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An Automated Trapping Column Exchanger for High Throughput Nanoflow Liquid Chromatography Sandra Elizabeth Spencer, Thomas N. Corso, James G. Bollinger, Clark M. Henderson, Andrew N. Hoofnagle, and Michael J. MacCoss Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.6b04227 • Publication Date (Web): 12 Jan 2017 Downloaded from http://pubs.acs.org on January 16, 2017

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Analytical Chemistry

An Automated Trapping Column Exchanger for High Throughput Nanoflow Liquid Chromatography Sandra E. Spencer,1 Thomas N. Corso,2 James G. Bollinger,1† Clark M. Henderson,3 Andrew N. Hoofnagle,3 Michael J. MacCoss1* 1

Department of Genome Sciences, University of Washington School of Medicine, Box 355065, Seattle, WA, 98195-5065

2

CorSolutions, LLC., Cornell Business and Technology Park, 95 Brown Road, Box 1007, Ithaca, NY, 14850-1294

3

Department of Laboratory Medicine, University of Washington School of Medicine, Box 357110, Seattle, WA, 98195-7110

ABSTRACT: As compared to conventional high performance liquid chromatography (HPLC) techniques, nanoflow HPLC exhibits increased sensitivity and limits of detection. However, nanoflow HPLC suffers from low throughput due to instrument failure (e.g. fitting fatigue and trapping column failure), limiting the utility of the technique for clinical and industrial applications. To increase the robustness of nanoflow HPLC, we have developed and tested a trapping column exchanging robot for autonomous interchange of trapping columns. This robot makes reproducible, automated connections between the active trapping column and the rest of the HPLC system. The inter-trapping column retention time is shown to be sufficiently reproducible for scheduled selected reaction monitoring assays to be performed on different trapping columns without rescheduling the selection windows.

Over the past few decades, high performance liquid chromatography (HPLC) coupled to mass spectrometry (MS) has become an indispensable analytical technique in the clinical laboratory.1,2 Improvements in assay performance for these applications can be attained by miniaturization of HPLC to nano-HPLC via reduction of the analytical column inner diameter. Here, nano-HPLC is defined as HPLC using solvent flow rates of less than 1 µL/min3 and analytical column inner diameters of 20-100 µm4. Miniaturization of HPLC in this manner decreases the chromatographic dilution of the analyte band, improving the overall sensitivity and limit of detection.3,4 The sensitivity of nano-HPLC-MS is further improved by directly coupling the nano-HPLC output to a nanoelectrospray ionization source (nano-ESI).5 In comparison to conventional ESI, nano-ESI results in an increased ionization efficiency due to decreased droplet size.6 In addition, the reduced flow rate of nanoflow HPLC is compatible with nano-ESI and as such the HPLC column output does not need to be split as for conventional HPLC,7 further increasing the sensitivity of the analysis. The innately high sensitivity of nano-HPLC results in improved detection limits and decreased sample loading requirements, making nanoflow HPLC-MS desirable for sample limited applications such as clinical analyses. High throughput implementation of nano-HPLC-MS in the clinical laboratory is currently instrumentation limited.1,8 One specifically impactful limitation of the robustness of nano-HPLC is system clogging due to packing material fouling (an example of new packing material is

shown in Figure 1A and fouled packing material is shown in Figure 1B). In these situations, an experienced operator is required since capillary breakage (Figure 1C and 1D) or leaks in the pressurized system can arise from over- or under-tightening of zero-dead-volume fittings, respectively. In an effort to reduce the primary limitations of nano-HPLC and increase the amenability of the technique to high throughput clinical applications, we have designed and implemented an autonomous trapping col-

Figure 1. Example of A. new column packing material B. used column packing material C. damaged trapping column frit and D. damaged open trapping column end.

