B American Society for Mass Spectrometry (outside the USA), 2013DOI: 10.1007/s13361-013-0749-z

J. Am. Soc. Mass Spectrom. (2014) 25:30Y36

RESEARCH ARTICLE

Picoelectrospray Ionization Mass Spectrometry UsingNarrow-Bore Chemically Etched Emitters

Ioan Marginean,1 Keqi Tang,1 Richard D. Smith,1 Ryan T. Kelly2

1Biological Sciences Division, Pacific Northwest National Laboratory, Richland, WA 99352, USA2Environmental Molecular Sciences Laboratory, Pacific Northwest National Laboratory, Richland, WA 99352, USA

Abstract. Electrospray ionization mass spectrometry (ESI-MS) at flow rates below~10 nL/min has been only sporadically explored because of difficulty inreproducibly fabricating emitters that can operate at lower flow rates. Here wedemonstrate narrow orifice chemically etched emitters for stable electrospray atflow rates as low as 400 pL/min. Depending on the analyte concentration, weobserve two types of MS signal response as a function of flow rate. At lowconcentrations, an optimum flow rate is observed slightly above 1 nL/min,whereas the signal decreases monotonically with decreasing flow rates at higherconcentrations. For example, consumption of 500 zmol of sample yielded signal-to-noise ratios ~10 for some peptides. In spite of lower MS signal, the ion

utilization efficiency increases exponentially with decreasing flow rate in all cases. Significant variations inionization efficiency were observed within this flow rate range for an equimolar mixture of peptide, indicatingthat ionization efficiency is an analyte-dependent characteristic for the present experimental conditions.Mass-limited samples benefit strongly from the use of low flow rates and avoiding unnecessary sampledilution. These findings have important implications for the analysis of trace biological samples.Key words: Nanoelectrospray, nano-ESI, Quantitation, Mass-limited analysis

Received: 15 July 2013/Revised: 3 September 2013/Accepted: 5 September 2013/Published online: 12 October 2013

Introduction

E arly mass spectrometric measurements usingelectrospray ionization (ESI) established that for a

wide range of liquid flow rates, the signal depends primarilyon analyte concentration and is largely independent of flowrate. In this concentration-sensitive response regime [1], theelectrospray efficiency is greatest at the lowest achievableflow rate, where the smallest amount of analyte is consumed[2]. Poor ionization/desolvation efficiency at higher flow rates,together with the limited ion sampling capacity of the MS inletcapillary/orifice, are mainly responsible for a plateau in signalintensity despite increasing amounts of analyte delivered. Asteep signal decline has been also noted at liquid flow ratesclose to the mL/min range [3, 4], which could be alleviated tosome extent by pneumatic nebulization [5].

Low-flow electrosprays were recognized [6–9] for theirability to generate gas-phase ions with high efficiency becauseof smaller primary droplets generated at the electrospraysource. At sufficiently low liquid flow rates, depending onsample composition, the ESI-MS response becomes mass/

flow-sensitive in that the signal decreases with decreasing flowrate. Several studies show that MS signal decreases monoton-ically with decreasing flow rate [10–17], whereas others showan optimum flow rate at which MS signal reaches maximumbefore a steep decline at lower flow rates [18–24]. However,even with lower corresponding signal, smaller flow ratesalways display higher ion utilization efficiencies (the propor-tion of analyte molecules in solution converted to gas-phaseions) [14–18]. Minimal charge competition [24] and ionizationsuppression [14] effects have also been demonstrated at lowflow rates.

Low-flow electrosprays can be operated in two basicscenarios, self-fed or pump-driven. The term nanoelectrospraywas originally applied to self-fed electrosprays [25], but it isnow commonly applied to all electrosprays in the flow rangebetween a few to a few hundred nL per min. In self-fed mode,the sample-containing liquid is loaded inside the emittercapillary and the high voltage is applied via a wire in directcontact with the liquid or a conductive emitter coating. In theseconditions, the liquid flow rate is determined by the balanceamong the capillary forces, surface tension, and electrical shearstress. This self-fed electrospray configuration allowsextended analysis time even for minute amounts of liquidCorrespondence to: Ryan T. Kelly; e-mail: ryan.kelly@pnnl.gov

samples [25–28] and has become part of standard modusoperandi following gel electrophoresis separation of proteins[29]. Despite evident benefits, self-fed electrosprays have majordrawbacks that include poor reproducibility and signal stability,as well as incompatibility with online separation techniques.MSsignal variation due to electrophoretic effects is also observed asthe sample is consumed [9]. In addition, the flow rate dependson both the emitter geometry and the applied voltage, making itdifficult to ascertain. The flow rate dependence on geometry canbe avoided by using a pump to deliver liquid to the emitter whileintroducing high voltage contact at a metal union or elsewhere inthe flow path. Online coupling with liquid chromatographic(LC) [30–32] and electrophoretic [17, 26] separations at flowrates below 20 nL/min have been demonstrated.

