Atmospheric Pressure Drift Tube Ion Mobility-Orbitrap Mass

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Atmospheric Pressure Drift Tube Ion Mobility-Orbitrap Mass Spectrometry: Initial Performance Characterization. Joel D Keelor, Stephen Zambrzycki, Anyin Li, Brian H Clowers, and Facundo M Fernandez Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.7b01866 • Publication Date (Web): 11 Oct 2017 Downloaded from http://pubs.acs.org on October 16, 2017

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Analytical Chemistry

1

Atmospheric Pressure Drift Tube Ion Mobility-Orbitrap Mass Spectrometry: Initial

2

Performance Characterization.

3 4

Joel D. Keelor1, Stephen Zambrzycki1, Anyin Li1, Brian H. Clowers2, and Facundo M.

5

Fernández1

6 7

1

8

30332, United States

9

2

10

School of Chemistry and Biochemistry, Georgia Institute of Technology, Atlanta, Georgia

Department of Chemistry, Washington State University, Pullman, Washington 99164, United

States

11 12 13 14 15 16 17 18 19 20 21 22

*Corresponding

author.

Phone:

23

[email protected].

404-385-4432,

Fax:

ACS Paragon Plus Environment

404-385-3399.

E-mail:


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Abstract

2

Atmospheric pressure drift tube ion mobility spectrometry (AP-DTIMS) was coupled

3

with Fourier transform Orbitrap mass spectrometry. The performance capabilities of this

4

versatile new arrangement were demonstrated for different DTIMS ion gating operation modes

5

and Orbitrap mass spectrometer parameters in regard to sensitivity and resolving power.

6

Showcasing the optimized AP-DTIMS-Orbitrap MS system, isobaric peptide and sugar isomers

7

were successfully resolved and the identities of separated species validated by high-energy

8

collision dissociation (HCD) experiments.

9

2 ACS Paragon Plus Environment

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1


Introduction

2

Ion mobility (IM) spectrometry provides an additional analytical separation dimension

3

when combined with mass spectrometry (MS), as well as the ability for ion selection and

4

filtering based on “size-to-charge” ratios, ultimately enabling increases in instrument specificity,

5

peak capacity, and dynamic range. By expanding spectral coverage across two or more

6

dimensions (e.g. drift time and m/z), IM-MS analysis facilitates spectral interpretation by

7

lessening spectral congestion,1 with different compounds with distinct structural motifs – e.g.

8

lipids, carbohydrates, peptides, etc. – mapped to unique molecular trend lines.2 Data extracted

9

from this type of multidimensional space has immensely enriched chemical classification and

10

molecular identification in various “omics” research fields. Further expansion of the IM-MS

11

approach with time-nested liquid chromatography (LC)-IM-MS, or IM-IM-MS systems further

12

increases peak capacity, recovering more of the IM-MS dimensional space obscured by the

13

pseudo-orthogonal dependence between ion mobility (K) and m/z, and also removing ambiguity

14

in regions of CCS-m/z overlap.3, 4 Such nested IM-MS platforms are capable of providing greater

15

information density all the while reducing spectral complexity without a significant cost to

16

analysis time.

17

Currently, the most successful commercial implementations of IMS paired with MS

18

employ time-dispersive drift tube (DTIMS) or traveling wave (TWIMS) mobility techniques.

19

These particular techniques, for which ion-neutral collision cross-section (CCS) determination is

20

relatively straightforward, have proven exceptionally useful in the burgeoning fields of

21

proteomics, metabolomics, and structural MS. The majority of traveling wave or drift tube IM-

22

MS arrangements, best exemplified by the Waters Synapt G2-S or Agilent 6560 instruments,

23

respectively, feature mobility cells embedded within the MS system that are thereby restricted to

24

operation at reduced pressures by the vacuum constraints of the mass analyzer.2,

25

separations rely on ion/gas interactions that scale with pressure, and such reduced-pressure

26

configurations inevitably limit the achievable mobility resolving power as levels of ion diffusion

27

are more appreciable than at atmosphere. Ion trapping approaches, however, can partially or 3 ACS Paragon Plus Environment

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All IMS


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completely offset this limitation. Enhancements to resolution and sensitivity have also been

2

made in reduced pressure systems by exploiting different gas polarizabilities,1,

3

additional electrodynamic RF field focusing to reduce diffusional broadening,7, 8 or electrostatic

4

lens implementations that can restore IMS operation at atmospheric pressure.9 In this pressure

5

regime, the resolving power of DTIMS is maximized as diffusion is reduced, yielding peak

6

capacities that may even surpass LC separation efficiencies.10,

7

atmospheric pressure operation is the assurance that the ion mobilities measured are from fully

8

thermalized ions interacting with a neutral gas under the low field limit.12, 13 However, very few

9

implementations of atmospheric pressure (AP)-DTIMS-MS are commercially developed, with

10 11

11

6

or applying

An additional benefit of

the TOFWERK IMS-TOF system recognized as the prime example.14 Traditionally, time-dispersive ion mobility has been paired with quadrupole ion traps 16

15

12

and time-of-flight (ToF) analyzers

13

resolutions and scan speeds ideally suited for time-nested analysis. Contemporary commercial

14

IM-Q-ToF MS instruments are designed to maximize sensitivity, and commonly incorporate ion

15

trapping and transfer stages in order to alleviate the intrinsically low IM duty cycles.16-18 Spatial

16

and temporal DTIMS-MS multiplexing approaches, which encompass either arrays of mobility

17

analyzers coupled to a single detector or time-multiplexing of multiple ion injection pulses per

18

IM acquisition, have also been used to improve IM duty cycle.