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umn exchanging robot. The device is conceptually based upon a previously published Plug-and-Play HPLC-MS ion source that was designed to automate the formation of zero-dead-volume connections between the capillary trapping column, analytical column, and nano-ESI emitter.9 In this system, linear compression is used to make zero-dead-volume connections between the trapping column and the autosampler or analytical column, eliminating complications associated with making connections manually or implementing common LC hardware such as a multiport valve. The trapping column is autonomously exchanged by calling a Windows batch script from the mass spectrometry vendor software. Here we demonstrate the reproducibility and utility of the trapping column exchanger for scheduled selected reaction monitoring (SRM) assays. Experimental Section Reagents. HPLC grade isopropyl alcohol, acetonitrile, methanol, and water, laboratory grade formic acid (FA), and ammonium bicarbonate (AmBic) were purchased from Fisher Scientific (Waltham, MA). PierceTM Peptide Retention Time Calibration Mixture (PRTC) was from ThermoFisher Scientific (San Jose, CA). Human apolipoprotein A1 (ApoA1) uniformly 15N-labeled and enriched (isotopic enrichment >98% 15N by LC/MS, purity > 90% by SDS-PAGE) was purchased from Cambridge Isotope Laboratories (Tewksbury, MA). Trifluoroethanol (TFE), dithiothreitol (DTT), iodoacetamide (IAA), and ammonium hydroxide were purchased from Sigma Aldrich (St. Louis, MO). Hydrochloric acid was from JT Baker (Phillipsburg, NJ). Worthington trypsin was used for proteolytic digestion (Lakewood, NJ). Sample Collection. The collection and use of human specimens was approved by the Human Subjects Division at the University of Washington. Capillary dried blood spots (DBS) were acquired from human donors using sterile incision lancets [Tenderfoot (ITC Med) or Quickheel (BD)]. The lancet was used to prick the subject’s finger of choice to produce an initial drop of blood that was discarded. The second drop of blood was placed directly onto Whatman 903 filter paper (GE Healthcare BioSciences Corp., Westborough, MA) without touching the finger to the paper. Five DBS specimens were collected in this manner from each volunteer five times in one day. DBS samples were dried for 3 hours at 25 °C, placed in a re-sealable plastic bag pre-charged with 300 grams of calcium sulfate desiccant, and transferred to -80 °C until processing. Sample Preparation. DBS samples were equilibrated to room temperature. A BSD700 semi-automated dried sample puncher (BSD Robotics, Brisbane, QLD, Australia) was used to take a 3.2 mm (1/8”) diameter punch from each DBS. Each punch was transferred to a 96-well microtiter plate (Grenier Bio-One, Monroe, NC) and the plate was sealed using a silicon plate mat (Grenier Bio-One, Monroe, NC) and stored at -80°C until digestion. Prior to digestion, DBS punches were equilibrated to

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room temperature. Stainless steel forceps were used to transfer a single 3.2 mm DBS punch to a 1.5 mL Eppendorf Safe-Lock low protein binding microfuge tube (Fisher Scientific, Waltham, MA). Each DBS punch was wetted with 70 µL of 14 ng/µL 15N-labeled ApoA1 in 100 mM AmBic. TFE (70 µL) was added to the solution and the sample was vortexed then incubated at 65 ℃ and 1400 RPM for 1 hr to physically denature and extract proteins from the paper. DTT was added to the analyte solution (2 µL of 0.5 M in 100 mM AmBic) and the mixture was incubated at 65 ℃ and 1400 RPM for 1 hr to reduce disulfide bonds. Alkylation was performed by adding 8 µL IAA (0.5 M in 100 mM AmBic) and incubating the solution in the dark at room temperature for 30 minutes without shaking. DTT (2 µL, 0.5 M in 100 mM AmBic) was added to the sample to quench the alkylation reaction. TFE was diluted to 5.5% (v/v) by adding 1200 µL of 27.5 mM AmBic with 0.1 µM of internal standard peptides to the mixture. Trypsin (20 µL, 2 mg/mL in 1 mM HCl) was added to the mixture and digestion was performed at 37 ℃ and 1400 RPM. After 2 hr of digestion, a second aliquot of 20 µL of 2 mg/mL trypsin was added to the mixture. Digestion was halted after a total of 20 hr by the addition of 3 µL of 88% FA. To remove non-volatile salts and other contaminants that may interfere with HPLC-ESI-MS analysis, solid phase extraction (SPE) was performed using Waters Oasis 60 µm/30 mg MCX plates (Milford, MA) on a Biotage 96+ Positive Pressure Manifold (Charlotte, NC) with house nitrogen gas. The SPE resin was washed and conditioned with 1 mL of methanol at 2 psi, 1 mL 2.8% ammonium hydroxide in water at 3 psi, 2 mL of methanol at 2 psi, and 3 mL of 0.1% FA in water at 9 psi. Equilibration of the SPE resin was performed using 1 mL of 0.1% FA in water at 3 psi and 0.1% FA in methanol at 2 psi. Digested DBS samples were centrifuged at 16,000 RCF and room temperature for 10 minutes. A 1.25 mL aliquot of the supernatant was loaded at 0.5 psi onto SPE resin. Peptides were eluted with 1 mL of 2.8% ammonium hydroxide in methanol into a 1.5 mL 96-well plate at 0.5 psi for 2 min and 3 psi for an additional 5 min. Eluates were transferred to 1.5 mL Eppendorf Safe-Lock low protein binding microfuge tubes and desolvated under vacuum via centrifugal evaporation. Samples were reconstituted in 250 µL of 95:5 water/acetonitrile with 0.2% TFA for 2 hr at room temperature in a thermomixer at 1400 RPM. Digests were diluted to 500 µL with 0.2% TFA in water. The final analytical sample consisted of a pool of 25 reconstituted DBS samples donated on the same day by a single individual. PRTC was spiked into the mixture at a final concentration of 17 mM. HPLC-Tandem Mass Spectrometry (MS/MS). NanoHPLC-MS/MS was performed using a NanoAcquity LC system (Waters, Milford, MA) coupled to a TSQ Vantage triple quadrupole mass spectrometer (ThermoFisher Scientific, San Jose, CA). Solvent A was 0.1% formic acid in water and solvent B was 0.1% formic acid in acetonitrile. Kasil frits were made in fused silica microcapillary tubing


