Orbitrap Mass


Apr 29, 2019 - Harnessing that power depends on high resolution in both dimensions. The ultimate mass resolution and accuracy are delivered by Fourier...
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High-Resolution Differential Ion Mobility Separations/ Orbitrap Mass Spectrometry without Buffer Gas Limitations Matthew A. Baird, Pavel Vyacheslavovich Shliaha, Gordon A. Anderson, Eugene Moskovets, Victor Laiko, Alexander A. Makarov, Ole Nørregaard Jensen, and Alexandre A. Shvartsburg Anal. Chem., Just Accepted Manuscript • Publication Date (Web): 29 Apr 2019 Downloaded from http://pubs.acs.org on April 29, 2019

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is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

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Analytical Chemistry

High-Resolution Differential Ion Mobility Separations/ Orbitrap Mass Spectrometry without Buffer Gas Limitations Matthew A. Baird,† Pavel V. Shliaha,‡ Gordon A. Anderson,§ Eugene Moskovets,¶ Victor Laiko,¶ Alexander A. Makarov,Ξ Ole N. Jensen,‡ Alexandre A. Shvartsburg†,* Department of Chemistry, Wichita State University, 1845 Fairmount, Wichita, Kansas 67260, United States Department of Biochemistry and Molecular Biology, VILLUM Center for Bioanalytical Sciences, University of Southern Denmark, DK-5230 Odense M, Denmark † ‡

§

GAACE, 101904 Wiser Parkway Ste 105, Kennewick, Washington 99338, United States



MassTech Inc., 6992 Columbia Gateway Dr., Columbia, Maryland 21046, United States

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Thermo Fisher Scientific, Hanna-Kunath Strasse 11, Bremen 28199, Germany

* [email protected] ABSTRACT: Strong orthogonality between differential ion mobility spectrometry (FAIMS) and mass spectrometry (MS) makes their hybrid a powerful approach to separate isomers and isobars. Harnessing that power depends on high resolution in both dimensions. The ultimate mass resolution and accuracy are delivered by Fourier Transform MS increasingly realized in Orbitrap MS, whereas FAIMS resolution is generally maximized by buffers rich in He or H2 that elevate ion mobility and lead to prominent non-Blanc effects. However, turbomolecular pumps have lower efficiency for light gas molecules and their flow from the FAIMS stage complicates maintaining the ultra-high vacuum needed for Orbitrap operation. Here we address this challenge via two hardware modifications: (i) a differential pumping step between FAIMS and MS stages and (ii) reconfiguration of vacuum lines to isolate pumping of the Orbitrap ultra-high vacuum region. Either greatly ameliorates the pressure increases upon He or H2 aspiration. This development enables free optimization of FAIMS carrier gas without concerns about MS performance, maximizing the utility and flexibility of FAIMS/MS platforms.

Ion mobility spectrometry (IMS) separates and characterizes ions based on properties of their transport through gas driven by an electric field.1,2 Both IMS and mass spectrometry (MS) date from the early 1900-s. The original method of linear IMS is based on absolute mobility (K) at moderate field intensity (E).1,3,4 Linear IMS and MS were initially joined in the 1960-s to explore the fundamentals of ion transport for mass-selected small ions.4,5 The first analytical application (demonstrated in the 1970-s) was distinguishing isomers of organic compounds,6 and isomer separations have been central to IMS/MS since. Structural elucidation of atomic clusters and, with the advent of softionization MALDI and electrospray (ESI) sources, gas-phase biomolecules ensued in the 1990-s.7-9 The field blossomed in the 2000-s as IMS/MS interfaces employing rf focusing in electrodynamic ion funnels greatly enhanced sensitivity and turned IMS/MS into a versatile analytical tool.10-12 The introduction of multiple commercial instruments since 2006 has hugely expanded the user community and augmented the impact of IMS/MS technology.12,13 Nonetheless, linear IMS/MS is fundamentally limited by low orthogonality between the dimensions: ions of larger size (and accordingly lower K) tend to be heavier, particularly for chemically close compounds with