19

pseudo-random sequence multiplexing methods have enabled favorable increases in signal-to-

20

noise ratios and sensitivity, while also retaining the higher theoretical resolving powers,

21

enhancing instrument efficiency for more practical applications of AP-DTIMS.22,

22

advances in coupling atmospheric pressure ion mobility with FT ion cyclotron resonance (ICR)

23

mass spectrometry are pushing the boundaries of achievable peak capacity.24 Initial cost,

24

however, still prevents FTICR instrumentation from becoming mainstream.

, with modern ToF technology now providing mass

19-21

Hadamard transform and

23

Recent

25

Presented in this work is the combination of atmospheric pressure DTIMS with Orbitrap

26

FT-MS, aimed at both increasing drift tube IMS separation power and providing higher mass

27

accuracy and stability for better annotation of unknowns. One of the challenges with combining 4 ACS Paragon Plus Environment

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DTIMS with Orbitrap is that the analyzer scan times are rather long in comparison with TOF,

2

with only a single report on a custom ion mobility drift cell paired with a modified Orbitrap MS

3

currently in the literature.25 In contrast, we here present the coupling of an unmodified Thermo

4

Q-Exactive Orbitrap mass spectrometer to a commercially developed, fully modular

5

EXCELLIMS MA3100 atmospheric pressure drift tube ion mobility spectrometer. The DTIMS

6

module is equipped with dual ion gates, which not only serve to define the boundaries of the drift

7

region, but also enable several different modes of mobility selection and filtering,

8

accommodating the Orbitrap scan speeds. Several key mobility parameters, including drift tube

9

temperature, drift gas flow rate, and electric field strength in both positive and negative ion

10

modes were optimized. The effect of DTIMS gate pulse width on IM-MS resolving power,

11

together with the impact of fixed-gate versus scanned-gate acquisition modes on system

12

sensitivity, was also explored. Primary consideration was given to understanding Orbitrap

13

variables that determine maximum detector scan rate, such as injection time (IT) and automatic

14

gain control (AGC) settings, while investigating system limits of detection. Envisioned

15

applications for this system include rapid screening of complex combinatorial libraries and rapid

16

metabolomics phenotyping.

17

Experimental

18

Chemicals and Materials

19

Positive mode ion mobility calibration standards including 2,6-ditertbutyl pyridine

20

(≥97%), nicotinamide (≥99.5%), trihexylamine (≥96%), and negative mode standard citric acid

21

(≥99.5%) were purchased from Sigma Aldrich (St. Louis, MO). Positive mode standards were

22

mixed in a 1:1:1 ratio in 80:20 methanol/water at 25 ppm w/v (i.e. 130 pmol µL-1, 204 pmol µL-

23

1

24

concentration series of negative mode citric acid standard, ranging from 100 ppb to 100 ppm w/v

25

(~0.52–520 pmol µL-1), was prepared by serial dilution of a 1000 ppm w/v stock solution

26

dissolved in 80:20 methanol/water. Solutions containing isomeric Gly-Asp-Gly-Arg-Ser and Ser-

, and 93 pmol µL-1 of 2,6-ditertbutyl pyridine, nicotinamide, and trihexylamine, respectively). A



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Arg-Gly-Asp-Gly peptide sequences were prepared in 50:50 acetonitrile/water with 0.1% formic

2

acid to 1 mg mL-1 following the protocol provided with the Waters reverse peptide kit

3

(P#700005089), obtained from Waters Corporation (Milford, MA), and then mixed and diluted

4

to 100 ppm w/v (204 pmol µL-1). Sugar analytes D-(+)-raffinose pentahydrate (≥98%), D-(+)-

5

melezitose hydrate (≥99%), and D-(+)-melibiose (≥98%) were also procured from Sigma

6

Aldrich, and dissolved in 80:20 methanol/water as a 100 ppm w/v mixture (~168 pmol µL-1 for

7

D-(+)-raffinose and D-(+)-melezitose hydrates, and 292 pmol µL-1 for D-(+)-melebiose) with

8

≥2.5 molar excess of NaCl. HPLC grade methanol or acetonitrile organic solvents (Sigma

9

Aldrich) and ultra-pure 18.2 MΩ cm deionized water (Barnstead Nanopure Diamond, Van Nuys,

10

Ca) were used for all analyte solutions. High-purity nitrogen (99.998%) and ultra-zero grade

11

compressed air (99.998%) were acquired from Airgas Inc. (Atlanta, GA) and used as DTIMS

12

buffer gases.

13 14

Instrumentation and System Parameters

15

The EXCELLIMS MA3100 drift tube ion mobility spectrometer uses a traditional

16

stacked-ring electrode construction, with the cell body divided into a desolvation and a drift

17

segment, 6.25 cm and 10.55 cm long, respectively. For reference, the electrodes are thin steel

18

bands (width: ~4.5 mm, spacing: ~1 mm), all resistively-coupled to one another via printed

19

circuit board connections and secured to a rectangular ceramic support (50 mm x 25 mm). The

20

insulated ceramic block is temperature-regulated (≤250 ̊C) using resistive heating elements

21

positioned along the cell body and at the drift gas inlet. Operation potentials up to ±10,000 V

22

were applied to the desolvation cell inlet, resulting in a maximum linear electric field gradient of

23

~570 V cm-1 across the entire drift space.