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(150 µm i.d., Phenomenex, Torrence, CA) using Kasil 1 (29.1% potassium silicate solution, PQ Corporation, Valley Forge, PA) mixed 4:1 with formamide (Bethesda Research Labs, Gaithersburg, MD). The mixture was centrifuged at 10,000 rpm for 2 min then the microcapillary tubing was dipped into the solution. Capillaries were dried flat overnight at 80 °C and frits were cut to approximately 2 mm. To ensure reproducible packing, fritted trapping columns were self-packed in batches with 4 µm Jupiter Proteo 90 Å C12 particles in 80/20 isopropyl alcohol/acetonitrile and cut to 5.0 ± 0.2 cm. Helium gas was used (99.995% purity, Praxair, Danbury, CT) at a back pressure of 500 psi. The polyimide coating was removed from a portion of a fused silica microcapillary tube (75 µm i.d., 18 cm) and a micropipette puller (Sutter Instruments Model P-2000, Novato, CA) was used to create a pulled tip at one end of the tubing (heat = 260, fil = blank, vel = 45, del = 200, pul = blank; heat = 210, fil = blank, vel = 25, del = 200, pul = blank; heat = 180, fil = blank, vel = 20, del = 128, pul = blank). The pulled tip silica capillary analytical column was self-packed with Dr. Maisch GmbH ReproSil-Pur 120 C18-AQ 3 µm C18 particles (Ammerbuch, Germany) in 80/20 isopropyl alcohol/acetonitrile (back pressure 1000 psi, 99.995% He) and maintained at 50.0 ± 0.1 ℃ throughout the analysis. After a trapping column was replaced, 1 µL of 0.1% formic acid in water was injected onto the column and the system was washed for 2 min with 80% B. The solvent composition was changed to 50% B over the course of 1 min and maintained for 5 min. To equilibrate the system, the solvent composition was ramped to 2% B over the course of 1 min and this solvent composition was maintained for 11 min. Washing and equilibration was performed at a flow rate of 0.500 µL/min. Analyte (1 µL) was subsequently injected into the system and trapped on the trapping column for 5 min, during which time hydrophilic compounds such as buffer salts were rinsed to waste using 2% B at a flow rate of 2 µL/min. The analytical separation was performed at a flow rate of 0.250 µL/min. A 30 min linear gradient from 2 – 30% B was performed. The solvent composition was increased to 60% B over the course of 5 min and maintained at 60% B for 5 min. The solvent composition was then increased to 95% B over the course of 1 min and the flow rate was increased to 0.500 µL/min to wash the system for 5 min prior to decreasing the solvent composition to 2% B over the course of 1 min to equilibrate the system for 13 min. MS data were collected throughout the analytical gradient, wash, and equilibration steps. Analyte ions were formed by nano-ESI by applying 2.0 kV to the capillary inlet of the mass spectrometer. The capillary temperature was maintained at 275 °C. A scheduled SRM mass spectrometry experiment to monitor 1338 transitions was performed using a cycle time of 1.5 seconds. Tuned s-lens values were used and the full width half maximum (FWHM) resolution for Q1 and Q3 was set to 0.70. The collision gas pressure was 1.5 mTorr. Scheduling was performed in Skyline using a custom retention time database and 5 min retention time windows. The