analogous density and structural features usually probed by IMS.14-16 Therefore, most of the 2-D separation space normally stays empty. That outcome appeared inherent until the invention in the 1980-s of differential or field asymmetric waveform IMS (FAIMS) relying on ∆K - the increment of K between two E levels.2,17 The ∆K is captured in a strong periodic asymmetric field created by loading a waveform of some amplitude (dispersion voltage, DV) across a gap between two electrodes.2,18 Ions carried through by gas flow are deflected to either electrode and lost on contact, but a dc compensation voltage (CV) superimposed on the waveform can balance one species and let it pass the gap at a certain CV governed by the K(E) function. Scanning CV reveals the spectrum of entering ions. For uniformity across devices with all gap widths (g), CV is expressed as the compensation field (EC).19 The ion mass (m) correlates to ∆K much more weakly than to K, bringing about far greater (sometimes nearperfect) orthogonality of FAIMS to MS.14,20-22 Thus, FAIMS resolves isomers and isobars better than linear IMS at equal resolving power (R), by ~3 - 4× for lipids and peptides.21-23 Until a decade ago, that intrinsic gain was negated by the low R of FAIMS (RFAIMS): then <15 compared to >100 for

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linear IMS.24,25 Hence, FAIMS resolution was wanting despite superior orthogonality. With the development of novel planar-gap analyzers using a homogeneous electric field and extended separation time, buffers rich in He or H2, and high-definition waveforms, RFAIMS rose to >100 (>200 for multiply-charged ions).26-28 While linear IMS has since improved to similar R values, especially for the trapped IMS (TIMS),29,30 the orthogonality advantage of FAIMS allows disentangling species merged in linear IMS: isotopomers are the prime example.31 High-resolution FAIMS effectively separates lipid and peptide isomers23,32-34 and protein conformers,35 including peptides with variant positions of posttranslational modifications (PTMs).33,34 The utility of high-resolution FAIMS was constrained by its combination with MS instruments of modest resolving power (RMS), mainly quadrupole ion traps26-28,31-33,35 with RMS ~ 103. Frontline analyses of complex biological and environmental samples demand much greater RMS (105 106) and mass measurement accuracy (mma) under 1 ppm provided by Fourier Transform (FT) MS: FT Ion Cyclotron Resonance and OrbitrapTM MS.36,37 These metrics permit distinguishing nominal isobars and deducing stoichiometries based on “exact” m/z up to ~1 kDa, which is crucial for studies in proteomics,38-40 petroleomics,41-44 and other areas. An increasingly prominent tool in proteomics is electron transfer dissociation (ETD) that severs the peptide backbone bonds without abstracting the weakly bound PTMs.33,34,38-40,45,46 The ETD reaction time (here 15 ms) easily fits the long ion storage times mandatory for sought FTMS resolution (~1 s).34 Hence, ETD is available in many Orbitrap MS platforms but rarely in those without ion trapping such as time-of-flight (ToF) MS. On the other hand, ETD can process continuous ion beams output by FAIMS,33,34,39,40 but not short packets from dispersive linear IMS techniques of drift-tube and traveling-wave IMS. That makes FAIMS an obvious match to FTMS, even though ToFs now deliver RMS ~ 5 × 104 (at the floor of FTMS range). High-resolution FAIMS generally required mixtures of 40 - 85 % He or H2 with N2 or CO2. The He/N2 compositions are most common,26-28,32,33 but H2/N2 maximize RFAIMS for proteins and large peptides35,47 whereas He/CO2 reveal the structurally informative isotopic shifts.48 All turbomolecular (turbo) pumps evacuate light molecules with faster thermal speed less efficiently. The vacuum systems of commercial MS instruments are not designed to pump He or H2, causing a pressure rise throughout the manifold with losses of MS resolution, mma, and sensitivity due to collisional scattering, accelerated wear of turbos, or shutdown by the safe pressure interlock. A destructive internal arc discharge can also happen, as He and H2 have lower electrical resistances than N2 or air, and here (to the left of Paschen curve bottom) the elevated pressure reduces them further.49 This aspect is universal to MS, but is worse for FTMS that requires ~10−10 Torr pressure versus ~10−5 Torr for transmission quadrupoles or traps and ~10−7 Torr for ToFs.50 That has obstructed adding high-resolution FAIMS to Orbitrap MS. Lower He or H2 fractions can often be offset by higher ED. By raising DV from the previous maximum of 5 kV to 6 kV,

