Carsten Stoermer

Structural analysis of intact monoclonal antibodies by electron transfer dissociation mass spectrometry.

Improving qualitative and quantitative characterization of monoclonal antibodies is essential, because of their increasing popularity as therapeutic drug targets. Electron transfer dissociation (ETD)-based top-down mass spectrometry (MS) is the method of choice for in-depth characterization of post-translationally modified large peptides, small- and medium-sized proteins, and noncovalent protein complexes. Here, we describe the performance of ETD-based top-down mass spectrometry for structural analysis of intact 150 kDa monoclonal antibodies, immunoglobulins G (IgGs). Simultaneous mass analysis of intact IgGs as well as a complex mixture of ETD product ions at sufficiently high resolution and mass accuracy in a wide m/z range became possible because of recent advances in state-of-the-art time-of-flight (TOF) mass spectrometry. High-resolution ETD TOF MS performed on IgG1-kappa from murine myeloma cells and human anti-Rhesus D IgG1 resulted in extensive sequence coverage of both light and heavy chains of IgGs and revealed information on their variable domains. Results are superior and complementary to those previously generated by collision-induced dissociation. However, numerous disulfide bonds drastically reduce the efficiency of top-down ETD fragmentation within the protected sequence regions, leaving glycosylation uncharacterized. Further increases in the experiment sensitivity and improvement of ion activation before and after ETD reaction are needed to target S-S bond-protected sequence regions and post-translational modifications.

Electron transfer dissociation in the hexapole collision cell of a hybrid quadrupole‐hexapole Fourier transform ion cyclotron resonance mass spectrometer

Electron transfer dissociation (ETD) of proteins is demonstrated in a hybrid quadrupole-hexapole Fourier transform ion cyclotron resonance mass spectrometer (Qh-FTICRMS). Analyte ions are selected in the mass analyzing quadrupole, accumulated in the hexapole linear ion trap, reacted with fluoranthene reagent anions, and then analyzed via an FTICR mass analyzer. The hexapole trap allows for a broad fragment ion mass range and a high ion storage capacity. Using a 3 T FTICRMS, resolutions of 60 000 were achieved with mass accuracies averaging below 1.4 ppm. The high resolution, high mass accuracy ETD spectra provided by FTICR obviates the need for proton transfer reaction (PTR) charge state reduction of ETD product ions when analyzing proteins or large peptides. This is demonstrated with the ETD of ubiquitin and apomyoglobin yielding sequence coverages of 37 and 20%, respectively. We believe this represents the first reported successful combination of ETD and a FTICRMS.

Data‐dependent electron transfer dissociation of large peptides and medium size proteins in a QTOF instrument on a liquid chromatography timescale

Liquid chromatography (LC) electron transfer dissociation (ETD) tandem mass spectrometry (MS/MS) of protein digests is demonstrated in a hybrid quadrupole-hexapole orthogonal time-of-flight (OTOF) mass spectrometer. Analyte ions are selected in a mass-analyzing quadrupole, accumulated in the hexapole linear ETD reaction cell and mutually stored with ETD reagent anions. Product ions are collected in an ion cooler and then analyzed by an OTOF mass analyzer. The hexapole structure of the ETD reaction cell allows for a broad fragment ion mass range distribution and a high ion storage capacity. Analytically useful ETD OTOF-MS/MS spectra could be obtained at a rate of faster than 2 Hz. When used in conjunction with LC this high speed allows for several MS and MS/MS spectra to be obtained across each LC peak. An MS scan is used to select the precursor ions. With a 1 m flight tube and single reflection, resolutions of about 10 k and a mass accuracy of 5 ppm were achieved. When analyzing a 100 fmol solution of a tryptic digest of bovine serum albumin (BSA) by LC/ETD MS/MS, 27 unique peptides were identified with a summed Mascot score of 1316 using the Swiss Prot database. In addition, we explored the capability for analyzing small proteins with the present hybrid instrument. ETD MS/MS of intact ubiquitin ([M+12H](12+)) leads to the identification of the protein with a Mascot score of 264.

Identification of hemoglobin variants by top-down mass spectrometry using selected diagnostic product ions

Hemoglobin disorder diagnosis is a complex procedure combining several analytical steps. Due to the lack of specificity of the currently used protein analysis methods, the identification of uncommon hemoglobin variants (proteoforms) can become a hard task to accomplish. The aim of this work was to develop a mass spectrometry-based approach to quickly identify mutated protein sequences within globin chain variants. To reach this goal, a top-down electron transfer dissociation mass spectrometry method was developed for hemoglobin β chain analysis. A diagnostic product ion list was established with a color code strategy allowing to quickly and specifically localize a mutation in the hemoglobin β chain sequence. The method was applied to the analysis of rare hemoglobin β chain variants and an Aγ-β fusion protein. The results showed that the developed data analysis process allows fast and reliable interpretation of top-down electron transfer dissociation mass spectra by nonexpert users in the clinical area.

Evaluation of a New Reagent-Ion Source and Focusing Ion–Molecule Reactor for Use in Proton-Transfer-Reaction Mass Spectrometry

We evaluate the performance of a new chemical ionization source called Vocus, consisting of a discharge reagent-ion source and focusing ion-molecule reactor (FIMR) for use in proton-transfer-reaction time-of-flight mass spectrometry (PTR-TOF) measurements of volatile organic compounds (VOCs) in air. The reagent ion source uses a low-pressure discharge. The FIMR consists of a glass tube with a resistive coating, mounted inside a radio frequency (RF) quadrupole. The axial electric field is used to enhance ion collision energies and limit cluster ion formation. The RF field focuses ions to the central axis of the reactor and improves the detection efficiency of product ions. Ion trajectory calculations demonstrate the mass-dependent focusing of ions and enhancement of the ion collision energy by the RF field, in particular for the lighter ions. Product ion signals are increased by a factor of 10 when the RF field is applied (5000-18 000 cps ppbv-1), improving measurement precision and detection limits while operating at very similar reaction conditions as traditional PTR instruments. Because of the high water mixing ratio in the FIMR, we observe no dependence of the sensitivity on ambient sample humidity. In this work, the Vocus is interfaced to a TOF mass analyzer with a mass resolving power up to 12 000, which allows clear separation of isobaric ions, observed at nearly every nominal mass when measuring ambient air. Measurement response times are determined for a range of ketones with saturation vapor concentrations down to 5 × 104 μg m-3 and compare favorably with previously published results for a PTR-MS instrument.