Nanowell-mediated multidimensional separations combining nanoLC with SLIM IM-MS for rapid, high-peak-capacity proteomic analyses

Abstract

Mass spectrometry (MS)-based analysis of complex biological samples is essential for biomedical research and clinical diagnostics. The separation prior to MS plays a key role in the overall analysis, with separations having larger peak capacities often leading to more identified species and improved confidence in those identifications. High-resolution ion mobility (IM) separations enabled by Structures for Lossless Ion Manipulation (SLIM) can provide extremely rapid, high-resolution separations and are well suited as a second dimension of separation following nanoscale liquid chromatography (nanoLC). However, existing sample handling approaches for offline coupling of separation modes require microliter-fraction volumes and are thus not well suited for analysis of trace biological samples. We have developed a novel nanowell-mediated fractionation system that enables nanoLC-separated samples to be efficiently preconcentrated and directly infused at nanoelectrospray flow rates for downstream analysis. When coupled with SLIM IM-MS, the platform enables rapid and high-peak-capacity multidimensional separations of small biological samples. In this study, peptides eluting from a 100-nL/min nanoLC separation were fractionated into ~60 nanowells on a microfluidic glass chip using an in-house-developed robotic system. The dried samples on the chip were individually reconstituted and ionized by nanoelectrospray for SLIM IM-MS analysis. Using model peptides for characterization of the nanowell platform, we found that at least 80% of the peptide components of the fractionated samples were recovered from the nanowells, providing up to ~tenfold preconcentration for SLIM IM-MS analysis. The combined LC-SLIM IM separation peak capacities exceeded 3600 with a measurement throughput that is similar to current one-dimensional (1D) LC-MS proteomic analyses.

Graphical Abstract

A nanowell-mediated multidimensional separation platform that combines nanoLC with SLIM IM-MS enables rapid, high-peak-capacity proteomic analyses.

Proteomics, which seeks to understand cellular function and dysfunction and the overall composition, structure, and activity of proteins within complex biological samples, has become essential for nearly all fields of biomedical research and is making inroads as a tool for clinical diagnostics [1,2]. Liquid chromatography (LC) coupled to mass spectrometry (MS) comprises an unrivaled bioanalytical platform for proteomic analysis of complex biological samples [3,4]. Enhancing proteome coverage, or identifying more species, is a key challenge for proteomics, which increases as sample sizes decrease [5]. Similarly, increasing measurement throughput and reducing the cost per analysis is crucial for enabling studies comprising large numbers of samples. Large sample sets are necessary to distinguish meaningful differences between experimental groups from the large biological variability often present within each group [6]. Failure to design experiments having sufficient numbers of biological samples and technical replicates due to practical resource constraints has been a key short-coming of “omics” technologies and is a contributing factor in, e.g., the limited success of proteomics for biomarker discovery [7].

The liquid-phase separation used in conjunction with MS plays a key role in determining both proteome coverage and analytical throughput [8]. For example, the flow rate of the separation typically dictates the electrospray source ionization efficiency and impacts the resulting sensitivity [9]. Similarly, slower separations having larger peak capacities have been found to lead to more identified species [10]. in part because enhanced separations limit the number of analytes transferred to the MS at any given time. Improved separations increase the MS dynamic range [11], minimize the potential for ionization suppression [12], and reduce spectral complexity and congestion effects complicating identifications. Unfortunately, increased peak capacity is typically accompanied by decreased throughput. For example, increasing the column length and/or gradient time is typically used to increase the peak capacity of a one-dimensional (1D) LC separation [13]. Multidimensional separations having orthogonal separation mechanisms can potentially have far greater resolving power, as the peak capacities of the constituent separations can achieve near perfect orthogonality (from unrelated separation mechanisms) [14,15]. To date, the most commonly used multidimensional separations involve two different modes of LC, typically strong cation exchange combined with reversed-phase (RP)LC [16] or RPLC performed under different conditions (e.g., pH) [17,18]. While two-dimensional (2D) LC has enabled dramatic increases in proteome coverage, it has several limitations. A loss of resolution often accompanies the transfer of analytes from one separation to another, and while this can be minimized by performing online LC-LC separations, the MS and MS/MS cycle times are constrained by the rapid gradient of the second dimension separation [19]. In addition, the throughput of LC-LC separations is very low, whether performed online or offline via fraction collection, often requiring a full day or more to analyze a single sample. Finally, there is only partial orthogonality across the LC modes, so the overall peak capacity never approaches that of the product of the individual separations [20]. Capillary electrophoresis (CE) is another liquid-phase technique, which separates analytes based on their electrophoretic mobilities under an applied electric field. Hybrid LC-CE analyses have been demonstrated previously, most notably by the Jorgenson and Ramsey groups [21–23]. In all cases reported by these groups and others, the coupling mechanism between LC and CE involved transferring only a small portion of the sample from LC to CE, while the rest of the LC eluent was wasted. As such, the peak capacity gains achieved in the multidimensional separation were offset by a large loss in sensitivity resulting from inefficient analyte utilization.