we separated peptide isomers in N2 with mean resolution of ~70% that in He/N2 mixtures.34 The upgraded FAIMS system was mounted on an Orbitrap XL instrument and is readily attachable to any mass spectrometer without influencing its operation.34 However, one desires the resolving power of Orbitrap MS with ETD while retaining full FAIMS resolution, lower DV, and flexible buffer choice unrestricted by the MS instrument. Here we present a high-resolution FAIMS/Orbitrap MS platform that achieves these goals by largely decoupling the FAIMS carrier gas from the Orbitrap stage in two distinct ways. The performance is shown for our established standard of monomethylated complete histone tail variants.33,34 Experimental methods All tested configurations connect an ambient-pressure FAIMS device24 via an ion funnel interface to an LTQ Orbitrap XL (Thermo Fisher Scientific, San Jose, CA) with

Figure 1. Schematic representation of the FAIMS/LTQ XL/Orbitrap XL/ETD platform. The new tandem ion funnel interface in front with associated vacuum pumping is delineated in red. the ETD option (Figure 1). The MS instrument (not counting the ETD stage) has five turbo pumps: one on the linear ion trap (LTQ) region and four on ultra-high vacuum (UHV) regions including the Orbitrap cell. All are backed by two mechanical forevacuum (fore) pumps. The pressures are measured by ion gauge (in the LTQ region), Penning gauge (in the Orbitrap region), and Pirani and convectron gauges for rough vacuum in the backing line and front MS chamber (Figure 1). Ions generated by an ESI source using a chemically etched glass emitter of 20 μm internal diameter (i.d.) enter the FAIMS analytical gap via a curtain plate/orifice inlet on the side of one electrode.24 The He/N2 or H2/N2 mixture formulated by digital flowmeters (MKS Instruments) from UHP grade gases is passed through a universal filter and supplied to that inlet at defined volume flow rate (Q).24,47 The larger part (~70%) desolvates entering ions, and the rest pulls them through the gap (g = 1.88 mm). The residence time (t) scales as 1/Q, here t ~ 140 - 70 ms at relevant Q = 2 - 4 L/min.51 The optimum bisinusoidal waveform28,31 has 2:1 harmonics ratio, a frequency of 1 MHz, and DV = 4 kV wherein the electrical breakdown caps the He fraction26 to 65%. The CV is scanned at 0.1 - 0.3 V/min to record FAIMS spectra or fixed for ETD.33,34 The CV

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and waveform are loaded on the inlet and opposite electrodes respectively, with both biased to stay above the MS inlet voltage at any used CV.24 The FAIMS device sits in a plastic bracket in front of the funnel chamber. The spacing between electrodes and MS inlet capillary should be minimized for best ion transmission, but exceed g to preclude arcing from the highvoltage rf electrode to the capillary at low dc voltage. With g = 1.88 mm, a reasonable distance is ~2.5 mm. That area is open, decoupling the FAIMS cell from the MS vacuum suction. As ions need no desolvation after FAIMS, the inlet heating devised for that in ESI/MS systems is reduced to minimize diffusional ion losses, in all systems to ~100 oC from the regular 200 - 300 oC without FAIMS. We first assessed each arrangement based on highvacuum pressures measured by cathode ionization gauges. Both are calibrated for N2, but the gas identity matters and true pressures (P) of He or H2 are far above52 the raw “N2 equivalent” values PNit. The correction factors S = PNit/P somewhat depend on the pressure and gauge construction, but the values of 0.18 for He and 0.48 for H2 are typical.52 The pressure for a mixture with known component fractions (f) is:51 𝑛 𝑛 𝑃 = 𝑃𝑁𝑖𝑡∑𝑖 = 1𝑓𝑖 ∑𝑖 = 1𝑆𝑖𝑓𝑖

inlets, while the 2 mm exit fits within the acceptance diameter of a standard quadrupole lens - here Q00 of LTQ. The funnel chamber is pumped through the instrument (Figure 1). Ions enter through a thin metal sheet with a slit of 11 holes spread over a 4-mm segment approximating the shape of beams emerging from the FAIMS gap.24 The open area of this non-contiguous aperture equals that of standard LTQ capillary with 0.43 mm i.d. and gas conductance QIN of ~0.6 L/min.24 Substituting an aperture for the capillary raises QIN to ~0.8 L/min, which matches or slightly exceeds the outflow from FAIMS gap at Q = 2 L/min. The resulting funnel pressure (for air or N2) is 1.7 Torr, at which the peak rf voltage of 50 V at 1 MHz frequency and dc gradient of ~15 V/cm efficiently convey ions over a broad m/z range. The 5

We used eq (1) to project true pressures in the MS manifold. The rough vacuum pressures measured by thermal conductivity gauges depend on the gas nature less, and for He specifically S = 1 within the error margin.52 Therefore, we accept P = PNit for those quantities.

Instrument development and evaluation Original system with single-funnel interface (SFI) Ion funnels (electrode stacks with internal bore narrowing along the axis) transport and compress large ion plumes with virtually no losses.21 The Dehmelt pseudopotential of rf field repels ions from electrodes, while the axial dc field and MS vacuum suction drag them along to the exit. In the in-line SFI adopted in our FAIMS/MS work to date (Figure S1),24,31 the MS inlet is on the funnel axis with the capillary replaced by a wide tube (to ameliorate ion losses to the walls). The 100-mm long funnel comprises 100 metallized 0.5 mm thick PCB electrodes, layered with insulating plates of same thickness.24 The 25 mm opening is wide enough to encompass ion beams expanding from MS

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The actual analyses involved H3.1 tails (ARTK4QTARK9STGG KAPRKQLATK23AARKSAPATGGVKKPHRYRPGTVALRE,monois otopic mass 5338.1 Da) with me on K4, K9, or K23 site.33,34 Mixtures of these isomers in 10+ charge state (m/z = 536.22) yield characteristic FAIMS spectra in He/N2 buffers, which we picked to benchmark the resolution of PTM localization variants and sensitivity of their identification by FAIMS/ETD.33,34 These peptides assembled by Fmoc solid-state synthesis and native chemical ligation were purified by liquid chromatography (LC).33 The variant mixtures were dissolved to 5 μM in 99.9:0.1 water/formic acid and infused to the ESI source at 0.5 μL/min. The Orbitrap MS was operated in the magnitude mode with the transient duration controlled by set RMS value.53

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As He is added, the PNit in funnel, LTQ, and Orbitrap regions rise - to respectively 2.7, 2.1 × 10−5, and 4.9 × 10−10 Torr at 100% He (Figure 2a). The increase of 7× in Orbitrap versus ~30% in LTQ regions illustrates the outsized consequence of He for UHV. Given the inefficiency of turbos for He, the pressure must increase in LTQ much more than Figure 2. Pressures in the funnel, LTQ, and Orbitrap regions with SFI interface depending on the He content in FAIMS buffer: (a) raw measurements and (b) adjusted for LTQ and Orbitrap per eq (1). The range relevant to tandemfunnel interface is filled. in the funnel, i.e., by >>60%. That happens once we apply eq (1), with P rising by 7× (to 1.1 × 10−4 Torr) in LTQ and yet greater ~40× (to 2.7 × 10−9 Torr) in the Orbitrap region (Figure 2b). The gas flow to the LTQ region (and hence to the Orbitrap analyzer) may contain less He than the FAIMS buffer because of ambient air sucked into the open MS inlet.

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However, both enclosures are continually enriched in He by preferential removal of N2 molecules. Thus, the real pressure in either may be below or (except at 100% He) above the calculations in Figure 2b, but they convey the magnitude of elevation. Nonetheless, we obtained quality FAIMS and FAIMS/ETD data in this regime. The m/z and widths of MS peaks do not materially differ from those34 with N2 buffer at equal RMS = 30,000 (Figure 3a). In the FAIMS spectrum for K4me/K23me with K9me trace34 (Figure 3b), we assigned features A, B, B1 to K4me; C, C1 to K23me, and D to K9me based on redundant data for individual variants and mixtures under identical conditions with LTQ read-out.33 All species are perfectly separated at major conformers B, C, D Figure 3. Data for K4me/K23me/K9(trace) with SFI: (a) MS spectral window for z = 10; (b) FAIMS spectrum over shown m/z range. The minor feature D is scaled by 100×, peaks selected for ETD are labeled in red. with peak widths at half maximum (w) of 1.2 V/cm, matching earlier findings.33 With N2 buffer, the same peaks had w = 2.4 V/cm at equal Q and t and still 1.6 V/cm at t > 300 ms (with Q = 0.9 L/min) and marginal signal too low for ETD analysis.34 The present ion signal compares to that with N2 buffer. (a)

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The data for K9me versus K4me or K23me (Figure 3b) for the first time demonstrate the ability to detect <1% variant 5+ concentrations. z31 c194+ Such dynamic range is critical for practical A proteomics. In RMS particular, existing 15 K middle-down histone analysis workflows discover most abundant B isoforms, but others RMS may have major epigenetic roles.54,55 15 K The ion counts in Figure 4. ETD spectral windows A, B, and C suffice for c194+ and z315+ obtained at peaks for confident ETD A, B, C in Figure 3b, the RMS identification Cvalues are labeled. (Figure 4). The RMS 30 K

dominant informative fragments remain34 complementary c194+ and z315+ with s/n = 40 - 70 upon 10 min averaging (at RMS = 15,000 - 30,000). The parallel spectra with N2 buffers evidenced contamination by K23me at B and K4me at C in ~3% amounts,34 but none is seen here. As present peak heights for major isoforms are similar at RMS = 30,000 and higher at RMS = 15,000, this verifies the superior variant resolution in He/N2 media. We again encounter34 the z437+ product of K23me, with m/z peaks differing from the Figure 5. ETD spectral window for c194+ fragment obtained at peak B in Figure 3b with RMS = 100,000. adjacent for z315+ of K4me by 0.07 Th at most. That is enough for baseline resolution with present RMS = 30,000 (Figure 4), but indistinguishable using the LTQ. The interferences may cluster even closer,34 e.g., some isotopic peaks of z437+ and z315+ split by m/z = 0.01 Th. Resolving those requires RMS ~ 100,000, achievable for the present precursor signal with slightly lower s/n ~ 30 (Figure 5). The absence of fragments for isomeric impurities at peak apexes prevents leveraging them to evaluate sensitivity as with N2 buffer.34 However, the presently similar signal and s/n for fragments indicate close inter-isoform detection limits of ~500 fmol with a priori assignment.34 In the

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accurate mass and time (AMT) tag approach, proteolytic peptides are identified by the LC elution profile in LC/MS (rather than MS/MS) for higher sensitivity.56 Within that paradigm, one may assign variants using the catalogued EC values. Then the LoDs are just ~2 fmol, judging by feature D (Figure 3b). The much greater edge of AMT in this context derives from lower informative fragment yields in ETD (~0.5 %)34 compared to collisional dissociation in bottomup proteomics. Some ubiquitin conformers selected by FAIMS exhibit distinct ETD fragment patterns, likely controlled by backbone accessibility in different folds.57 This can mean varying informative fragments across conformers and/or variants. That is not noted here, possibly as peptides cannot conceal sites internally as well as larger proteins and/or conformational differences are smaller here because of more extensive unfolding upon stronger field heating of ions by presently greater field in He/N2 buffers.58 In general, one should not assume same informative fragments to be most pronounced for all FAIMS peaks. While these data prove high-resolution FAIMS/FTMS analyses employing up to (at least) 65% He feasible in the arrangement validated with N2 buffer,34 the pressure rise is a hardware reliability risk. In some newer Orbitrap models (e.g., Q-Exactive and Orbitrap Fusion), the FTMS region is less isolated from the MS inlet and thus would experience

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Analytical Chemistry faster pressure rise upon He addition, perhaps preventing operation at the same fractions. We did not attempt H2/N2 buffers with this system for instrument safety reasons, but the results with the upgraded interface below suggest a failure already at single-digit H2 percentages. To facilitate use of He/N2 buffers and try H2/N2 important for intact protein analyses,35 we have switched to a tandem-funnel interface. Differentially pumped tandem-funnel interface (TFI) In TFI,59,60 the funnel terminating in the first MS vacuum region (as in SFI) is preceded by a separately pumped funnel facing the MS inlet. The pressure there can be raised up to ~25 Torr (versus 1 - 4 Torr in SFI) while maintaining the requisite MS vacuum. This permits opening the inlet in Figure 6. Scheme of the tandem funnel interface (photos in Figure S2). proportion (usually by ~5×), normally to improve sensitivity by better capturing wide ion plums that arrive from ESI or other atmospheric-pressure ion source.59 That is moot after FAIMS, where ions hit the inlet over a small area that can be covered by a slit as described.24 The purpose of the wider inlet here differs - diluting the FAIMS “eluent” by ambient air to cut the He or H2 fractions in gas flow to the instrument. With present QIN = 4 L/min, those should drop by ~5 - 2.5× with Q = 2 - 4 L/min (e.g., to ~13 26% from 65% and to ~20 - 40% from 100%). With SFI or TFI, the MS inlet can be perpendicular to the