24

Attached in front of the desolvation chamber is a source enclosure housing a “sheathless”

25

(i.e. without nebulizing gas) electrospray tip (50 µm i.d.), adjustable to a maximum potential of

26

±5000 V relative to the IMS inlet bias. The MA3100 design features two Bradbury-Nielsen ion


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shutters, situated at the entrance and the exit of the drift cell. The first and second ion gates are

2

floated at ~62.9 % and ~7.7 % of the operation potential, respectively. The first ion gate is pulsed

3

with a tunable symmetric potential of ±70 V during conventional drift mode operation. At the

4

end of the drift region and positioned directly behind the second ion gate/aperture grid is a

5

Faraday anode disk with a 6.4 mm diameter opening allowing partial ion transmission to the

6

entrance of the Orbitrap’s ion transfer capillary. Compressed air or nitrogen drift gas (≤3.0 L

7

min-1) is delivered through a heated input line at the drift cell terminus, passed as a symmetric

8

sheath-flow across the anode, and pumped out (≤3.5 L/min) through an exhaust port located at

9

the front of the desolvation cell via a diaphragm pump. Gas feed lines, high voltage electronics,

10

and multifunction data acquisition DAQ hardware are contained in the MA3100 peripheral

11

controller box connected to the DTIMS and Orbitrap computer system. Controller box

12

commands were issued using the EXCELLIMS VisIon software (version 1.2.0.31). Known drift

13

tube dimensions and experimentally-determined optimal settings for key mobility parameters in

14

positive and negative ion mode are outlined in Table 1.

15

The MA3100 module was mounted to the atmospheric pressure interface of a Thermo

16

Fisher Q-Exactive Orbitrap mass spectrometer. A schematic of the system configuration is

17

depicted in Figure 1. Ion transport past the DTIMS Faraday detector was governed by the

18

combined effect of the vacuum pull within the interface preceding the mass spectrometer inlet

19

and the electrostatic fields applied. The Q-Exactive MS Tune source voltages and sheath gas

20

flows were assigned “0” values (switched off) and the S-Lens RF level was held at 50 V. The ion

21

transfer capillary temperature was set to match the drift tube temperature (60-240 °C). Parameter

22

space experiments focused on the Orbitrap detector automatic gain control (AGC), injection time

23

(IT), and resolution settings that influenced the effective analytical cycle time. Unless otherwise

24

specified, the mass resolution was set at 17,500 to afford the fastest possible analyzer scan rate

25

(~12 Hz). During IM-MS analysis, AGC threshold was tuned to a minimum of 2.0E+04 ion

26

counts or a maximum of 5.0E+06 ion counts, while IT was varied from 100-2000 ms. The

27

number of microscans setting was left at 1, and the mass range was typically set between 50-500 7 ACS Paragon Plus Environment


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Da. For fragmentation studies with peptides using the high-energy collision dissociation (HCD)

2

cell, the normalized collision energy (NCE) was 20-45 V for an all ion fragmentation (AIF)

3

range centered at m/z = 400 Da. Orbitrap analysis was conducted using Thermo Xcalibur 2.6

4

software. ProteinProspector software version 5.17.1 (University of California, San Francisco,

5

USA) was used for assignment of peptide ion fragments. Most relevant MS settings are

6

summarized in Table 1.

7 8

5.3.3. DTIMS Ion Gating Schemes

9

The MA3100 is equipped to perform several analysis modes based on various trigger

10

configurations of the dual ion shutters. For “Faraday mode” acquisitions, the second ion gate was

11

held open at a set potential, thereby serving as an open aperture grid, while the first ion gate was

12

pulsed open/closed once per sweep scan and signal current was recorded at the DTIMS anode.

13

When operating with MS detection, the second ion gate was also utilized to select (or filter)

14

target ions using several distinct modes (Figure 2, Table 1). In “gated mode,” the second ion gate

15

was pulsed open following a delay after the first gate pulse, and signal for ions transmitted

16

through the fixed-gate time window was measured by the Orbitrap MS detector. Using the

17

alternative “scan mode,” the second gate pulse was scanned in sequential time bins across the

18

defined drift period in order to generate a complete mobility-mass dataset.

19

The gate pulse widths for each ion shutter ranged from 30 µs to the full mobility

20

acquisition period (≤50 ms). The first gate pulse width in all experiments was varied between 50-

21

600 µs; larger pulse widths resulted in a total loss of spectral resolution. In gated mode, the

22

second ion gate was typically pulsed open for a narrow 1-2 ms over the selected drift time

23

interval or mobility peak of interest. Scan mode functions required definition of the drift time

24

interval or scan duration, the second ion gate pulse width, and the step size of the scanned second

25

gate. Unless otherwise specified, the scan time window was set within a range of 4-16 ms over

26

the standard 20.3 ms mobility acquisition period, following a short delay (≤1 ms) from the first

27

ion gate trigger pulse (the period was defined as an uneven value to reduce periodic anode 8 ACS Paragon Plus Environment

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noise). DTIMS dwell time was set to 1015 ms, which equated to 50 Faraday acquisitions per gate

2

scan step for the set 20.3 ms scan period. Except during specific characterization experiments,

3

the first and second gate pulse widths were set equal to one another. The second gate was

4

stepped at time intervals ½ to ¼ the pulse width over the defined scan window to effectively

5

oversample, rendering scan-step-to-pulse-width ratios of 2:1 or 4:1. VisIon Faraday spectra were

6

generated from 20 second summations of full-period mobility scans, and mass spectra were

7

produced from total scan time averages of the Orbitrap TIC spectra. When ion mobility

8

separations were not desired, the DTIMS unit functioned as a passive guide for ion transmission

9

simply by floating both ion gates “open” at voltages defined by the drift cell potential gradient

10

(e.g. “open mode”).

11 12

Results and Discussion

13

AP-DTIMS Basic Performance Optimization

14

Performance of the AP-DTIMS unit was first characterized for key physical parameters

15

affecting ion mobility separation, namely drift electric field strength, drift gas temperature, and

16

drift gas flow rate. A systematic exploration of these parameters on attributes such as sensitivity

17

and resolution is presented in the supporting information (Figures S1-S3), but the main findings

18

are summarized here, with optimum parameters presented in Table 1. In positive ion mode, the

19

greatest resolving power (Rp≥70) and signal intensities were achieved using the highest

20

programmable operating potentials (9000-10,000 V). This resolving power is comparable to

21

some existing low-pressure drift tube instruments (i.e. Agilent 6560), but with a short, compact

22

drift tube. Maximum signal in negative mode was achieved at a slightly reduced DTIMS

23

operating potential (-7000 to -8000 V) to avoid signal distortion from electron currents on the

24

Faraday detector originating from ion source discharges. These distortions could also be

25

mitigated by decreasing the electrospray potential (-2200 V) relative to the positive mode value

26

(2600 V). Higher DTIMS temperatures (180-220 °C) assisted droplet desolvation while retaining 9 ACS Paragon Plus Environment


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resolving power, but temperatures in excess of 240 °C induced currents in the Faraday plate

2

detector and an artificial elevation of the spectral baseline by ~3 V. Drift gas flow rate, in

3

combination with DTIMS temperature, had the most influence on drift gas uniformity and signal

4

fidelity. Balancing a higher drift gas input (2.0-3.0 L min-1) with a lower exhaust pumping rate

5

(≤1.5 L min-1) had an optimal effect on signal, facilitating ion declustering and stabilizing gas

6

flow for ion transmission. High exhaust pump rates were prone to disrupt the electrospray

7

stability.

8 9

DTIMS Gate Pulse Width and Scan Step Functions

10

The contribution of various gate pulse widths on DTIMS-Orbitrap MS sensitivity and

11

resolving power was investigated to better understand the effect of scanning gate operation

12

parameters. Analysis of a citric acid standard was performed in negative ion mode using

13

different gate 1 and gate 2 pulse width ratios, and different scan step increments to control over-

14

or under-sampling. Figure 3A shows the Faraday responses for 4 ppm w/v (~21 pmol µL-1) citric

15

acid with gradually increasing gate #1 pulse widths. The mobility peaks showed increasing

16

abundances with near linearity until a gate #1 pulse width of approximately 300 µs. Beyond this

17

value, the peaks plateaued and broadened, further diminishing mobility resolving power. In

18

practice, increasing the gate #1 pulse width also extends the drift time by a commensurate

19

amount on the falling edge of the widening peak so the peaks in Figure 3 were aligned to a

20

centroid by subtracting ½ pulse width from the drift time. For Faraday mode, gate #1 pulse

21

widths of 75-150 µs were determined to provide the best balance between sensitivity and

22

resolving power.

23

The effect of gate #2 pulse operations relative to gate #1 was evaluated in scan mode.

24

Orbitrap parameters, further discussed in the next section, were set as follows: resolution=

25

17,500 [12 Hz], AGC= 5.0E+06, IT= 1000 ms. The gate #1 pulse width was set to 100 µs, while

26

gate #2 pulse widths were varied from 25-400 µs over a drift time scan window of 2.5 ms


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centered on the citric acid drift time (Figure 3B). For the shortest gate #2 pulse width (25 µs), no

2

signal was detected since the programmed pulse width was below the rated cut-off of the ion

3

shutter. Peak area was observed to increase with gate #2 width while resolution decreased for

4

gates wider than 100 µs, but resolving powers remained comparable to those observed from the

5

Faraday detector response (gate #1 and #2 at 100 µs: Rp ~60-70). For alignment, the citric acid

6

peaks were corrected for gate pulse width by subtracting ½ of the first gate pulse width and

7

adding ½ of the second gate pulse width. Improved timing correction for instances where the

8

pulse width of gate #2 was larger than gate #1 required addition of an extra adjustment factor

9

equal to (1/16)tgate#2, which is related to the scan step. As expected, shorter gate #2 pulse widths

10

relative to gate #1 resulted in lower intensity MS signals due to gate shutter “clipping” of ion

11

packets, with modest or no improvement to resolving power.

12

The impact of the scan step duration on signal was investigated independently, while

13

setting gate #1 and gate #2 pulse widths equal (100 µs). Gate scan steps were varied from 12.5

14

µs up to 200 µs for an IMS dwell time of 1015 ms, equating to 50 DTIMS acquisitions per scan

15

step over the 20.3 ms drift period. Orbitrap parameters were set as before (resolution: 17,500 [12

16

Hz], AGC: 5.0E+06, IT: 1000 ms). Figure 3C shows the citric acid MS signal using these

17

various scan steps, which corresponded to oversampling ratios of 8:1 (12.5 µs), 4:1 (25 µs), and

18

2:1 (50 µs), a matched sampling ratio of 1:1 (100 µs), and an undersampled ratio of 1:2.5 (250

19

µs). There was a minor drift time variance across peak apexes for the different scan steps, which

20

may be correlated with minute fluctuations in measured ion intensity over the different mobility

21

times. As expected, undersampling resulted in a dramatic loss of resolving power, with

22

oversampling being critical to refine peak shape when using gate #1 widths of 100 µs or larger.

23

An oversampling of 4:1 (25 µs) was observed to provide the best balance between adequate

24

sampling and overall speed of analysis.

25 26

Effect of Orbitrap Automatic Gain Control and Injection Time



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Apart from DTIMS gate pulsing schemes, the Orbitrap MS detector settings were the

2

other principal determinants of performance, as they potentially had a substantial influence on

3

sensitivity, resolution and analytical cycle time. Analyzer resolution, injection time, and

4

automatic gain control were examined while assessing DTIMS performance in both fixed-gate

5

and scan modes. The lowest available analyzer resolution (17,500) was chosen for all

6

characterization experiments in order to permit the fastest allowable acquisition rate (~12 Hz) for

7

the selected mass range (m/z = 50-500). The automatic gain control, which defined the threshold

8

for the target ion number, was set initially to the highest available value (5.0E+06) in order to set

9

a fixed injection time. The maximum injection time, or the nominal duration of ion accumulation

10

and the rate-limiting cycle step governing sensitivity, was adjusted between 100-2000 ms, values

11

that allowed for adequate analyte detection.

12

Figure S.4 shows a calibration curve for a citric acid concentration series as a function of

13

injection time for a fixed-gate acquisition, i.e. holding the DTIMS gate #2 open for a set window

14

at a specified drift time (gate #1 pulse width: 100 µs, gate #2 window: 2.5 ms). Interestingly, no

15

notable differences were observed when increasing Orbitrap injection times from 100 to 2000

16

ms, with the average signal intensities for ~110 total spectra being similar for each IT tested

17

(RSD ~3.6% excluding 100 ppb). It was initially expected that signal intensity would scale not

18

just with concentration, but also proportionately with the number of mobility peaks binned to the

19

C-trap prior to injection and mass analysis (see Supporting Information for more detail).

20

However, our results seem to indicate imperfect synchronization between mobility and mass

21

analysis. Presuming the Q-Exactive operated here by continuously scanning at ~12 Hz during

22

“zero” injection time bins, e.g. either measuring blank/incomplete spectra between injection

23

events for larger IT values or acquiring partial scans at lower IT values, a plausible consequence

24

would be the net signal average appearing equal across all injection times. Significantly, it must

25

be noted that despite an expected variation for extracted TIC peak intensity associated with each

26

injection event, the VisIon software reports this total average signal intensity derived from the

27

MS TIC data, as represented in Figure S.4. 12 ACS Paragon Plus Environment

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Based on the linear portion of the citric acid concentration curve (0.1-10 ppm), the limit

2

of detection was estimated to be approximately ~800 ppb w/v (~4 pmol µL-1). Setting a fixed

3

Orbitrap injection time (IT: 1000 ms), the same trend in the concentration curve was observed

4

when switching from DTIMS fixed-gate acquisition to scan mode acquisition, i.e. scanning

5

DTIMS gate #2 pulses across the full drift period (gate #1 & #2 pulse widths: 100 µs, scan step:

6

25 µs). In this mode, overall intensities were lower by 2-3 orders of magnitude. The loss in

7

sensitivity here while scan-stepping DTIMS gate #2 was expected, as the peak signal previously

8

measurable in a single Orbitrap scan using fixed-gate mode was now divided among smaller

9

sequential DTIMS time bin segments now defined by the gate #2 pulse width and scan step.

10

The AGC parameters had a more pronounced effect at higher sample concentrations and

11

specific injection times. Figure S.5 shows overlaid MS drift traces for a scan mode acquisition of

12

25 ppm w/v citric acid using low (2.0E+04 counts) or high (5.0E+06 counts) AGC values with

13

the nominal injection time set to IT = 1000 ms. With the high AGC setting, injection time

14

remained stable at 1000 ms, as the total measured ion intensity was always below the target AGC

15

threshold for the tested analyte concentration. However, for the same analyte concentration, it

16

was possible to surpass the lower AGC ion count threshold with an injection time under 1000

17

ms, which resulted in erratic IT fluctuations down to several hundred milliseconds during

18

measurements. This variable decrease in IT for the lower AGC setting corresponded to an

19

effective increase in the number of citric acid peak detection events during Orbitrap scan cycles,

20

indicated by the fine structure in the broad red MS trace. Despite essentially equivalent peak

21

areas for both signal traces, more Orbitrap scan cycles featuring analyte injection/detection

22

translated again to a greater average signal intensity reported for the lower AGC setting (red

23

trace) in Figure S.5.

24 25

DTIMS-Orbitrap MS Applications

26

Several test analyte sets were investigated to explore system performance in applications

27

involving isomer separation and identification. Figure 4A shows the Faraday response and 13 ACS Paragon Plus Environment


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associated mass spectrum for a 100 ppm w/v mixture of two reverse peptides (Ser-Asp-Gly-Arg-

2

Gly and Gly-Arg-Gly-Asp-Ser) collected using optimized mobility parameters and scan mode

3

acquisition (gate #1 and #2 pulse width: 150 µs, scan step: 50 µs, scan window: 5-15 ms). Three

4

species were detected in the Faraday trace, representing the singly charged peptide monomers

5

(m/z = 491.221) overlapping at td = 12.25 ms and the doubly protonated monomers (m/z =

6

246.114) of GRGDS and SDGRG at td = 7.12 ms and 7.45 ms, respectively. The [M+2H]2+

7

peptide species, having reported collision cross-section areas of Ω = 222.7 Å2 and Ω = 211.7 Å2

8

at 250˚C in N2,26 were clearly resolved to baseline by AP-DTIMS (Rp~72) with roughly double

9

the resolving power possible for these ions using TWIMS (Rp>36). Isomer identities were

10

verified by drift time measurements for single-component peptide standards, and were further

11

confirmed by high-energy collision dissociation (HCD) MS/MS experiments of the mobility-

12

resolved sequence isomers. Figure 4B-C shows the characteristic HCD MS/MS spectra for the

13

peptide sequences with their unique fragment assignments.

14

In addition to the reverse peptide sequences, a simple carbohydrate mixture of sugar

15

isomers and structural homologues was examined. Analyte solutions were concentrated at 100

16

ppm w/v with a 2.5x excess of NaCl salt to facilitate positive mode ionization via sodium adduct

17

formation. Figure 5A-D depicts VisIon spectral data for both Faraday and MS detectors obtained

18

using a DTIMS scan mode over a 4 ms scan acquisition window. The 200 µs gate pulse widths

19

used resulted in a small reduction in Rp to benefit sensitivity and a large solvent signal recorded

20

in the Faraday channel. Within the drift time zone of 9-13 ms, four distinct signals were

21

observed. In order of increasing td, the carbohydrate peaks were assigned to [M+Na]+ for the

22

disaccharide D-(+)-melibiose (m/z = 365.105) at td = 9.94 ms, and the trisaccharide isomers D-

23

(+)-melezitose (m/z = 527.158) and D-(+)-raffinose (m/z = 527.158) a td = 11.70 ms and td =

24

12.19 ms, respectively. The peak order was consistent with mobility distribution reports from the

25

literature 15, 27 and was verified by injecting single-component solutions of the standards. The one

26

peak not assigned to a sugar at td = 10.70 ms is a suspected contaminant tentatively identified as

27

the sodiated adduct of decamethylcyclopentasiloxane (m/z = 393.082), a common ESI 14 ACS Paragon Plus Environment

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1

background ion. The m/z vs. drift time plot conveyed an additional approach to visualize the

2

data, portraying the nested IM-MS distribution and precursor ion trend-lines. The ellipses drawn

3

on the plot demark peaks for the carbohydrates and contaminant ion, while filtering any other

4

background signal overlapping in the same mass/mobility region. This data show the power of

5

AP-DTIMS-Orbitrap MS for discriminating isobaric analytes in complex chemical systems.

6 7

Conclusion

8

This work involved characterization of a dual-gate AP-DTIMS-Orbitrap system,

9

showcasing its performance capabilities to date. The DTIMS unit afforded the greatest separation

10

efficiency near the maximum operation potential of 10,000 V while using gas temperatures ≥200

11

˚C and high drift gas flow rates of 2.0-3.0 L min-1 balanced with lower exhaust pump rates of

12

0.5-1.5 L min-1. The effects of different ion mobility gating schemes on sensitivity and resolving

13

power were thoroughly investigated. It was found that resolving power (Rp≤70) was mostly

14

determined by the initial DTIMS gate pulse width (50-150 µs), and during scan mode analysis,

15

the scan step bin width for the second ion gate if pulse width was set equal to the first gate.

16

Likewise, sensitivity and throughput were primarily dictated by DTIMS gate pulse width, while

17

Orbitrap parameters governing analytical cycle had a subtler and more complex influence.

18

Orbitrap variable injection times did not appear to significantly alter intensity levels at high gain

19

settings, but had a more pronounced impact on average signal intensity at the lowest gain settings

20

and higher analyte concentrations. The optimized AP-DTIMS-Orbitrap MS parameter settings

21

were used to successfully resolve simple mixtures of peptide and sugar isomers, and HCD

22

MS/MS was performed to further confirm peptide identities based on characteristic

23

fragmentation spectra. Future experiments will include collision cross section measurements and

24

peak capacity estimations for the IM-MS platform, with the ultimate outlook for this system

25

involving implementation of multiplexed sampling approaches. Further improvements in



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1

resolving power through the use of longer drift tubes, more effective ion gating, and higher drift

2

voltages, are also envisioned.