LC followed by a gas-phase ion mobility (IM) separation prior to MS analysis is a promising option for multidimensional separation [15,24], as the millisecond timescale of the IM separation can provide added peak capacity without decreasing measurement throughput. In addition, RPLC and IM separations are highly orthogonal as they are based on distinct separation mechanisms. RPLC is based on the hydrophobic interactions between analytes and a stationary phase, while IM separates gas-phase ions based on their mobilities through a buffer gas in the presence of an electric field.

Structures for Lossless Ion Manipulation (SLIM) is a new technology that enables efficient and extended gas-phase ion manipulations. SLIM devices utilize electric potentials applied to arrays of electrodes patterned on two closely spaced planar surfaces fabricated utilizing photolithography [25–29]. A combination of RF and DC potentials applied to the electrodes serve to establish electric fields that allow for the storage, manipulation and transfer of ions. The planar nature of single-level SLIM enables multiple features, including the ability to significantly increase pathlength in a small footprint, whether operating in drift-tube ion mobility (DTIM) [25] or traveling wave ion mobility (TWIM) [30] modes, and allowing the seamless integration of multiple ion manipulations [31], high sensitivity, as well as the ability to construct multilevel devices [32]. In the TWIM mode, the use of serpentine ultralong path with extended routing (SUPER) multi-pass separations has recently been demonstrated providing >100 m pathlengths for IM separations. TW SLIM have also enabled the trapping of extremely large ion populations (>10⁹ ions for use in separations, a 10² to 10³ increase over what was previously feasible), and compression ratio ion mobility programming (CRIMP) for reversing peak width increases in long separations and providing increased peak intensities [33,34]. These capabilities have demonstrated remarkable improvements in conjunction with mass spectrometry (MS) for the analysis of isomeric lipids, peptides, and metabolites. All of these features make SLIM IM an ideal platform for providing extremely rapid and high-resolution separations. As such, SLIM is well suited as a second dimension IM separation following nano-flow LC (nanoLC) to obtain high peak capacities with MS for enhanced proteome coverage.

A key challenge in implementing multidimensional separations is the efficient transfer of samples between dimensions. This is especially difficult when analyzing very small (e.g., nanogram) sample amounts and transferring nanoliter-volume sample fractions that are too small to be handled using conventional pipettes and well plates or vials. We have recently used an in-house-constructed robotic nanopipetting platform and microfabricated ‘nanowell’ chips to effectively handle low-nanoliter volumes while minimizing analyte losses to surfaces. The platform has enabled far smaller proteomic samples (extending to single mammalian cells) to be processed and analyzed using a workflow termed nanoPOTS (Nanodroplet Processing in One pot for Trace Samples) [35–38], and has been used to couple two different modes of LC for profiling several hundred cells with a depth of coverage of ~6,000 protein groups [18].

While the nanowell-mediated two-dimensional (2D) LC has achieved very deep proteome coverage for nanogram samples, the RPLC separations, each taking ~2 h, provided a challenge for measurement throughput. Here we report a nanowell-mediated platform that offline-couples nanoLC with SLIM IM-MS with minimal sample losses at the interface between the two separation modes. Peptides eluted from the nanoLC column were fractionated and preconcentrated into nanowells and injected as narrow bands for rapid SLIM IM separation with the incorporation of a highly sensitive nanoelectrospray ionization (nanoESI) interface, thus providing both large peak capacities and high sample utilization efficiencies. We analyzed 62 nanoLC fractions in ~90 min and obtained an overall peak capacity that exceeded 3600. Although the effective peak capacity from the first dimension nanoLC separation was reduced to some degree due to pooling of multiple peaks into a given nanowell, this approach provides a unique platform to achieve rapid and highly sensitive proteomic analyses with overall increased peak capacities for small samples. Offline preconcentration between multidimensional separations was previously not feasible for such samples. The implementation of this nanowell-mediated platform is anticipated to accelerate clinical diagnostics and biomedical research that often require the analysis of large numbers of samples and/or limited sample amounts for rapid, high-peakcapacity proteomic analyses.