funnel axis.60,61 This geometry (similar to the Z-spray concept)62 adopted here is anticipated to suppress chemical noise by directing neutrals and poorly desolvated ions away from the funnel exit.61 In our implementation (Figures 6, S2), both funnels are built from 0.5 mm-thick PCB electrodes with inside edges metallized to 1.5 mm margins and 0.45-mm thick insulating plates. The front funnel with 20-mm long straight and 105mm long conical sections has the 52 mm opening and 4.5 mm exit. The heated cone from LTQ is moved to the funnel side, with the original capillary (0.58 mm i.d.) replaced by a wider (1 mm i.d.) 108-mm long steel capillary ending at the 7 × 7 mm window in the straight section. Ions emerging therefrom are deflected 900 by the dc-only repeller

electrode. The gas jet shoots across through the holes in opposite wall (made by cutting out 45-mm long plate sectors) toward the intake of external forepump (Edwards 30). The pressure inside is measured by second Pirani gauge. The back funnel of the “keyhole” geometry lies 11 mm behind the front funnel with a lateral displacement of 3 mm to exclude direct gas jet penetration from the front funnel to the LTQ chamber. Ions traverse a 10-mm long straight section with a 20 mm opening, a 30-mm long conical section with a 4 mm exit, a 10-mm diverging section, a 15 mm-long straight section of 20 mm diameter, and 30-mm long final conical section with a 3.4 mm exit. The rf waveform in both funnels has 110 V peak voltage at 0.8 MHz frequency. The dc voltages are deployed at two Figure 7. Data for K4me/K23me/K9me with TFI: (a, b) MS spectral windows for z = 10 at RMS of 30,000 and 100,000; (c) FAIMS spectra over shown m/z range. and four points in the front and back funnels respectively, here adjusted to the gradient of 6 V/cm along both. The minimum pressures in the front and back funnels (with N2 or air) are 6 and 0.5 Torr; higher values are reachable by choking the external pump. Here we set a pressure of 13 Torr in the front funnel. Then the pressure in the back is 0.7 Torr - about 40% that in SFI. However, the area of exit aperture is ~3× that in SFI (9 vs 3 mm2), and the pressures in LTQ and Orbitrap regions are equivalent to those with SFI: 1.7 × 10−5 and 7 × 10−11 Torr. Adding He lifts all measured pressures more gradually than with SFI, as intended. With Q = 2 L/min, the raw PNit in the back funnel or LTQ region inch up by ~5 - 10% at 65 100% He. The reading in the Orbitrap region goes up more as with SFI, but still by just ~50 - 100% to 1.1 - 1.4 × 10−10 Torr. These observations support the above picture of He dilution at MS inlet: use of 13 - 20% He with SFI increased PNit by ~5 - 10% in the funnel or LTQ and ~70 - 120% in the Orbitrap region (Figure 2a). The pressures per eq (1) are only a bit higher, 1.9 - 2.1 × 10−5 Torr in LTQ and 1.2 - 1.7 × 10−10 Torr in the Orbitrap. The dramatic difference of these from those for SFI (Figure 2b) reflect both lower raw pressures and lower He fractions in TFI. This performance enabled us to investigate greater He/N2 flows and H2/N2 buffer. With Q = 4 L/min (the realistic limit for present FAIMS device), the PNit in Orbitrap region increased to 1.8 - 2.1 × 10−10 Torr at 65 - 100% He. This compares to 1.7 - 2.4 × 10−10 Torr with SFI at 26 - 40% He (Figure 2a), confirming our proposed dilution 536.22