collision energy (CE) was determined as: CE = 0.0334(m/z) + 3.141 or CE = 0.0291(m/z) + 3.9543 for doubly and triply charged ions, respectively. The average retention time of six injections of the PRTC peptides in DBS matrix was used to schedule the SRM experiment. Trapping Column Exchanger. A CAD illustration for the trapping column exchanger is shown in Figure 2. The trapping column cartridge A holds up to four trapping columns and is mounted on translatable stage B. Servo motor C controls the axial movement of the stage for trapping column selection. Servo motor D controls the perpendicular motion of the mount for fitting E, making or breaking the zero-dead-volume connection between the active trapping column and the rest of the system. A mounting bracket (F) was designed such that the trapping column exchanger mounts directly on the capillary column heater (G) attached to the atmospheric inlet of the mass spectrometer. A video of the robot exchanging the trapping column in real speed is shown in Supporting Information Movie S1. Servo motor D draws fitting E back, separating the trapping column cartridge A from the flow path. A different trapping column is moved into

Figure 2: CAD drawing of the trapping column exchanging robot. Key parts discussed in the text are A. trapping column cartridge B. translatable stage C. axial servo motor D. perpendicular servo motor E. autosampler line fitting F. mounting bracket G. capillary column heater and H. analytical column fitting.



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alignment with the flow path by servo motor C and servo motor D drives fitting E forward toward fitting H, reforming the zero-dead-volume connection between the new trapping column, the autosampler and analytical column. Provided in Supporting Information Figures S1 – S3 are CAD drawings of a side view of the column exchanger-heater assembly, a top view of the column exchanger, and an angled view of the column exchanger, respectively. The design of the trap changer incorporates linear slides that accurately align the column ends to the bores of the inlet and outlet fittings during the compression sealing process. As this sealing process is completely linear, any twisting or rotational motion of the capillary column is eliminated. Furthermore, the compression force is precisely controlled by the motion drive system. The resulting connections are reproducible and circumvent any risk of damaging the column ends (such as shown in Figures 1C and 1D). Results and Discussion System Conditioning. Consecutive 1 µL injections of PRTC in DBS were performed for seven days. The extracted ion current trace for nano-HPLC-MS of five PRTC peptides is shown in Figure 3A. Injections 1-5 were quality control standards and blank injections and these injections are not shown. The retention times of the PRTC peptides SSAAPPPPPR and ELASGLSFPVGFK are displayed in Figure 3B and 3C, respectively, from the 6th to the 138th injection. The height of the shaded box represents the full width at the base of the chromatographic peak and the horizontal line through the box represents the apex of the peak. The vertical bars inside the shaded box indicate the peak FWHM. As can be seen in Figure 3B for the PRTC peptide SSAAPPPPPR, multiple injections of

Figure 3: A. representative chromatogram for the separation of PRTC peptides in reconstituted dried blood spot B. plot of the retention time of the standard peptide SSAAPPPPPR and C. ELASGLSFPVGFK over the course of 7 days of constant analysis. In panels B-C the height of the grey box represents the peak full width at base, the vertical line represents the full width of the peak at half maximum, and the horizontal line through the shaded box represents the peak apex.