EXPERIMENTAL SECTION

Chemicals and reagents

Fluorescent peptides (HiLyte™ Fluor 488-labeled [pSer5]-kemptide and ß-Amyloid) were purchased from AnaSpec (Fremont, USA). HPLC peptide standards (H2016), methionine enkephalin and leucine enkephalin, were purchased from Sigma-Aldrich (St. Louis, USA). The HeLa digest (HeLa Protein Digest Standard) was purchased from Thermo Fisher Scientific (Waltham, USA). Unless otherwise noted, all other chemicals and reagents were purchased from Sigma-Aldrich (St. Louis, USA). Deionized water (18.2 MΩ) was produced using a Barnstead Nanopure Infinity system (Los Angeles, USA).

Fabrication of the nanowell microfluidic chip

The microfabrication of the nanowell microfluidic glass chip is based on photolithography, with detailed information described previously [35]. The surface of each nanowell pedestal was hydrophilic, while the background surrounding the nanowell pedestals was rendered hydrophobic by treating with 2% (heptadecafluoro-1,1,2,2-tetrahydrodecyl) dimethylchlorosilane (PFDS) in 2,2,4-trimethylpentane.

LC separation and fractionation

The LC column (50 μm i.d., 360 μm o.d., 50 cm long) was packed with 3-μm C18 packing material (300-Å pore size, Phenomenex, Torrance, USA) [39]. The LC separation flow rate was 100 nL/min on a binary pump system (Thermo Scientific Dionex UltiMate 3000 pump). A linear 60 min gradient of 5–22% Buffer B (0.1% formic acid in acetonitrile) was used for the nanoLC separation followed by a gradient of linear increase to 40% in 10 min and a 10 min wash in 90% B, and a final 20 min equilibration with Buffer A (0.1% formic acid in water).

The LC eluent was fractionated into each of the 62 nanowells for 1 min during the gradient time 21–83 min using our in-house-developed robotic system [18] and allowed to dry.

SLIM IM-MS separation and analysis

The dried peptide samples on the chip were individually reconstituted and electrosprayed into the SLIM IM-MS separation and analysis platform using a robotic nanopipetting system equipped with a rotating interface housing a chemically etched capillary tip [40] (20 μm i.d., 360 μm o.d., 4 cm long) that served as both pipet tip and electrospray emitter. A photograph of the robotic platform is shown in Electronic Supplementary Material (ESM) Fig. S1. Peptides were electrosprayed at a flow rate of 30 nL/min into a heated capillary inlet (120 °C) before entering an ion funnel operated at ~2.45 Torr. The ions then passed through a rectangular ion funnel [41] to the SLIM module, which was maintained at ~2.50 Torr in nitrogen buffer gas. The SLIM module had a 12.3 m length serpentine separation path, which was segmented into two separate regions to allow for compression ratio ion mobility programming (CRIMP) and/or ‘in-SLIM’ ion accumulation [42,43]. To maximize both gains in signal-to-noise ratio and acquisition rate for these samples, ions were accumulated for 1 s inSLIM using the first 6.15 m region as described previously [43]. Following accumulation, ions underwent TWIM separation through the second 6.15 m region, which resulted in <2 sec acquisition time for each separation. The TW was operated using a square waveform at 25 V amplitude and a frequency of 120 kHz, with a lateral guard voltage of 10 V. The confining RF was operated at 360 V_pp and a frequency of 900 kHz. After the SLIM module, ions traveled through a rear ion funnel and short quadrupole before entering an Agilent 6538 QTOF mass spectrometer (Agilent Technologies, Santa Clara, CA) for mass analysis. Mass accuracy was calibrated prior to the experiments using Agilent tune mix ions m/z 622.02896 and 1221.99064. Instrument control and data acquisition were performed using in-house-developed software tools. Data processing was also performed using in-house tools combined with IM-MS Browser B.08.00 and Mass Profiler (Agilent Technologies, Santa Clara, CA).