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Analytical Chemistry mechanism. The corresponding true pressures are 2.2 - 2.9 × 10−10 Torr. The mass spectra for K4me/K9me/K23me mix33 obtained at Q = 2 L/min and 65% He (Figure 7 a, b) exhibit the requested RMS of 30,000 or 100,000, correct m/z, and signal similar to that with SFI. This shows proper Orbitrap MS operation with TFI, expected from the results at higher pressures with SFI. The FAIMS spectra (Figure 7c) follow that in Figure 3b, save for a much larger peak D resulting from greater amount of K9me in the sample. Slightly varying peak height ratios at two RMS (Figure 7c) are common for acquisitions on different days. The peaks seem Figure 8. Schematic representation of the FAIMS/LTQ XL/Orbitrap XL/ETD platform with single ion funnel interface in front. The vacuum pumping reconfiguration is depicted in red lines. marginally wider than with SFI (here meanS3, w= 1.4 V/cm for tandem funnel interface (photos in Figures S4). B, C, D), possibly as stronger suction from TFI inlet diminishes t, but are substantially narrower than with N2 buffer at similar Q and signal.34 In sum, the increase of true pressure in Orbitrap region is within 2.5× at our default Q = 2 L/min and 4× at maximum Q = 4 L/min with neat He, and lower 2× - 3× at 65% He. This should not cause problems even in the long term. However, H2/N2 buffers remain prohibited: PNit in Orbitrap rocketed by ~25× (to 1.7 × 10−9 Torr) already at 20% H2. To fix that, we have taken an alternative direction of reconfiguring the vacuum pumping. Vacuum pumping reconfiguration The residual pressure of any gas achievable by a turbo is the compression ratio (c) times the pressure in backing line (Pback). While a low c for He and (more so) H2 is inevitable,

unmonitored). While this is feasible with any front MS interface, here we used SFI as only one suitable external pump was available. In this arrangement, the pressures (with N2 buffer) are 1.7 Torr in the funnel, 1.6 × 10−5 Torr in LTQ, and 5 × 10−11 Torr in Orbitrap region (Figure 9a). While the first two values equal those prior to reconfiguration, the reduction of the last reflects PBack plummeting to 0.01 Torr. The PBack and PNit in the Orbitrap region do not move up even at 100% He (with Q = 2 L/min), although P presumably does. The pressures in the funnel and LTQ edge up to 2.1 Torr and 1.8 × 10−5 Torr at 100% He (Figure 9a). Both increase less than prior to reconfiguration because of more efficient pumping of the funnel and backing of LTQ turbo by dedicated forepumps. Encouraged by this success, we retried H2/N2 buffers (Figure 9b). With Q = 2 L/min, we now get to 60% H2 before PNit in LTQ becomes a concern at ~6 × 10−5 Torr. At that point, the values in the funnel and Orbitrap are fair (2.8 Torr and 3 × 10−10 Torr, respectively): the H2 fraction is now limited by the pressure in LTQ. The true pressures in the LTQ and Orbitrap regions are again higher, though by less than with He because of greater S for H2: ~8 × 10−5 and 4 × Figure 9. Pressures measured in the funnel, LTQ, and Orbitrap regions with SFI interface, reconfigured pumping, and HCD gas on (solid lines) or off (dashed lines) for (a) He/N2 at Q = 2 L/min, (b) H2/N2 at Q = 2 L/min, (c) H2/N2 at Q = 1.4 L/min. The ranges relevant to TFI are filled. 10−10 Torr, respectively. This result is promising, but the ultimate resolution of protein conformers necessitated35 H2 fractions up to 84%. As raising Q increases the load of light gas and thus the pressures in all stages, we can decrease Q to reverse the effect. The optimum Q in FAIMS drops for larger ions that diffuse slower and thus can stay in the gap longer at equal sensitivity. For macromolecules for which H2/N2 buffers are most pertinent, Q < 2 L/min is appropriate. At Q = 1.4 L/min, we can go to 100% H2 with PNit of <6 × 10−5 Torr in LTQ and 3 × 10−10 Torr in the Orbitrap region (Figure 9c). At any H2 fraction with either Q, we still see PBack = 0.01 Torr. This

