one hand, this approach provides efficient fragmentation in both ionization modes, positive as well as negative. On the other hand, it enables an additional previously not achieved post ionization of the fragments. While the first results in atypical fragment patterns and, thus provides orthogonal information, the second is crucial especially to identify low abundant ions. T2 - EMSC 2018 CY - Saarbruecken, Germany DA - 4.3.2018 KW - Tandem MS PY - 2018 AN - OPUS4-45671 LA - eng AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER - TY - JOUR A1 - Ickert, Stefanie A1 - Schwaar, Timm A1 - Springer, A. A1 - Grabarics, M. A1 - Riedel, Jens A1 - Beck, S. A1 - Pagel, K. A1 - Linscheid, M. W. T1 - Comparison of the fragmentation behavior of DNA and LNA single strands and duplexes N2 - DNA and locked nucleic acid (LNA) were characterized as single strands, as well as double stranded DNA-DNA duplexes and DNA-LNA hybrids using tandem mass spectrometry with collision-induced dissociation. Additionally, ion mobility spectrometry was carried out on selected species. Oligonucleotide duplexes of different sequences – bearing mismatch positions and abasic sites of complementary DNA 15-mers – were investigated to unravel general trends in their stability in the gas phase. Single stranded LNA oligonucleotides were also investigated with respect to their gas phase behavior and fragmentation upon collision-induced dissociation. In contrast to the collision-induced dissociation of DNA, almost no base loss was observed for LNAs. Here, backbone cleavages were the dominant dissociation pathways. This finding was further underlined by the need for higher activation energies. Base losses from the LNA strand were also absent in fragmentation experiments of the investigated DNA-LNA hybrid duplexes. While DNA-DNA duplexes dissociated easily into single stranded fragments, the high stability of DNA-LNA hybrids resulted in predominant fragmentation of the DNA part rather than the LNA, while base losses were only observed from the DNA single strand of the hybrid. KW - Oligonucleotide fragmentation KW - Locked nucleic acids KW - Collision induced dissociation (CID) KW - Double strands KW - Ion mobility spectrometry PY - 2019 U6 - https://doi.org/10.1002/jms.4344 VL - 54 IS - 5 SP - 402 EP - 411 PB - Wiley AN - OPUS4-47485 LA - eng AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER - TY - CONF A1 - Ickert, Stefanie A1 - Riedel, Jens A1 - Beck, S A1 - Linscheid, M.W. T1 - Collision Specific Fragmentation of Oligonucleotides in Tandem Mass Spectrometry N2 - Tandem MS experiments allow the fast acquisition of spectral datasets with enormous size and unprecedented content of information. The most commonly used method is undoubtedly collision induced dissociation (CID). The relatively young technical adaptation of CID to modern day Orbitrap experiments needs a higher confinement voltage and has accordingly been named higher-energy collisional dissociation HCD. Despite the name the absolute value of transferred energy per collision in HCD is lower than in CID. Since for many proteins CID and HCD result in comparable fragmentation patterns the two techniques are often treated as interchangeable. This approach is, however, not without pitfalls as other classes of biomolecules often exhibit strongly pronounced fragmentation specificity. As an example, MS/MS experiments on several 4-mer oligonucleotides were conducted employing both collision methods and the results were thoroughly compared. It is apparent that not only the total fragment yield but also the identity of the observed fragments differ significantly between the two methods. T2 - Jahrestagung der Deutschen Gesellschaft für Massenspektrometrie DGMS CY - Kiel, Germany DA - 05.03.2017 KW - Tandem MS KW - DNA KW - Mass Spectrometry PY - 2017 AN - OPUS4-42328 LA - eng AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER - TY - CONF A1 - Ickert, Stefanie A1 - Hofmann, J. A1 - Riedel, Jens A1 - Beck, S. A1 - Pagel, K A1 - Linscheid, M.W. T1 - Collision Induced Dissociation of DNA Oligonucleotides Studied by Tandem Mass Spectrometry and Ion Mobility N2 - Multiply negatively charged DNA oligonucleotides of small sizes (n=15-40) have been subjected to a comprehensive tandem mass spectrometric study (MS/MS). Collision induced dissociation (CID) mass spectrometry (MS) was applied as a tool to break down isolated DNA oligonucleotides with a defined number of charges. Various lenghts and sequences were analyzed and all of them showed a comparable direct correlation regarding the threshold collision energy for fragmentation and dependence on number of charges on the precursor ions. It was also revealed that the increase in charges and thus Coulomb repulsion results in the transition from a folded, compact form to an elongated structure of the precursor ions. T2 - Jahrestagung der Deutschen Gesellschaft für Massenspektrometrie DGMS CY - Hamburg, Germany DA - 28.02.2016 KW - Ion Mobility KW - DNA KW - Mass Spectrometry PY - 2016 AN - OPUS4-42329 LA - eng AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER - TY - CONF A1 - Ickert, Stefanie A1 - Hoffmann, J. A1 - Riedel, Jens A1 - Beck, S. A1 - Pagel, K A1 - Linscheid, M.W. T1 - DNA Oligonucleotide Fragmentation in Tandem Mass Spectrometry Experiments N2 - Multiply negatively charged DNA oligonucleotides of small sizes (n=15-40 bases) have been subjected to a comprehensive tandem mass spectrometric study (MS/MS). Collision induced dissociation (CID) mass spectrometry (MS) was applied as a tool to break down isolated DNA oligonucleotides with a defined number of charges. Various lenghts and sequences were analyzed unravelling a comparable direct correlation between the threshold collision energy for fragmentation and the charge-per-base density in the precusor ion. It was also revealed that the increase in charges and thus Coulomb repulsion results in the transition from a folded, compact form to an elongated structure of the precusor ions. T2 - International Mass Spectrometry Conference IMSC CY - Toronto, Canada DA - 20.08.2016 KW - Mass Spectrometry KW - DNA KW - Tandem MS PY - 2016 AN - OPUS4-42330 LA - eng AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER -