Pulsed Nano-ESI Atmospheric-Pressure Ion ... - ACS Publications

Subscriber access provided by CORNELL UNIVERSITY LIBRARY

Article

Pulsed nano-ESI Atmospheric-Pressure Ion Mobility Mass Spectrometry with Enhanced Ion Sampling William P McMahon, Arjuna Subramanian, Carina S. Minardi, Rohan Dalvi, and Kaveh Jorabchi Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.6b03395 • Publication Date (Web): 26 Oct 2016 Downloaded from http://pubs.acs.org on October 26, 2016

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a free service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are accessible to all readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

Analytical Chemistry 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.

Page 1 of 29

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Analytical Chemistry

Pulsed nano-ESI Atmospheric-Pressure Ion Mobility Mass Spectrometry with Enhanced Ion Sampling William P. McMahon, Arjuna Subramanianǂ, Carina S. Minardi†, Rohan Dalvi‡, and Kaveh Jorabchi*

Department of Chemistry, Georgetown University, Washington, DC 20057

*

Corresponding author: [email protected]

ǂ

Currently at Princeton University, Princeton, NJ

†

Currently at Yale University School of Public Health, New Haven, CT

‡

Currently at Montgomery Blair High School, Silver Spring, MD

ACS Paragon Plus Environment


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ABSTRACT Ion mobility-mass spectrometry (IM-MS) has gained considerable attention for detection of clusters and weakly-bound species created by electrospray ionization (ESI). Atmosphericpressure (AP) IM-MS offers an advantage in these studies compared to its low-pressure counterpart, owing to soft introduction of ions into the mobility cell with minimal ion activation. Here, we report new approaches to improve the sensitivity and soft ion introduction in AP-IMMS. For the former, we demonstrate enhanced aerodynamic sampling of ions from the mobility cell into the MS using pulsed-field sampling. In this approach ions are driven toward the MS and the field is shut down once the ions reach the vicinity of the MS inlet orifice. The pulsed-field operation provides arrival times without the need for an exit ion gate in the mobility cell and leads to improvements in sensitivity of up to one order of magnitude. For soft ion generation, we report a pulsed nano-ESI source to introduce a packet of ions into the room-temperature mobility cell without induced desolvation. Further, we demonstrate the application of the pulsed nano-ESI AP-IM-MS with enhanced ion sampling for detection of solvent clusters of amines and peptide aggregates.

2


Page 2 of 29

Page 3 of 29

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


INTRODUCTION Gentle formation of gas-phase ions by electrospray ionization (ESI) continues to inspire development of new analytical strategies. In this arena, ion mobility-mass spectrometry (IM-MS) has garnered substantial attention in recent years.1 The rapid growth of this technique stems from its ability to provide information on both size (collisional cross section) and m/z of ions, offering insights into the conformation of biomolecules and formation of clusters and aggregates. The majority of ESI-IM-MS techniques utilize ion mobility separations in the low-pressure region located downstream of the atmospheric-pressure (AP) ion sampling orifice of the MS. Ions are typically formed in atmospheric pressure and desolvated during transmission into the low-pressure region, where ion lenses focus the ions into and out of the mobility cell. The transfer from atmospheric pressure to low pressure, however, can alter ion properties. Clustering with solvent can occur during supersonic expansion of the ion beam into the low-pressure region.2 Moreover, acceleration of ions during ion transfer and ion injection into the mobility cell results in ion heating, and can induce cluster fragmentation.3 In contrast, mobility separations at atmospheric pressure offer insights into ion properties prior to the MS, thereby minimizing ion sampling effects. Multiple ESI-AP-IM-MS platforms have been developed to explore this advantage.4-6 In particular, a dual-gate operation mode has enabled ion arrival time measurements independent of the acquisition speed of the MS, allowing coupling of AP-IM spectrometry to a variety of mass spectrometers with minimal instrumental modifications.4,7 Recent efforts to improve the sensitivity in ESI-AP-IM-MS have focused on enhancing duty cycle by multiplexing techniques.8-10 The transfer of ions from IM cell to the MS, however, has received less attention. It is of note that the ion acceptance diameter for AP-IM cell is significantly larger than those offered by MS orifices. The minimal loss of ions between the ESI 3



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

and the AP-IM cell enables sensitivity improvements by operation of ESI at higher flow rates in ESI-AP-IM spectrometry.11,12 However, the wide radial distribution of ions within the mobility cell creates challenges for efficient transfer of ions from AP-IM cell into the MS. Ideally, the ions should be focused into the MS inlet at the exit of the IM cell. Yet, electrostatic and electrodynamic ion focusing techniques are not effective at atmospheric pressure because of the high ion-neutral collision frequency. Therefore, a significant portion of the ions are lost in transfer from the AP-IM cell to the MS. Focusing by flow dynamics has been shown to be more effective than electrostatic focusing for ion sampling from atmospheric pressure.13-15 Accordingly, a flared MS inlet has been used in one study to improve the sensitivity of AP-IM-MS.4 The extent of sensitivity improvement was not characterized for mobility-separated ions in this study, although a factor of 2-5 improvement was expected based on performance of the inlet with a range of other ions sources.16 It is of note that a later investigation using a Venturi-assisted ESI did not show a significant improvement in sensitivity with the flared inlet relative to the standard tube inlet,17 highlighting the difficulties in broad application of these methods. An alternative approach for enhancement of ion transmission from atmospheric pressure to low pressure is pulsed-field sampling. Using AP-matrix assisted laser desorption ionization-MS, it has been shown that limiting the duration of ion extraction under a strong electric field leads to enhanced entrainment of ions in the aerodynamically sampled flow and improved sensitivities.18,19 Here, we report pulsed-field ion sampling of mobility separated ions and investigate sensitivity enhancements with this approach. In our experiments, ion packets are driven toward the MS in the AP-IM cell and the electric field in the mobility cell is turned off once the ions are in the vicinity of the MS inlet. The efficient aerodynamic sampling in these 4


Page 4 of 29

Page 5 of 29

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


conditions leads to major improvements in sensitivity. Pulsed and non-linear electric fields in the IM cell have been used in the past to induce axial and radial compression of ions.20 However, high electric fields are needed in this approach, resulting in reduction of ion transmission efficiency from the IM cell into the MS. In contrast, our approach relies on aerodynamic effects at low-electric fields to improve ion transmission to the MS. We also demonstrate that the pulsed-field sampling offers a new approach for measuring ion arrival times without the use of an ion exit gate in the mobility cell, further eliminating sources of ion loss. Moreover, to capitalize on softness of AP-IM, we utilize a pulsed nano-ESI for generating ion packets. In conventional ESI-AP-IMS, a desolvation area is incorporated for ESI droplets and the ions are introduced as a packet into the IM cell using an injection ion gate.21,22 The desolvation reduces ion clustering and leads to more compact ion packets. However, weaklybound species may also be dissociated as a result of desolvation, particularly when thermal desolvation at higher temperatures is used. Furthermore, the presence of water has been shown to help preserve some non-covalent interactions as demonstrated for ubiquitin dimerization.23 In our experiments, a pulsed nano-ESI replaces the ion injection gate, creating a soft ion generation scheme without induced thermal desolvation. The pulsed nano-ESI AP-IM-MS technique in conjunction with improved ion sampling is applied to investigate formation of clusters from peptides and amines.

Experimental AP-IM-MS. The experimental setup is depicted in Figure 1. A 0.5 mL polypropylene sample tube (Eppendorf, Hamburg, Germany) was sealed inside a 4 mL screw threaded glass vial 5



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

(VWR, Radnor, PA, USA) with a cap containing a septum. The vial was pressurized (10 psig) to ensure a constant supply of solution to the spray. A platinum wire (0.25mm dia - 99.997%, Alfa Aesar) was immersed into the sample to charge the solution. Sample was fed through a 40-cm fused silica capillary (id:100 µm, od: 360 µm, IDEX, Lake Forest, IL, USA) into a borosilicate pulled glass tip (id: 5 µm ±20%, WPI, Sarasota, FL, USA). The 3-cm mobility region consisted of 5 stainless steel rings (id: 1.0 cm, od: 3.0 cm, thickness: 1.5 mm) separated by 3.0 mm Delrin spacers. The last ring was positioned 11 mm from the end of the cell to allow for introduction of nitrogen into the cell. The orthogonally introduced nitrogen flow was distributed across the cell using a 24-mm id, 27-mm od Delrin guard spacer terminating 2.5 mm prior to the end of the cell. The rings were resistively coupled with 200 kΩ resistors while the last ring connected to the ground via a 600 kΩ resistor. An acrylic outer casing (id: 3.05 cm, od: 3.79 cm) held the rings and spacers in place and sealed the mobility region to the ESI emitter, positioning the spray tip flush with the downstream surface of the first ring. The desolvation plate of the q-TOF MS (QStar XL MS/MS, Sciex, Concord, ON, Canada) was removed and the mobility cell was sealed to the flat sampling plate of the MS (0.25 mm sampling orifice) using a Viton O-ring. 0.7 L/min nitrogen gas (99.999%) was introduced at the end of the mobility region. 0.5 L/min of the flow was sampled into the MS while 0.2 L/min exited through the rear of the cell, serving as the mobility buffer gas. For corona discharge experiments, the ion source region was modified by replacing the ESI emitter with a stainless steel needle as shown in Figure S1. A grounded ring electrode was positioned between the tip of the needle and the first ring of the mobility region to assist in production of discharge at relatively low voltages applied to the needle (-1.3 kV).

6


Page 6 of 29

Page 7 of 29

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Pulsing Electronics and Data Acquisition. As shown in Figure 1, three high voltage solidstate relays (Panasonic AQV258) were used to control skimmer, solution, and mobility cell voltages. The timing sequence was generated by a microprocessor (Arduino Due - Torino, Italy). The digital outputs from the microprocessor were conditioned using a variable resistor followed by an operational amplifier to drive the solid state relays. The actual voltages applied to all elements were monitored using digital oscilloscopes. The mobility cell voltage showed rise and fall times of 25 and 60 µs, respectively, while the skimmer pulse had rise and fall times of 13 and 490 µs, respectively. Data were collected by measuring mass spectra as a function of ion arrival times. Spectra were collected with an MS integration time of 1 s per spectrum. The arrival time was scanned with 100 µs steps and 5-15 seconds of spectra collection for each step. Ion intensities were then extracted from the spectra and averaged across the spectra collection time to produce an ion intensity value for each arrival time. The plots of ion intensity versus arrival time were constructed to show arrival curves for ions. Chemicals and Reagents. Analytical solutions were made in 1 mM HCl as solvent. Details regarding chemicals and sample preparation can be found in Supporting Information. SAFETY CONSIDERATIONS The use of high voltage presents an electrical shock hazard – care must be taken to properly shield from accidental exposure. Additionally, standard safety protocol must be followed for all chemicals. Particular care must be taken in handling of iodomethane and 1aminohexane as they both exhibit moderate to high toxicity.

RESULTS and DISCUSSION 7



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Ion Sampling into the MS and Arrival Time Measurements. In atmospheric-sampling mass spectrometers, ions are typically guided toward the MS inlet using electrostatic fields. However, the presence of a high electric field close to the MS inlet hampers entrainment of the ions in the sampled flow, resulting in loss of ions to the exterior surfaces of the MS orifice and the interior surface of the ion transfer channel.19 To improve the sensitivity of ESI-AP-IM-MS, we investigated the effectiveness of pulsed-field ion sampling by comparing the sensitivity between two operating modes for the experimental setup shown in Figure 1: 1) continuous-field ion sampling mode, and 2) pulsed-field ion sampling mode. The former represents conventional ion sampling in AP-IM-MS where ions are sampled under a continuous electric field at the MS inlet, while the latter incorporates a pulsed field in the sampling area to enhance aerodynamic ion sampling at reduced field strengths. To ensure that the observed effects are related to ion sampling, ions are generated and introduced into the IM cell in identical conditions using a pulsed nano-ESI in both modes. The pulse sequences for the two modes are illustrated in Figure 2 for one cycle at an operating frequency of 25 Hz. In continuous-field operation, the solution voltage is held constant. The high voltage relay for the mobility cell is opened for 500 µs, grounding the mobility cell and inducing a significant potential difference between the electrospray tip and the cell. A pulse of charged droplets is generated within this time period, creating ions, which are then separated once the mobility cell voltage is established by closing the relay. The skimmer is gated with a delay relative to the start of the mobility separations (Figure 2a) to measure the ion arrival times. The delay is scanned and reflects the arrival time of the ions because no ions are detected after the gate is closed. However, note that the skimmer gate passes all ions arriving prior to the set delay. Accordingly, the ion sampling occurs in a cumulative fashion and ion 8


Page 8 of 29

Page 9 of 29

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


arrival curves follow a sigmoidal shape in this mode with the inflection point marking the arrival time for the whole ion packet. Figure 3 illustrates the sigmoidal ion arrival curve in continuousfield mode for trimethylhexyl ammonium ion. We have selected this ion for characterizing the ion sampling because quaternary ammonium ions are not susceptible to significant solvent clustering,24,25 thereby minimizing clustering effects in conditions where no desolvation is applied. The pulse sequence for the pulsed-field mode is depicted in Figure 2b. Ion generation is similar to that in continuous-field mode. However, the electric field for ion-mobility separations is applied for a set amount of time, followed by a pulsed-field sampling time during which the mobility field is turned off. The solution voltage is also turned off during pulsed-field sampling time to prevent formation of a spray. In this mode, ions are driven toward the MS for the set duration of the mobility electric field. Once the mobility field is turned off, only ions that have travelled far enough to reach the aerodynamic sampling area of the MS are detected. Therefore, ion arrival curves are measured by monitoring ion intensity as a function of mobility field-on time after spray generation. The skimmer gate is closed after the pulsed-field sampling time and the mobility field is turned on for the remainder of the cycle to purge all ions out of the cell prior to the start of the next cycle. Figure 3 contrasts the ion arrival curves in pulsed-field mode to that in continuous-field mode. A number of differences between the two modes are observed. First, ions are detected earlier with pulsed-field sampling, evident from an earlier rise in intensity compared to that observed in continuous-field mode. For ions to be detected in the continuous-field mode, they have to travel to the skimmer before the voltage is raised to block ions (see Figure 2a). However, in the pulsed-field mode, ions only need to reach the vicinity of the sampling orifice of the MS 9



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

during the on time of the mobility field. The flow into the MS then samples the ions while the mobility field is off. In other words, the sampling point is extended into the IM cell and the effective travel path for detection of ions is shorter in the pulsed-field mode. The second and the more drastic difference between the two operating modes is observed in the shape of the ion arrival curves. In the pulsed-field mode, the ion arrival curves appear as a peak superimposed on a sigmoidal curve. To explain this observation, we consider the aerodynamic sampling of ions at the end of the IM cell. The gas flow into the MS orifice entrains ions located further from the orifice in both axial and radial directions.19 When the mobility field is turned off in the pulsed field-mode, a weak electric field (18-24 V/cm) in the opposite direction of the mobility field is established by the voltage on the sampling orifice and the grounded last ring of the mobility cell. Therefore, ions experience repulsion by the sampling plate while being carried by the flow into the MS. These opposing effects, along with the time allowed for sampling (pulsed-field time), define an aerodynamic sampling volume upstream of the MS orifice. Now we consider the ion arrival curves in pulsed-field mode (Figure 3). Ions are driven toward the aerodynamic sampling volume during the on time of the mobility field. An increase in the ion intensity is observed as the ion packet is moved into the sampling volume with longer field duration. The peak ion intensity is observed when the mobility field is turned off at the maximal spatial overlap between the ion packet and the sampling volume. At a longer field-on duration the ion packet is pushed out of the optimum sampling volume, leading to reduction in observed ion intensity. At sufficiently long field-on durations, the entire ion packet is sampled under the influence of the mobility electric field, similar to the continuous-field mode. Therefore, the ion intensity in the pulsed-field mode eventually converges to that of the continuous-field 10


Page 10 of 29

Page 11 of 29

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


mode at long field-on durations. In summary, the passage of the ions across the sampling volume in the pulsed-field mode provides partial sampling (in contrast to cumulative sampling) of the ion packet, leading to the appearance of a peak in arrival curves. The position of the peak apex reflects the arrival time of the ion packet in pulsed-field mode. It is noteworthy that the sampling volume in pulsed-field mode is analogous to a pulsed exit gate in the dual-gate IM-MS,7 but without a physical ion gate in the mobility region. Therefore, similar to the dual-gate IM-MS, pulsed-field ion sampling offers an avenue for measuring the arrival time of ions at the end of the IM cell, facilitating AP-IM coupling to MS. This capability is particularly useful for MS instruments where the data acquisition time is longer than the time scale of ion mobility separation (e.g. ion trap analyzers). Effect of Pulsed-field Sampling Time. Figure 3 also demonstrates that the peak heights increase and the ions are detected earlier as the pulsed-field sampling time is increased from 0 ms (continuous-field) to 8 ms. However, the arrival curves show minimal differences between 8 and 11 ms pulsed-field times. Longer ion sampling durations with longer pulsed-field times allow ions located farther from the MS orifice (both radially and axially) to travel with the sampled flow and be detected. In other words, the aerodynamic sampling volume grows with the pulsed-field time, leading to sampling of a larger portion of the ion packet. However, the aerodynamic pull towards the MS is weakened with the distance from the orifice. Therefore, if the ion is too far, the repulsive force of the weak electric field overcomes the aerodynamic pull and the ion is driven away from the orifice regardless of the sampling time. Consequently, the balance between the repulsion by the weak field and attraction by the flow field into the MS creates a maximum sampling volume which appears to be reached between 8 and 11 ms pulsedfield sampling time in our experiments. It is of note that the peak intensity in Figure 3 for the 11



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 12 of 29

pulsed-field mode with 11 ms sampling time is about 10 fold higher than that at the plateau section of the sigmoidal arrival curve for continuous-field mode, highlighting a dramatic improvement in sensitivity as a result of more efficient ion sampling. Role of Desolvation in Pulsed-field Ion Sampling. A question that may arise regarding sensitivity enhancements in the pulsed-field mode is: does a longer sampling time simply provide more time for desolvation, leading to greater ion intensity? We do not expect desolvation to play a major role in the experiments above because the quaternary ammonium ion is not susceptible to extensive solvent clustering.25 To further verify that the observed effects are mainly related to ion sampling, we modified the ion source by replacing the ESI emitter with a stainless steel needle to use a corona discharge as ion source. A schematic of this configuration and the corresponding pulse sequence are shown in Figures S1 and S2, respectively. The ion arrival curves for H(NO2)2¯ are contrasted in pulsed-field and continuous-field sampling modes in Figure S3. Similar to ions generated by ESI, the pulsed-field sampling leads to a drastic enhancement in sensitivity. It is of note that the counter flow gas prevents the neutrals generated by corona discharge from traveling toward the MS.26 As a result, the ion-molecule chemistry with corona-discharge-produced neutrals is avoided during the pulsed-field sampling time. Moreover, even though clustering with trace water may still occur in dry nitrogen counter flow gas, the ions rapidly reach equilibrium composition after their generation in the source. In other words, desolvation or other ion-molecule reactions are not expected to increase the concentration of H(NO2)2¯ during the pulsed-field sampling time. Therefore, observation of sensitivity enhancement with pulsed-field sampling for the corona discharge ion confirms that aerodynamic sampling, rather than desolvation, is responsible for improved sensitivity. H(NO2)2¯ is selected among other major corona discharge ions (e.g. NO3-, NO2-) for the above studies because it is 12