Sample recovery from nanowells

Sample recovery from nanowells was evaluated by first dispensing 100 nL of 10 μM fluorescently labeled peptides into each nanowell. The resulting droplet was then covered with mineral oil to minimize lensing of the rounded nanowell droplets for fluorescence imaging. On a separate chip, 100 nL of the same 10 μM peptide solution was dispensed into each nanowell and evaporated to dryness for 5 min in open air. Ten nanoliters of 899:100:1 water:methanol:acetic acid solvent was then added to the dried nanowells, aspirated from the nanowell and discarded. This process was the same as extracting and collecting the fractions from nanowells into capillaries. Finally, 100 nL of H₂O was added to the nanowell, covered with oil as before, and the fluorescence was measured. One hundred nanoliters of H₂O was also dispensed into unused nanowells, covered with mineral oil and the fluorescence was recorded as a blank for subtraction.

Sample preconcentration in nanowells

To investigate the preconcentration effect from nanowells, we first employed a series of peptide samples containing leucine enkephalin and methionine enkephalin with various concentrations (i.e., 50, 100, 200, 300, 400, 500 and 600 ng/μL) for direct MS analysis, and plotted their corresponding average intensities at a stable electrospray stage versus concentrations to generate a standard calibration curve. We further loaded serial volumes (i.e. 10, 20, 40, 60, 80, 100, and 120 nL) of peptide samples containing leucine enkephalin and methionine enkephalin at a concentration of 50 ng/μL into nanowells and reconstituted them in 10 nL of 899:100:1 water:methanol:acetic acid solvent for MS analysis. By recording the average intensities at a stable electrospray stage, the effective concentrations of the reconstituted peptides at different ratios of loading to reconstitution volumes could be calculated using the standard calibration curve. Finally, the corresponding effective concentration factors as ratios of the effective concentrations to the original concentration (i.e. 50 ng/μL) at different ratios of loading to reconstitution volumes could be obtained.

RESULTS AND DISCUSSION

Nanowell-mediated nanoLC-SLIM IM-MS platform

We previously reported a nanowell-mediated 2D LC platform [18] in which peptides eluting from a high-pH nanoLC column were fractionated into nanowells, dried, reconstituted in the low-pH buffer and collected from nanowells into capillaries for manual low-pH nanoLC injections and analyses. Despite the dramatically enhanced proteome coverage, the lack of automated sample injection and long total analysis time (>24 h for a 12-fraction analysis) limited the throughput of the nanowell-mediated 2D LC platform. In this work, we developed a new robotic nanopipetting platform with rotational capabilities to directly inject sample from a nanowell and electrospray into a SLIM IMMS platform for rapid and automated high-peak-capacity multidimensional peptide separations.

The entire workflow of the offline nanowell-mediated nanoLC-SLIM IM-MS platform is shown in Fig. 1. Peptides eluted from a nanoLC separation operating at 100 nL/min were fractionated into 62 nanowells on a microfabricated glass chip at 1 min intervals using an in-house-developed robotic system (ESM Fig. S1a and Video S1). The fractions were allowed to dry automatically in ~1 min without requiring a time-consuming SpeedVac that is commonly used in two-dimensional LC separations. The low-flow nanoLC allowed the use of 0.6-mm-diameter nanowells with total surface exposure of just ~0.28 mm², effectively minimizing adsorptive surface losses. The dried peptides on the chip were individually reconstituted using 10 nL of electrospray solvent for ionization at nanoelectrospray flow rates for SLIM IM-MS separation and analysis using the robotic system (ESM Fig. S1b and Video S2). In this way, the nanoLC-eluted peptides were preconcentrated by a factor of ~10 in the nanowells. As a result, the nanowell platform not only minimized sample losses at the interface between the two separation modes, but also provided concentrated samples with high sample utilization efficiencies to achieve both increased peak capacities and high sensitivity.

Fig. 1.

Workflow for the nanowell-mediated nanoLC-SLIM IM-MS platform. (a) Samples are separated using LC, fractionated into nanowells and allowed to dry. (b) Samples in each nanowell are reconstituted and electrosprayed for subsequent IM-MS analysis. The insert images illustrate the reconstitution (1) and aspiration (2) process for reconstituting and collecting samples, and nanoESI (3) for subsequent SLIM IM-MS separation and analysis

Sample recovery from nanowells

We first evaluated the sample recovery from nanowells by using two different fluorescently labeled peptides, [pSer5]-kemptide and ß-amyloid. Fluorescence intensities of the original peptide solutions were compared to those of remaining peptides in nanowells following sample loading, evaporation, reconstitution and extraction as shown in Fig. 2a and ESM Fig. S2a. The difference in signal intensity after blank subtraction indicates the percent recovery from the wells (Fig. 2b and ESM Fig. S2b), which was found to be at least 84% using this method, which is in close agreement with our previous evaluations based on MS signal intensities [18]. The results indicate that the nanowell platform enabled a high sample recovery between the nanoLC separation mode and the SLIM IM separation mode.