3 4 5 6

Acknowledgements This work was supported by the National Science Foundation and the NASA Astrobiology Program, under the NSF Center for Chemical Evolution (CHE-1504217).

7


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Ruotolo, B. T.; McLean, J. A.; Gillig, K. J.; Russell, D. H. J. Mass Spectrom. 2004, 39, 361-367. May, J. C.; Goodwin, C. R.; Lareau, N. M.; Leaptrot, K. L.; Morris, C. B.; Kurulugama, R. T.; Mordehai, A.; Klein, C.; Barry, W.; Darland, E.; Overney, G.; Imatani, K.; Stafford, G. C.; Fjeldsted, J. C.; McLean, J. A. Anal. Chem. 2014, 86, 2107-2116. Valentine, S. J.; Kulchania, M.; Barnes, C. A. S.; Clemmer, D. E. Int. J. Mass Spectrom. 2001, 212, 97-109. Valentine, S. J.; Kurulugama, R. T.; Bohrer, B. C.; Merenbloom, S. I.; Sowell, R. A.; Mechref, Y.; Clemmer, D. E. Int. J. Mass Spectrom. 2009, 283, 149-160. Pringle, S. D.; Giles, K.; Wildgoose, J. L.; Williams, J. P.; Slade, S. E.; Thalassinos, K.; Bateman, R. H.; Bowers, M. T.; Scrivens, J. H. Int. J. Mass Spectrom. 2007, 261, 1-12. Matz, L. M.; Hill Jr, H. H.; Beegle, L. W.; Kanik, I. J. Am. Soc. Mass Spectrom. 2002, 13, 300-307. Allen, S. J.; Giles, K.; Gilbert, T.; Bush, M. F. Analyst 2016, 141, 884-891. Gillig, K. J.; Chen, C.-H. Mass Spectrometry 2014, 3, S0032. Gillig, K. J.; Ruotolo, B. T.; Stone, E. G.; Russell, D. H. Int. J. Mass Spectrom. 2004, 239, 43-49. Hill Jr., H. H.; Siems, W. F.; St. Louis, R. H.; G., M. D. Anal. Chim. Acta 1990, 62, 1201A-1209A. Kanu, A. B.; Gribb, M. M.; Hill, H. H., Jr. Anal. Chem. 2008, 80, 6610-6619. Revercomb, H. E.; Mason, E. A. Anal. Chem. 1975, 47, 970-983. Siems, W. F.; Wu, C.; Tarver, E. E.; Hill, H. H.; Larsen, P. R.; McMinn, D. G. Anal. Chem. 1994, 66, 4195-4201. Kaplan, K.; Graf, S.; Tanner, C.; Gonin, M.; Fuhrer, K.; Knochenmuss, R.; Dwivedi, P.; Hill, H. H. Anal. Chem. 2010, 82, 9336-9343. Clowers, B. H.; Hill, H. H., Jr. Anal. Chem. 2005, 77, 5877-5885. Henderson, S. C.; Valentine, S. J.; Counterman, A. E.; Clemmer, D. E. Anal. Chem. 1999, 71, 291-301. Clowers, B. H.; Ibrahim, Y. M.; Prior, D. C.; Danielson, W. F.; Belov, M. E.; Smith, R. D. Anal. Chem. 2008, 80, 612-623. Ibrahim, Y. M.; Baker, E. S.; Danielson, W. F.; Norheim, R. V.; Prior, D. C.; Anderson, G. A.; Belov, M. E.; Smith, R. D. Int. J. Mass Spectrom. 2015, 377, 655-662. May, J. C.; McLean, J. A. Anal. Chem. 2015, 87, 1422-1436. Belov, M. E.; Clowers, B. H.; Prior, D. C.; Danielson Iii, W. F.; Liyu, A. V.; Petritis, B. O.; Smith, R. D. Anal. Chem. 2008, 80, 5873-5883. Kwasnik, M.; Caramore, J.; Fernández, F. M. Anal. Chem. 2009, 81, 1587-1594. Clowers, B. H.; Belov, M. E.; Prior, D. C.; Danielson, W. F.; Ibrahim, Y.; Smith, R. D. Anal. Chem. 2008, 80, 2464-2473. Zhang, X.; Knochenmuss, R.; Siems, W. F.; Liu, W.; Graf, S.; Hill, H. H. Anal. Chem. 2014, 86, 1661-1670. Tang, X.; Bruce, J. E.; Hill, H. H. Rapid Commun. Mass Spectrom. 2007, 21, 1115-1122. Ibrahim, Y. M.; Garimella, S. V. B.; Prost, S. A.; Wojcik, R.; Norheim, R. V.; Baker, E. S.; Rusyn, I.; Smith, R. D. Anal. Chem. 2016, 88, 12152-12160. Wu, C.; Siems, W. F.; Klasmeier, J.; Hill, H. H. Anal. Chem. 2000, 72, 391-395. 17 ACS Paragon Plus Environment


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Figure 1: Illustration of the AP-DTIMS-Orbitrap MS instrument configuration.



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TABLE 1: Summary of key system parameters

2 20 ACS Paragon Plus Environment

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Figure 2: Illustration of the DTIMS dual-gate modes of operation for ion selection and filtering. The corresponding MS TIC traces for gate #2 operations using gated mode and scan mode IMMS acquisition schemes are shown.