Page 13 of 29

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


less likely to be the product of declustering during ion sampling. The effects of declustering on ion arrival curves are further discussed below. Cluster Detection. To assess the applicability of pulsed nano-ESI pulsed-field IM-MS for investigation of weakly-bound species, we first examine solvent clustering using 1-aminohexane and its N-methylated homologs as a model system. Ion arrival curves of the four analytes are shown in Figure 4 using an aqueous solution with 2.5-µM total analyte concentration (see sample preparation details in Supporting Information). The primary amine shows the greatest extent of peak tailing because of unresolved late arriving ions. With increasing methylation, we observe that the population of late arriving ions diminish. Note that significant declustering is implemented within the MS (11 V difference between Q0 and Q2) to improve detection sensitivity. Therefore, all ions are detected in their fully desolvated form even though they may have travelled through the IM cell as a cluster. The diminishing population of late arriving ions with methylation suggests relation to hydrogen bonding and the formation of solvent clusters around the amine group.27,28 However, we were not able to detect the solvent clusters of analytes in the mass spectra even at softest operating conditions for the MS. To investigate the contribution of clustering to broadening and tailing of the ion arrival curves, we minimized declustering in nozzle-skimmer area by utilizing a potential difference of 4 V and implemented m/z filtering in Q1 for the completely desolvated ion. Ions were accelerated into the Q2 with a potential difference of 11 V for collisional focusing and improved sensitivity. In this operating mode, only ions fully desolvated prior to Q1 are detected by the MS. Figure 5a shows the comparison of the arrival curves with and without Q1 filtering for protonated 1-aminohexane. The ion arrival curve with no Q1 filtering is significantly broader 13



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 14 of 29

than the one where Q1 filtering is applied, confirming that the broadness arises from release of the analyte ions from late arriving clusters as a result of collisional activation in Q2. A similar experiment was performed using trimethylhexyl ammonium as a control ion. Figure 5b illustrates that the normalized arrival curves are identical between the two modes of Q1 filtering and no Q1 filtering as expected for an ion that is not prone to significant solvent clustering. This observation further supports our conclusions regarding solvent clustering as the cause of broad ion arrival curves. Peptide Aggregates. To further investigate the potential of pulsed nano-ESI pulsed-field IM-MS in detection of weakly-bound species, we have applied the method to analysis of ions generated from a 3-µM solution of substance P fragment (D-Pro-Gln-Gln-D-Trp-Phe-D-TrpLeu-Nle-NH2). The ion arrival curve for the doubly charged peptide ([P+2H]2+) is shown in Figure 6 by plotting the ion intensity for the monoisotopic peak (558.76 m/z) of the peptide as a function of field-on duration. A peak in arrival curve is observed for this ion as expected from aerodynamic sampling discussed above. Interestingly, when the singly charged ion ([P+H]+) intensity is plotted using its monoisotopic mass (1116.51 m/z), a series of peaks in arrival curve are observed. Considering the arrival of the doubly charged ion at 5.8 ms, the peak at 10.3 ms for [P+H]+ is attributed to the arrival of the singly charged peptide generated simultaneously with the doubly charged ion during the pulsed spray. The early arriving peaks are consequently attributed to multiply charged clusters, which travel through the IM cell and dissociate upon sampling by the MS. Figure S4 shows the average MS spectrum for arrival times 7.6-8.1 ms. Isotopic peaks at 1117.02 and 1116.86 m/z reveal presence of doubly and triply charged ions with the same monoisotopic m/z as the singly charged peptide. These peaks are attributed to the doubly charged 14


Page 15 of 29

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


dimer ([2P+2H]2+) and triply charged trimer ([3P+3H]3+) of the peptide. To highlight the arrival of aggregate species, the intensities of the second isotopic peaks of [2P+2H]2+ (1117.02 m/z) and [3P+3H]3+ (1116.86 m/z) are shown in Figure 6 as a function of time. The results confirm that multiply charged aggregates emerge from the spray and travel through the IM cell at room temperature and atmospheric pressure, arriving at the MS earlier than the monomer due to their higher charge. However, they dissociate upon sampling by the MS and produce the singly charged monomer. Such aggregates have also been observed for other peptides.29,30 Figure 6 also shows that a significant amount of singly charged monomer is produced from a cluster arriving at 6.1 ms, slightly after the doubly charged peptide. We were not able to identify the chemical composition of this species even with softest MS operation. We infer that the cluster is not stable enough to endure the ion sampling process of the MS. However, we note that [2P+2H]2+ is detected during the arrival of the unidentified species, suggesting the presence of higher-order peptide aggregates that produce the dimer, in addition to the monomer, upon sampling by the MS.