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PRTC in DBS are required to fully condition the trapping and analytical columns. The retention time of the PRTC peptide SSAAPPPPPR is more than two standard deviations higher than the average retention time for all injections prior to the 11th injection and more than three standard deviations higher than the retention time average for all injections prior to the 10th injection. These results are displayed graphically in Supporting Information Figure S4A. As a second example, the retention time over the course of the continual seven-day analysis of the PRTC peptide ELASGLSFPVGFK is plotted in Supporting Information Figure S4B. For ELASGLSFPVGFK, 9 injections of DBS spiked with PRTC are required for the retention time to fall within three standard deviations of the average retention time (41.7 ± 0.1 min) and 11 injections are required before the retention time is within two standard deviations of the arithmetic mean. This conditioning period is of note for experiments requiring trapping column exchange, especially for scheduled SRM experiments, and should be evaluated for each HPLC system prior to sample analysis. Assessing the Chromatographic Reproducibility of the System. As an example of the retention time reproducibility for the analysis of an endogenous peptide from the reconstituted DBS sample, the measurement of the retention time of the peptide DLATVYVDVLK from endogenous ApoA1 is shown in Figure 4A. The average apex of the chromatographic peak is 42.7 min with a standard deviation of 0.8 min (48 s). The range of values of the peak apex is 42.5 – 42.9 min and the 95% confidence interval in the mean (N = 129, t = 1.98) is ± 0.01 min (0.6 s). A box-and-whisker plot depicting the overall retention time reproducibility for the measurement of the endogenous ApoA1 peptide DLATVYVDVLK is shown in Figure 4B. The vertical lines represent the minimum and maximum retention time, the horizontal line represents the median retention time, and the boundaries of the upper and lower portions of the box represent the first and third quartile of the retention time measurements, respectively. In summary, the data shown in Figures 3 and 4 highlight the reproducibility of this system after a period of conditioning; by using the trapping column as a rapid, in-line sample clean-up and filtration step the analytical column is protected.

Figure 4: For the endogenous peptide DLATVYVDVLK from ApoA1 A. a plot showing the retention time for each injection over the course of seven days and B. a box-andwhisker plot of the average retention time over the seven days of analysis. Chromatographic Reproducibility of Different Trapping Columns. To qualitatively assess the reproduc-


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ibility of the system when the trapping columns are exchanged, the total ion current (TIC) trace for the analysis of DBS spiked with PRTC using three different trapping columns is overlaid in Figure 5. An expanded 2.5 min region of the chromatogram is displayed in the inset of Figure 5 showing the reproducibility of peak shape when different trapping columns are used. A more quantitative measure of the reproducibility of the HPLC system is shown in Figure 6. Each column was equilibrated and conditioned as described previously and DBS spiked with PRTC was injected six times onto each column. Shown in Figure 6A – 6C are the peak areas for the PRTC peptide ELASGLSFPVGFK injected onto three different trapping columns in sets of six injections (number 19 – 36). Shown in Figure 6D is the average peak area and standard deviation for each set of six injections on the three separate columns. A one-way ANOVA of the data presented in Figure 6 show that there is not a significant difference between the peak areas observed for a set of injections on different trapping columns or for different sets of injections onto a single column (α = 0.01, p = 0.02).

Figure 5: Trace of the TIC from injections of PRTC in DBS on three separate trapping columns. Inset is an expanded 2.5 minute region of the chromatogram.

Utility of the Trapping Column Exchanger for Scheduled Assays. To assess the utility of the trapping column exchanger for the replacement of trapping columns during scheduled assays, a SRM method was scheduled using retention time values from reconstituted DBS samples spiked with PRTC standards as determined on Trapping Column 1. A 5 min retention time window was used for each transition and the scheduled SRM experiment was performed on 4 different trapping columns (Trapping Column 1-4). Of the original 1338 transitions monitored it was manually determined that 857 transitions (184 peptides, 63 of 72 proteins) were reproducibly detected within a 5-minute window for all four trapping columns. A draftsman plot summarizing the results of this experiment is shown in Figure 7. The peak area for each of the 184 peptides (determined as the sum of the peak areas for each transition measured for that peptide) as measured on one trapping column is plotted against

Figure 6. Peak area for the peptide ELASGLSFPVGFK from PRTC in reconstituted DBS for each injection on A. trap 1, B. trap 2, and C. trap 3. The average and standard deviation for each set of six injections on the three trapping columns is shown in D. A one-way ANOVA showed no statistical difference between the mean intensities observed from each trap (α = 0.01, p = 0.02).