Fig. 2.

Sample recovery from nanowells using 10 μM Fluor 488-labeled [pSer5]-kemptide. Fluorescence images of original peptides (a) and remaining peptides (b) in nanowells after extraction, as well as their corresponding fluorescence intensities (c) (n = 3)

Preconcentration effect in nanowells

We further investigated the sample preconcentration effect in nanowells. Various volumes (10–120 nL) of unlabeled leucine enkephalin and methionine enkephalin peptide samples, having a concentration of 50 ng/μL, were loaded into nanowells and reconstituted in 10 nL of electrospray solvent for nanoESI-MS analysis. The effective concentration factors at different ratios of loading to reconstitution volumes are shown in Fig. 3. As expected, the concentration factors were found to increase linearly when increasing the ratio between the loading and reconstitution volumes. The ability to concentrate nanoliter samples by a factor of nearly ten and with only modest sample losses should provide a significant increase in sensitivity for the overall analysis and provides a more focused band for injection into the subsequent separation dimension.

Fig. 3.

Preconcentration effect of the nanowells. Effective concentration factors at different ratios of loading/reconstitution volume by using peptides leucine enkephalin and methionine enkephalin (n = 3)

Nanowell-based nanoESI-MS performance

The stability performance of the nanowell-based nanoESI was evaluated by depositing 10 nL of 50 ng/μL leucine enkephalin and methionine enkephalin peptides into nanowells and evaporating to dryness. The dried peptides in nanowells were then reconstituted in 10 nL of electrospray solvent and collected in the same chemically etched capillary tip (20 μm i.d., 360 μm o.d., 4 cm long) used for dispensing. The robotic system then rotated the tip to the inlet of the SLIM IM-MS platform, a voltage was applied, and the analyte was infused at a flow rate of 30 nL/min. By analyzing the peptide samples from 6 nanowells, we found that stable and reproducible nanoESI-MS signals could be successfully achieved, as demonstrated in Fig. 4a and 4b. Such stable and reproducible nanoESI plays an important role in the successful coupling of nanoLC with SLIM IM-MS.

Fig. 4.

Nanowell platform for nanoESI-MS analysis. (a-b) Chromatography of reconstituted peptides leucine enkephalin (a) and methionine enkephalin (b) from nanowells for nanoESI-MS analysis (n = 6). (c) Recovery of the reconstituted peptides from nanowells

We also measured the generated MS signals by directly introducing the same peptide samples without involving nanowells (control), and compared with those reconstituted from nanowells (reconstituted) to further evaluate the recovery from nanowells. The recovery from nanowells was calculated to be 79%−83% (Fig. 4c and ESM Table S1), which was consistent with that obtained by using fluorescently labeled peptides.

NanoLC separation and fractionation

For initial nanoLC-SLIM IM-MS coupling, 100 ng of HeLa digest was separated by nanoLC at a flow rate of 100 nL/min. The LC separation is shown in Fig. 5. The peptides eluted from 21 to 83 min, indicating an effective separation time of 62 min. For offline fractionation, the peptides were eluted from the nanoLC column into 62 nanowells at 1 min intervals. The peak capacity of the nanoLC separation was calculated to be 201 [44], but the effective peak capacity from the nanoLC was clearly reduced due to pooling of multiple peaks into a given nanowell. While greater overall peak capacity could be achieved by matching the number of fractions to the peak capacity of the LC separation, this would come at a cost of decreased measurement throughput. The tradeoffs between achievable proteome coverage and measurement throughput using this platform should be further explored in future work.

Fig. 5.

Base peak chromatograph of triplicate nanoLC separations using 100 ng of HeLa digest

SLIM IM-MS separations and analysis

Finally, we performed SLIM IM-MS separation and analysis for nanoLC fractionated peptide samples from each of the nanowells. The total SLIM IM-MS analysis for all nanoLC fractionated samples was completed within 90 min. The peak capacity of the SLIM IM separation for each of the 62 sample injections was ~60. Overall, the combined nanoLC-SLIM IM separation peak capacities was ~3600, which was ~18-fold higher than the 1D nanoLC separation alone.