CONCLUSIONS We have demonstrated that pulsed-field ion sampling leads to drastic improvements (up to one order of magnitude) in sensitivity of AP-IM-MS. The momentary shutdown of the mobility field in the vicinity of the MS orifice leads to aerodynamic transportation of ions into the MS from a larger volume upstream of the MS orifice compared to the situation where the mobility field is sustained continuously in the vicinity of the orifice. Notably, the voltage applied to the sampling plate of the MS in our experiments has the same polarity as the ions while the last electrode in the mobility cell is grounded during pulsed-field ion sampling. This repulsive 15



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 16 of 29

voltage within the plate-to-disk-electrode geometry of the ion sampling area leads to a potential hill in radial direction, causing ion defocusing. Therefore, the applied voltage reduces the size of the aerodynamic sampling volume in our experimental setup but it is necessary for transmission of the ions within the QStar MS. In contrast, ion sampling interfaces that utilize resistive capillaries allow independent control of the voltages applied to the atmospheric pressure and vacuum sides of the ion sampling capillary. We expect that the larger parameter optimization space offered by these interfaces would further improve the sensitivity in pulsed-field sampling. Another important aspect of the presented pulsed-field sampling is its utility in arrival time measurements. Passage of the ion clouds across the aerodynamic sampling volume with the sweep of the mobility field duration leads to the appearance of ion arrival peaks. Accordingly, the arrival times are measured using the on-time of the mobility field without the need for an exit gate. Therefore, the pulsed-field sampling offers a facile approach for coupling atmospheric pressure ion mobility devices to atmospheric-sampling mass spectrometers. It is noteworthy that the effective ion sampling position is pushed into the IM cell in the pulsed-field sampling mode. In our experiments, the arrival time of trimethylhexyl ammonium ion is reduced by 10% at pulsed-field sampling times of 8 ms and larger compared to that at continuous-field sampling. Considering the cell length of 3 cm, this shift translates to 3 mm reduction in effective cell length. Typically, the exact length of the IM cell is needed to measure the ion transport properties (e.g. collisional cross section) from the arrival times. To account for effective cell length, arrival time calibration would be required for accurate measurements of transport properties. A range of calibrants with well-established mobilities have been developed in recent years and can be used to calibrate the arrival times and to measure the effective length of the cell.25 16


Page 17 of 29

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


We have also demonstrated that a pulsed nano-ESI can be operated as a source of ion packets for mobility separations. Importantly, the spray is operated in ambient temperature and pressure without any induced thermal desolvation. These soft ion generation conditions along with soft ion injection into the IM cell preserve the weakly-bound species such as solvent clusters and aggregates. The delicate non-covalent clusters may be fragmented in the ion sampling process of the MS, however, arrival times of the declustering products offer insights into cluster formation by the pulsed-ESI. For substance P fragment as a model peptide, the arrival of earlier-than-expected singly charged ions indicate the formation of peptide aggregates. These results highlight the potential of the pulsed nano-ESI pulsed-field ion mobility-MS for studying weakly-bound species.

ACKNOWLEDGEMENTS The authors would like to thank the financial support from Georgetown University. CSM is grateful to the National Science Foundation Graduate Research Fellowship Program (DGE0903443). Additionally, the authors would like to thank Salman Americianaki for his assistance in early experiments.

Supporting Information Supporting Information Available: Experimental setup and pulse sequence for corona discharge ion source coupled to pulsed-field IM-MS, Ion arrival curves for a typical corona discharge ion, Mass spectrum of peptide aggregates. This material is available free of charge via the Internet at http://pubs.acs.org.