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Analytical Chemistry again testifies to superb UHV backing, with the pressure limitation moved to LTQ stage. The instrument normally also receives gas (N2) through the higher-energy collision dissociation (HCD) cell disposed downstream of the Orbitrap region. The MS1 and ETD MS/MS modes employed here do not involve HCD, so the gas supply to HCD can be turned off to reduce high-vacuum pressures (the pressure in funnel is naturally not affected). We see constant PNit shift down with any He/N2 or H2/N2 buffer: 7 - 8 × 10−6 Torr in LTQ and 2 - 3 × 10−11 Torr in the Orbitrap region (Figure 9 a - c), while PBack falls below the measurable range starting at 0.01 Torr. This makes less of a difference at elevated pressures with high H2 fractions, but can help in borderline situations. Conclusions and Future Directions We have integrated high-resolution FAIMS employing any buffer with Orbitrap MS while overcoming steep pressure rise in UHV regions upon substantial inflows of He or H2 not evacuated well by turbo pumps. This was attained by (i) inserting a differentially pumped tandem-funnel interface (TFI) after the FAIMS stage to dilute its eluent by ambient air or (ii) rearranging the vacuum system to provide turbo pumps on UHV regions with dedicated backing apart from that for pumps on LTQ stage. The 1st modification allows all He/N2 buffers at practical flow rates with only a modest pressure increase in any stage, but not H2/N2 mixtures with significant H2 fraction. The 2nd modification is more efficient, handling He/N2 buffers with negligible pressure increase and H2/N2 with up to 60% H2 at the “normal” flow rate or 100% H2 at reduced rate. In analyses not exploiting the HCD cell, the gas supply to HCD can be switched off as the last step to minimize the pressures. The performance is demonstrated in analyses of benchmark histone tails. The resolution of variants with methylation on different residues matches that obtained using same He/N2 buffers with low-resolution MS and exceeds that achieved in the FAIMS/Orbitrap MS employing N2 buffer. The sensitivity is similar to that with N2 buffers, with isoform LoD of several fmol if based on FAIMS separation and hundreds of fmol by ETD fragments. While the full EC spectra shown here took ~30 min to acquire, sufficient precursor ion signal at known peak EC in targeted analyses is measurable in a few seconds. This work addresses the He/N2 and H2/N2 mixtures that are common to FAIMS and most difficult because of the lowest molecular masses of the constituents. Hence, present developments ought to cover other buffers, including He/CO2 preferred for isotopologue separations48 and He/SF6 that yields extreme non-Blanc effects.63 One can now optimize FAIMS separations without regard for compatibility with ultra-high resolution MS performance. The persisting pressure increase with H2/N2 buffers can be mitigated by combining our two modifications. The outcome can be predicted by inspecting Figure 9 within the TFI ranges (that at Q = 1.4 L/min is narrower because of greater dilution of slower FAIMS eluent). The Figure 9b indicates the possibility of using H2/N2 with up to 100% H2 at Q = 2 L/min and higher, while staying within the allowed

LTQ pressure range. A further reserve is fitting the TFI with a valve leaking N2 into the front funnel59,60 while augmenting the pumping (using the spare capacity) to dilute the FAIMS eluent yet more. Those options may be warranted for Orbitrap models with UHV region more open to the MS inlet (e.g., the latest Fusion with resolving power of 500,000).64 Efforts along those directions are underway. While this research has focused on a planar-gap FAIMS device, the buffers rich in He or H2 also benefit resolution and/or sensitivity in FAIMS systems with curved2,18 or multichannel microscopic65 gaps. Also, linear (in particular, drift-tube) IMS strongly benefits from He buffer that (i) allows most robust and accurate first-principles ion mobility calculations for structural elucidation purposes66,67 and (ii) accelerates separations while improving sensitivity and/or resolution.68 Present developments facilitate coupling of both linear and nonlinear IMS analyzers to Orbitrap MS. CONFLICT OF INTEREST DISCLOSURE:

A.A.S. has an interest in Heartland MS that makes the ion funnel and FAIMS systems for mass spectrometers used in this work. SUPPORTING INFORMATION AVAILABLE: Photos of single and tandem ion funnel interfaces, the plot of backing line pressure with SFI.

AUTHOR INFORMATION Corresponding Author *E-mail: [email protected]

ACKNOWLEDGMENT The work at WSU was supported by NSF CAREER (CHE1552640), with P.S. stay at WSU funded by Lundbeck Foundation and Danish Cancer Society. We thank Julia Kaszycki for experimental help.

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