each other trapping column. The values of the slope of the line and R2 values are shown for each pair of trapping columns. The inset of each panel in Figure 7 shows an enlargement of the low peak area region of each comparison. The slope of the line of best fit for each plot was found to be 1.0 with R2 values greater than 0.999, indicating that the measurement of peptide peak area is reproducible between trapping columns. The average difference between peak areas on any two columns is less than 10% and the average standard deviation of the percent difference in peak area between any two columns is not greater than the average standard deviation of the percent difference in the peak area on a single column (Figure S5). These results suggest that for reproducibly packed trapping columns, a fouled trapping column may be exchanged during a scheduled assay without having to reschedule the assay. Conclusions The trapping column exchanging robot designed for high throughput nano-LC assays results in reproducible retention times for both standard and endogenous peptides after a period of conditioning (typically 10 or 11 injections of complex sample) for at least 7 days of continuous analysis. The peak area is reproducible after the conditioning period and if trapping columns are reproducibly packed, different trapping columns may be used for scheduled SRM assays without the need to re-schedule between columns. The high reproducibility in peak area and retention time could be used to further increase the throughput of nano-LC for clinical assays by conditioning and loading the trapping columns offline, effectively decreasing the analysis time for each sample.



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This work was supported, in whole or in part, by NIH Grants R01 GM107142, P41 GM103533, R44 RR024628, and R44 GM103386. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.

REFERENCES

Figure 7: Draftsman plot for the comparison of the peak area for each of 184 peptides measured on four different trapping columns. Each point represents the average of the inter-trap replicates. The numbers correspond to the slope and standard deviation from the regression analysis. The displayed in the inset of each panel is an expansion of the low peak area region.

ASSOCIATED CONTENT

1. Hoofnagle, A.N., Laha, T.J., Rainey, P.M., Sadrzadeh, S.M.H. Am. J. Clin. Pathol. 2006 126, 880-887. 2. Grebe, S.K., Singh, R.J. AACB. 2011, 32, 5-31. 3. Sneekes, E.-J., Rieux, L., Swart, R. http://www.dionex.com/en-us/webdocs/114984-WP-RSLCnanoWP70817.pdf. 2016, 1-6. 4. Rieux, L., Sneekes, E.-J., Swart, R., Swartz, M. LC GC N. Am. 2011, 29, 926. 5. Shen, Y., Zhao, R., Berger, S.J., Anderson, G.A., Rodriguez, N., Smith, R.D. Anal. Chem. 2002, 74, 4235-4249. 6. Wilm, M.S., Mann, M. Int. J. Mass Spectrom. Ion Processes. 1994, 136, 167-180. 7. Kumar, P.R., Dinesh, S.R., Rini, R. WJPPS. 2016, 5, 377-391. 8. Hoofnagle, A.N., Wener, M.H. J. Immunol. Methods. 2009, 347, 3-11. 9. Bereman, M.S., Hsieh, E.J., Corso, T.N., Van Pelt, C.K., MacCoss, M.J. MCP. 2013, 12, 1701-1708.

Supporting Information The Supporting Information is available free of charge on the ACS Publications website. Movie S-1. Video of the robotic trapping column exchanger moving between trapping columns. (trapexchange.mp4). Figure S-1. Side view of the trapping column exchangerheater assembly. Figure S-2. Top view of the trapping column exchanger. Figure S-3. Angled view of the trapping column exchanger. Figure S-4. Plot of the retention time for A. SSAAPPPPPR or B. ELASGLSFPVGFK from PRTC in reconstituted DBS. Figure S-5. Bland Altman-style plots to compare each of four trapping columns.

AUTHOR INFORMATION Corresponding Author *Michael J. MacCoss: [email protected]

Present Addresses †Jim G. Bollinger: Campus Box 8111, 660 S. Euclid Ave, St. Louis, MO, 63110

Author Contributions The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript.

Notes The device described in this work is currently being marketed by CorSolutions under the product name “CorConneX” and Thomas N. Corso is the Chief Technical Officer of CorSolutions. The MacCoss laboratory has a sponsored research agreement with ThermoFisher.

ACKNOWLEDGMENT


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