Development and Evaluation of a Reverse-Entry Ion Source Orbitrap Mass Spectrometer

Abstract

As a step towards development of a high-resolution ion mobility mass spectrometer using the orbitrap mass analyzer platform, we describe herein a novel reverse-entry ion source (REIS) coupled to the higher-energy C-trap dissociation (HCD) cell of an orbitrap mass spectrometer with extended mass range. Development of the REIS is a first step in the development of a drift tube ion mobility-orbitrap MS. The REIS approach retains the functionality of the commercial instrument ion source which permits the uninterrupted use of the instrument during development as well as performance comparisons between the two ion sources. Ubiquitin (8.5 kDa) and lipid binding to the ammonia transport channel (AmtB, 126 kDa) protein complex were used as model soluble and membrane proteins, respectively, to evaluate the performance of the REIS instrument. Mass resolution obtained with the REIS is comparable to that obtained using the commercial ion source. The charge state distributions for ubiquitin and AmtB obtained on the REIS are in agreement with previous studies which suggests that the REIS-orbitrap EMR retains native structure in the gas phase. Graphical Abstract ᅟ.

Selective binding of a toxin and phosphatidylinositides to a mammalian potassium channel

“…b Mass spectrum of GIRK2 in DDM after DHPC treatment, which reveals a well-resolved mass spectrum with the lipid adducts removed. DHPC treated GIRK2 in the charge-reducing detergent, C 10 E 5 acquired under minimal energy regimes on a c Waters Synapt G1 and d Thermo Exactive plus EMR modified with a rear-entry source 49 . The mass spectra resolves small molecule adducts bound to the tetrameric complex …”

“…We also acquired data on a higher mass resolution Orbitrap mass spectrometer 48 equipped with a custom reverse-entry ion source (REIS-Orbitrap) 49 , 50 that yielded sharp mass spectral peaks with addition of mass as small as 80 Da resolved (Fig. 1d ).…”

Generation of Charge-Reduced Ions of Membrane Protein Complexes for Native Ion Mobility Mass Spectrometry Studies

“…The addition of TMAO appears to promote fewer structure(s) thus yielding a tighter ATD consistent with the increase in mobility resolution. However, further investigations are warranted to probe protein structure upon binding of lipids and other molecules to membrane protein complexes, such as G protein coupled receptors (GPCR's), in the presence of TMAO [11,38]. Charge reduction with TMAO provides an attractive route to elucidate the structural effects that arise from a larger number of bound lipid at the resolution of individual binding events.…”

Fourier Transform-Ion Mobility-Orbitrap Mass Spectrometer: A Next-Generation Instrument for Native Mass Spectrometry

“…For these experiments, the nano-ESI source previously described was mounted onto a PF DT. 35 The nano-ESI uses pulled borosilicate glass capillaries, prepared in-house, that are either gold coated or contain a platinum wire (300 μ m) inserted into the capillary. ESI potentials of 1.50–2.00 kV were used for all studies.…”

“…We have previously shown the importance of improved R p in native MS using an Orbitrap over IM-ToF MS to characterize heterogeneous lipid binding events to the trimeric ammonia transport channel (AmtB), an integral membrane protein. 35 More specifically, IM-ToF MS simply does not possess sufficient R p to separate the individual lipids bound, whereas the Orbitrap MS successfully separated 46 different combinations of lipids bound to AmtB. Notably, R m/z of native mass spectra are markedly lower than those of small molecules (viz.…”