The peak capacity obtained from a conventional high-performance 1D LC separation is typically <500, even when employing extended (e.g., 10 h) gradients and long columns (e.g., 100 cm) [45]. Multidimensional separation methods such as 2D LC have enabled the enhancement of the peak capacities to over 1000 [46,21]. However, the combination of long analysis times and/or complicated instrumentation for those multidimensional separation methods result in a high cost per analysis, impeding studies with large numbers of samples, and imposing a tradeoff between proteome coverage and measurement throughput. In contrast to the routine lab methods, the nanowell-mediated nanoLC-SLIM IM-MS multidimensional separations reported here enable very high peak production rates (40 peaks per min), which, with further refinement of the SLIM interface (particularly as related to improvements in the in-SLIM ion accumulation process), data acquisition and analysis tools, should make in-depth proteome profiling a far faster and higher signal-to-noise yielding endeavor.

CONCLUSIONS

In this study, we developed a nanowell-mediated nanoLC-SLIM IM-MS multidimensional separation platform. This nanowell-mediated platform demonstrated a significant preconcentration effect and a high sample recovery of 80% or more by minimizing surfaces losses from nanowells for sample transferring between the multiple nanoLC-SLIM IM-MS separations, which is especially beneficial for deep proteome analysis for trace samples. Combined nanoLC-SLIM IM separation peak capacities of ~3600 were obtained within 90 min, provided an order of magnitude improvement in peak capacity over existing LC-MS approaches, and large further gains are anticipated.

This novel approach will broadly impact biological analyses from dramatically improved throughput and peak capacities such as selected reaction monitoring MS for targeted proteomics. By accelerating studies with large numbers of samples, this nanowell-mediated nanoLC-SLIM IM-MS multidimensional separation system will promote broad applicability to routine implementation in clinical diagnostics and biomedical research community.

Biography

Maowei Dou is Postdoctoral Research Associate at Pacific Northwest National Laboratory. He is currently working on developing bioanalytical technologies combining droplet-based biological sample preparation with ultrasensitive nanoLC-MS/MS for deep proteome profiling of trace biological samples.

Christopher D. Chouinard is Assistant Professor of Chemistry at the Florida Institute of Technology. His research focuses on ion mobility-mass spectrometry, including developing improved methods for metabolomics and lipidomics with gas-phase chemistry. He was previously a postdoctoral research associate at PNNL, where he worked on development of the Structures for Lossless Ion Manipulations (SLIM) platform.

Ying Zhu is a scientist in the Environmental Molecular Sciences Laboratory at Pacific Northwest National Laboratory with over ten years’ experience in ultrasensitive bioanalysis using microfluidic techniques and mass spectrometry. His current research focuses on the development of nanodroplet sample processing systems and its application to single cell typing of mammalian and plant cells, in-depth proteome mapping of tissue heterogeneity, and understanding microbial-plant interactions with high spatial and temporal resolution.

Gabe Nagy is Postdoctoral Research Associate at Pacific Northwest National Laboratory. He is currently working on developing bioanalytical separations methods using ion mobility-mass spectrometry (IM-MS) in structures for lossless ion manipulations (SLIM).

Andrey V. Liyu is Senior Electrical Engineer in the Instrument Development Laboratory at Pacific Northwest National Laboratory’s Environmental Molecular Sciences Laboratory. He develops hardware and software solutions for advanced scientific instrumentation, including robotic systems for nanoliter sample manipulation.

Yehia M. Ibrahim is a scientist in the integrative Omics group at Pacific Northwest National Laboratory. His interest has been in developing and applying advanced ion mobility and mass spectrometry-based technologies to solve challenges in proteomics and metabolomics. He is a co-inventor of SLIM, a new category of devices that provide unmatched ion mobility resolution.

Richard D. Smith is Battelle Fellow and Chief Scientist for the Biological Sciences Division at Pacific Northwest National Laboratory. Key research interests have involved the development of advanced separations and mass spectrometry technologies and approaches for improving biological analyses, and most recently the development of structures for lossless ion manipulations (SLIM) for achieving much higher resolution ion mobility separations with mass spectrometry.

Ryan T. Kelly is Associate Professor in the Department of Chemistry and Biochemistry at Brigham Young University with a joint appointment as a Senior Research Scientist at Pacific Northwest National Laboratory. His research interests focus on the development of microfluidic sample handling, advanced separations and ultrasensitive mass spectrometry to increase the sensitivity and throughput of biochemical analyses.