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Figure 3: (A) Faraday response for 4 ppm w/v citric acid in 80:20 methanol/water for increasing DTIMS gate #1 pulse widths. (B) MS signal for 10 ppm w/v citric acid for increasing DTIMS gate #2 pulse widths over a 2.5 ms scan window. The gate #1 pulse width was 100 µs and the scan step was 25 µs. (C) MS signal for 10 ppm w/v citric acid for increasing DTIMS gate #2 scan step ratios over a 2.5 ms scan window. Gate #1 and #2 pulse widths were set equal at 100 µs. The number of data points per peak is plotted with each curve trace. For (A-C), DTIMS operation potential was -7000 V, drift gas temperature was 200 ˚C, and drift gas flow rate and exhaust pump rate were 2.0 L min-1 and 0.5 L min-1, respectively. 22 ACS Paragon Plus Environment

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Figure 4: Scan mode averaged mass spectrum and Faraday response (inset) for 100 ppm w/v of mixture of [Ser-Asp-Gly-Arg-Gly] and [Gly-Arg-Gly-Asp-Ser] peptide sequence isomers in 50:50 acetonitrile/water with 0.1% formic acid (A). All ion fragmentation HCD of the separated peptide isomers using a normalized collision energy of 20 V, where individual [M+2H]+ species were isolated by ion mobility before fragmentation (B and C). Asterisks (*) denote identified sidechain fragments of serine, aspartic acid, and arginine. (DTIMS operation potential: 10,000 V, drift gas temperature: 220 ˚C, drift gas flow rate: 3.0 L min-1, exhaust pump rate: 1.5 L min-1; gate #1 and #2 pulse width: 150 µs; scan step: 50 µs). 23 ACS Paragon Plus Environment


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Figure 5: AP-DTIMS-Orbitrap MS analysis for 100 ppm solution (w/v in 50:50 methanol/water) of 3 saccharides: D-(+)-melibiose, D-(+)-melezitose, and D-(+)-raffinose. Panels show the extracted mass spectra for each mixture component (A), the corresponding Faraday response (B), a map of the chemical space with m/z plotted as a function of drift time (C), and the extracted MS total ion chronograms (D). (gate #1 & #2: 200 µs, scan step: 50 µs; AGC: 5.0E+06, IT: 1000 ms, Resolution: 35,000).


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Figure 1: Illustration of the AP-DTIMS-Orbitrap MS instrument configuration. 195x144mm (150 x 150 DPI)



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Figure 2: Illustration of the DTIMS dual-gate modes of operation for ion selection and filtering. The corresponding MS TIC traces for gate #2 operations using gated mode and scan mode IM-MS acquisition schemes are shown. 151x115mm (150 x 150 DPI)


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Figure 3: (A) Faraday response for 4 ppm w/v citric acid in 80:20 methanol/water for increasing DTIMS gate #1 pulse widths. (B) MS signal for 10 ppm w/v citric acid for increasing DTIMS gate #2 pulse widths over a 2.5 ms scan window. The gate #1 pulse width was 100 µs and the scan step was 25 µs. (C) MS signal for 10 ppm w/v citric acid for increasing DTIMS gate #2 scan step ratios over a 2.5 ms scan window. Gate #1 and #2 pulse widths were set equal at 100 µs. The number of data points per peak is plotted with each curve trace. For (A-C), DTIMS operation potential was -7000 V, drift gas temperature was 200 ˚C, and drift gas flow rate and exhaust pump rate were 2.0 L min-1 and 0.5 L min-1, respectively. 214x342mm (150 x 150 DPI)



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Figure 4: Scan mode averaged mass spectrum and Faraday response (inset) for 100 ppm w/v of mixture of [Ser-Asp-Gly-Arg-Gly] and [Gly-Arg-Gly-Asp-Ser] peptide sequence isomers in 50:50 acetonitrile/water with 0.1% formic acid (A). All ion fragmentation HCD of the separated peptide isomers using a normalized collision energy of 20 V, where individual [M+2H]+ species were isolated by ion mobility before fragmentation (B and C). Asterisks (*) denote identified sidechain fragments of serine, aspartic acid, and arginine. (DTIMS operation potential: 10,000 V, drift gas temperature: 220 ˚C, drift gas flow rate: 3.0 L min-1, exhaust pump rate: 1.5 L min-1; gate #1 and #2 pulse width: 150 µs; scan step: 50 µs). 157x191mm (150 x 150 DPI)


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Figure 5: AP-DTIMS-Orbitrap MS analysis for 100 ppm solution (w/v in 50:50 methanol/water) of 3 saccharides: D-(+)-melibiose, D-(+)-melezitose, and D-(+)-raffinose. Panels show the extracted mass spectra for each mixture component (A), the corresponding Faraday response (B), a map of the chemical space with m/z plotted as a function of drift time (C), and the extracted MS total ion chronograms (D). (gate #1 & #2: 200 µs, scan step: 50 µs; AGC: 5.0E+06, IT: 1000 ms, Resolution: 35,000). 228x189mm (150 x 150 DPI)



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Proposed TOC Graph 84x47mm (96 x 96 DPI)


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Atmospheric Pressure Drift Tube Ion Mobility-Orbitrap Mass

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