17



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 18 of 29

References (1) May, J. C.; McLean, J. A. Anal. Chem. 2015, 87, 1422-1436. (2) Rodriguez-Cruz, S. E.; Klassen, J. S.; Williams, E. R. J. Am. Soc. Mass Spectrom. 1999, 10, 958-968. (3) Merenbloom, S. I.; Flick, T. G.; Williams, E. R. J. Am. Soc. Mass Spectrom. 2012, 23, 553562. (4) Tang, X. T.; Bruce, J. E.; Hill, H. H. Rapid Commun. Mass Spectrom. 2007, 21, 1115-1122. (5) Kwasnik, M.; Fuhrer, K.; Gonin, M.; Barbeau, K.; Fernandez, F. M. Anal. Chem. 2007, 79, 7782-7791. (6) Guevremont, R.; Siu, K. W. M.; Wang, J. Y.; Ding, L. Y. Anal. Chem. 1997, 69, 3959-3965. (7) Clowers, B. H.; Hill, H. H. Anal. Chem. 2005, 77, 5877-5885. (8) Clowers, B. H.; Siems, W. F.; Yu, Z. H.; Davis, A. L. Analyst 2015, 14, 6862-6870. (9) Morrison, K. A.; Siems, W. F.; Clowers, B. H. Anal. Chem. 2016, 88, 3121-3129. (10) Zhang, X.; Knochenmuss, R.; Siems, W. F.; Liu, W. J.; Graf, S.; Hill, H. H. Anal. Chem. 2014, 86, 1661-1670. (11) Tang, X. T.; Bruce, J. E.; Hill, H. H. Anal. Chem. 2006, 78, 7751-7760. (12) Kanu, A. B.; Kumar, B. S.; Hill, H. H. Int. J. Ion Mobility Spectrom. 2012, 15, 9-20. (13) Zhou, L.; Zhai, L. L.; Yue, B. F.; Lee, E. D.; Lee, M. L. Anal. Bioanal. Chem. 2006, 385, 1087-1091. (14) Garimella, S.; Xu, W.; Huang, G. M.; Harper, J. D.; Cooks, R. G.; Ouyang, Z. J. Mass Spectrom. 2012, 47, 201-207. (15) Hawkridge, A. M.; Zhou, L.; Lee, M. L.; Muddiman, D. C. Anal. Chem. 2004, 76, 41184122. 18


Page 19 of 29

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


(16) Wu, S.; Zhang, K.; Kaiser, N. K.; Bruce, J. E. J. Am. Soc. Mass Spectrom. 2006, 17, 772779. (17) Dixon, R. B.; Muddiman, D. C. Rapid Commun. Mass Spectrom. 2007, 21, 3207-3212. (18) Tan, P. V.; Laiko, V. V.; Doroshenko, V. M. Anal. Chem. 2004, 76, 2462-2469. (19) Berkout, V. D.; Kryuchkov, S. I.; Doroshenko, V. M. Rapid Commun. Mass Spectrom. 2007, 21, 2046-2050. (20) Blanchard, W. C. Int. J. Mass Spectrom. Ion Processes 1989, 95, 199-210. (21) Matz, L. M.; Steiner, W. E.; Clowers, B. H.; Hill, H. H. Int. J. Mass Spectrom. 2002, 213, 191-202. (22) Chen, C.; Hou, K. Y.; Wang, W. G.; Li, J. H.; Li, H. Y. J. Chromatogr. A 2014, 1358, 192198. (23) Servage, K. A.; Silveira, J. A.; Fort, K. L.; Clemmer, D. E.; Russell, D. H. J. Phys. Chem. Lett. 2015, 6, 4947-4951. (24) Viidanoja, J.; Sysoev, A.; Adamov, A.; Kotiaho, T. Rapid Commun. in Mass Spectrom. 2005, 19, 3051-3055. (25) Fernandez-Maestre, R.; Harden, C. S.; Ewing, R. G.; Crawford, C. L.; Hill, H. H. Analyst 2010, 135, 1433-1442. (26) Sabo, M.; Okuyama, Y.; Kucera, M.; Matejcik, S. Int. J. Mass Spectrom. 2013, 334, 19-26. (27) Blades, A. T.; Klassen, J. S.; Kebarle, P. J. Am. Chem. Soc. 1996, 118, 12437-12442. (28) Demireva, M.; O'Brien, J. T.; Williams, E. R. J. Am. Chem. Soc. 2012, 134, 11216-11224. (29) Counterman, A. E.; Valentine, S. J.; Srebalus, C. A.; Henderson, S. C.; Hoaglund, C. S.; Clemmer, D. E. J. Am. Soc. Mass Spectrom. 1998, 9, 743-759.

19



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

(30) Counterman, A. E.; Hilderbrand, A. E.; Barnes, C. A. S.; Clemmer, D. E. J. Am. Soc. Mass Spectrom. 2001, 12, 1020-1035.

20


Page 20 of 29

Page 21 of 29

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Figure Captions Figure 1. Schematic of the experimental setup for pulsed nano-ESI pulsed-field ion mobility mass spectrometry. Figure 2. Pulse scheme for a) continuous-field mode, and b) pulsed-field mode at operating frequency of 25 Hz. A spray event duration of 500 µs is used for all experiments while the arrival time is scanned to generate the ion arrival curves. “Cleanup” refers to purging the cell of the remaining ions with the skimmer gate closed before the start of the next cycle. Figure 3. Effect of pulsed-field sampling on ion arrival curves for trimethylhexyl ammonium ion. Experimental parameters: solution voltage = 1285 V, mobility cell voltage = 1000 V, orifice voltage = 26 V, skimmer gate open = 22 V, skimmer gate closed = 89 V, Q0 DC potential = 20 V, Q2 DC potential = 9 V. The data were collected at a pulsed spray frequency of 15 Hz. Ion intensities were averaged for 8 s for each arrival time. Figure 4. Ion arrival curves for 1-aminohexane and its N-methylated homologs in pulsed-field mode with 8 ms sampling time. Experimental parameters: solution voltage = 1285 V, mobility cell voltage = 1000 V, orifice voltage = 26 V, skimmer gate open = 22 V, skimmer gate closed = 89 V, Q0 DC potential = 20 V, Q2 DC potential = 9 V. The data were collected at a pulsed spray frequency of 15 Hz. Ion intensities were averaged for 8 s for each arrival time. A normalization factor is applied to superimpose the curves. The normalization factors are noted in the legend and equalize the intensities for earliest arriving species of each homolog. Figure 5. Comparison of ion arrival curves with and without Q1 filtering for a) 1-aminohexane and b) trimethylhexyl ammonium in pulsed-field mode with 8 ms sampling time. Normalization factors of 14 and 4.5 are applied to Q1 fileted data in a and b, respectively, to superimpose the 21



