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.

Keywords: Exactive; Instrument development; Ion source; Membrane protein; Orbitrap.

Figure 1

(a) Solidworks rendering of the reverse entry ion source and configuration of the instrument. (b) Starting from left to right, ions enter the instrument through an 11.4 cm, 800 μm i.d. heated capillary and are radially focused by a 5.08 cm in length RF ion funnel. The RF ion funnel consists of 33 electrodes electrically coupled through a series of capacitors and resistors, wherein the first 15 electrodes have inner diameters of 2.54 cm and the last 18 electrodes linearly taper to 2 mm. Ions are then guided through an RF-only octupole ion guide (rod diameter of 1.59 mm and 4.76 mm inscribed diameter) prior to being loaded into the HCD Cell of the Exactive Plus EMR.

Figure 2

Mass spectra of ubiquitin acquired on the (a) commercial ion source and (b) REIS. Shown in the insets are the 5⁺ charge state giving isotopic resolution from both ion sources. Note the lower charge states observed using the REIS giving rise to the 3⁺.

Figure 3

Mass spectra collected from the unmodified orbitrap (a,d), Waters Synapt G1 (b,e), and the REIS (c,e). The full mass spectra are shown on the left, and the 16+ charge state is expanded on the right. The asterisks denote the adducted C8E4 detergent molecule bound to the AmtB in all three cases which was impossible to strip from the protein complex without dissociation of the complex itself.

Figure 4

(a) REIS-orbitrap fully resolves AmtB bound to different combinations of POPA, POPS, and TMCDL. A total of twenty-six distinct lipid bound species were observed with up to five total lipids bound to AmtB. (b) Notably, none of these individual lipids species were resolved on the Waters Synapt G1 instrument.

Figure 5

RESI-orbitrap can resolve complex mixtures of AmtB bound to four different lipid species: POPA, POPE, POPS, and TMCDL. Forty-five lipid species bound to AmtB were observed.