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 22 of 29

curves. Experimental parameters for both modes: solution voltage = 1270 V, mobility cell voltage = 1000 V, orifice voltage = 26 V, skimmer gate open = 22 V, skimmer gate closed = 89 V, Q0 DC potential = 20 V, Q2 DC potential = 9 V. Both modes are operated with the pulsed spray frequency of 15 Hz. Averaging time of 8 s is applied for no Q1 filtering while an averaging time of 15 s is used for Q1 filtering mode to improve S/N. Figure 6. Ion arrival curves in pulsed-field mode (8 ms sampling time) for species generated from substance P fragment at 3 µM. To facilitate comparison of arrival curves, normalization factors are applied to ion intensities and are noted within the Figure. Experimental parameters: solution voltage = 1270 V, mobility cell voltage = 1100 V, orifice voltage = 20 V, skimmer gate open = 15 V, skimmer gate closed = 66 V, Q0 DC potential = 12 V, Q2 DC potential = 9 V. The data were collected at a pulsed spray frequency of 25 Hz. Ion intensities were averaged for 10 s for each arrival time.

22


Page 23 of 29

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Figure 1

Pt Electrode Gas 10 psig

HV Relay

HV Power Supply (1.2-1.3 kV)

HV Relay

HV Power Supply (1000 V)

Pulled Glass Capillary

Q0

Q1

Q2

TOF Skimmer

Acrylic Case Nitrogen In

Nitrogen Out Resistively Coupled Steel Rings

DC Power Supply

Collision Cell HV Relay

23


QStar XL MS


Figure 2

a

Solution Voltage

1200 Spray Event

Voltage (V)

1000

Mobility Cell Voltage

Ion Arrival Time

Cleanup

Voltage (V)

0 Skimmer Voltage

125

Ions Sampled by MS

Ions Blocked

0 0

b

40

Time (ms)

Solution Voltage

1200 Spray Event

Voltage (V)

1000

Mobility Cell Voltage Pulsedfield Sampling Time

Ion Arrival Time

Cleanup

0 Voltage (V)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 24 of 29

Skimmer Voltage

125 Gate Open

Gate Closed

0 0

Time (ms)

24


40

Page 25 of 29

Figure 3

Continuous Field 2 ms Pulsed Field 2000

8 ms Pulsed Field 11 ms Pulsed Field

250 200

1600

150 100

1200

50 7.0

6.6

6.2

5.8

5.4

5.0

4.2

4.6

0

800

N+

400

Arrival Time (ms)

25


6.8

6.3

5.8

5.3

4.8

4.3

3.8

0 3.3

Ion Intensity (counts/s)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60



Figure 4

12x

13x

+H N 3

+H

2N

+

31x

H N

N+

2400

1800

1200

600

Arrival Time (ms)

26


9.0

8.5

8.0

7.5

7.0

6.5

6.0

5.5

5.0

4.5

4.0

3.5

0 3.0


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 26 of 29

Page 27 of 29

Figure 5


400

a

No Q1 Filtering

14 x Q1 Filtering +H

300

3N

200

100

b

9.0

8.5

8.0

7.5

7.0

6.5

6.0

5.5

5.0

4.5

4.0

3200

3.5

0

No Q1 Filtering

2400

4.5 x Q1 Filtering

1600 N+

800

Arrival Time (ms)

27


7.0

6.5

6.0

5.5

5.0

4.5

4.0

0 3.5


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60



Figure 6

35 2+

[P+2H] x 0.04

30

3+

[3P+3H] x 1.8 2+

[P+H]+

[2P+2H] x 2.6

25 20 15 10 5

28


12.5

11.5

Arrival Time (ms)

10.5

9.5

8.5

7.5

6.5

5.5

0 4.5


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 28 of 29

Page 29 of 29

Abstract Graphic

Ion Intensity

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Arrival Time (ms)

29


Pulsed Nano-ESI Atmospheric-Pressure Ion ... - ACS Publications

Recommend Documents