With both self-fed and pump-driven nanoESI, the standardmode of emitter fabrication has been to heat and pull glass orfused silica capillaries to a fine point with an opening as smallas 1 μm. The process offers limited reproducibility, and theinternal taper of the capillaries and narrow orifices can lead torapid fouling or clogging of the emitters. Faultless andreproducible emitter geometry becomes increasingly stringentto operate at lower flows, which has left the exploration of flowrates below ~10–20 nL/min extremely limited [18, 28]. Wehave developed a chemical etching process for fused silicacapillary emitters that improves reproducibility and avoids theclog-prone internal taper [33]. Because the capillary walls arevery thin at the orifice, a larger opening can support lower flowrates. For example, 20 μm i.d. etched emitters can support flowrates as low as 20 nL/min [34], which is not feasible usingheated and pulled emitters. Narrower bore etched emittersshould support even lower flow rates, but have not previouslybeen used to explore the subnanoflow regime.

A systematic investigation of ESI below the flow ratescommonly employed for nanoelectrospray is of both funda-mental and practical interest. It has been hypothesized that atsufficiently low flow rate, ionization efficiency will approach100 % for all analytes in solution, thus providing maximumpossible sensitivity as well as uniform response, in which allanalytes of a given concentration exhibit the same signalintensity [14, 24, 35, 36]. In addition, there is interest inultralow flow electrosprays for their potential to improveproteomic and metabolomic analysis of trace biologicalsamples, including single cells. MS has been used to profileproteins, peptides, and metabolites from single cells [37–43],but challenges in efficient sample preparation and detectionsensitivity have limited most studies to large or specialized celltypes. The small volume of a typical mammalian cell (a fewpL) implies that even at low nanoESI flow rates, the analysistime will either be extremely brief or the extent of dilution willbe very large. Sample dilution enables extended acquisitiontimes and the signal can be averaged to improve its signal-to-noise ratio (S/N); however, the extent of dilution enabling thebest overall data generation is not obvious. Exploration of thesub-nanoESI flow regime will thus inform the experimentaldesign to address both the potentially enhanced sensitivity andimproved volume compatibility with trace samples.

Here we utilize 2-μm-i.d. chemically etched emitters forreproducible electrospray operation in the largely unexploredultra-low flow rate range extending to 400 pL/min. Wedemonstrate that sample dilution should be minimized in manycases for improved analytical performance. Surprisingly,uniform response was not achieved with the set of analytes (amixture of peptides) considered in this study even at the lowestnanoESI flow rates. This set of experiments should inform notonly on the optimum flow rate but also on trace samplepreparation, delivery, and analysis.

ExperimentalReagents

All chemicals were purchased from Sigma-Aldrich (St. Louis,MO, USA) unless otherwise specified. Water was purifiedusing a Barnstead Nanopure Infinity system (Dubuque, IA,USA). A stock solution containing a 10 μM mixture of ninepeptides (angiotensin I, angiotensin II, bradykinin, fibrinopep-tide A, kemptide, mellitin, neurotensin, angiotensinogen, andSubstance P) was prepared from the corresponding 1mg/mL stocksolutions. Solutions of 1,000, 300, 100, 30, 10, 3, and 1 nM werefreshly prepared by successive dilution before each experimentin a 1:9 acetonitrile:water mixture with 0.1 % formic acid.

Infusion

Depending on the desired flow rate range (see experimentalconditions in Table 1), the solutions were loaded into 10, 25, or100 μL syringes (Hamilton, Reno, NV, USA) and infused by acomputer-controlled syringe pump (PHD 2000; HarvardApparatus, Holliston, MA, USA) through a fused silicacapillary (30 μm i.d., 360 μm o.d.; Polymicro Technologies,Phoenix, AZ, USA) transfer line. In all cases, the syringe/pumpcombination was operated at a flow rate at least 10 times higherthan the minimum recommended by the manufacturer. Toestablish the desired ultralow flow rates, we used metal unionsand ferrules for all connections, minimized the length of thetransfer line to 15 cm, and carefully eliminated air bubbles fromthe syringe. Before each set of measurements, the electrospraywas run overnight with the flow rate set at 1 nL/min. Toestablish optimum experimental conditions, the flow rate waschanged in 30-min steps of 100 pL/min down to 300 pL/min,then back to 1 nL/min. The response of the MS signal and thelack of hysteresis in these exploratory experiments suggested

Table 1. Experimental Conditions

Description Flow rate Emitter size Syringe Voltage Step

(nL/min) (μm) (μL) (V) (nL/min)

Ultralow 0.3–1 2 10 1,300 0.1Low 1–5 2 10 1,400 0.5Intermediate 2–40 5 25 1,700 1Large 20–400 10 100 1,900 10

I. Marginean et al.: Picoelectrospray Ionization Mass Spectrometry 31

that the flow rates set on the syringe pump were fullyestablished in less than 5 min. The bulk of measurements wereconducted in the range of 1 nL/min down to 100 pL/min in20-min steps of 100 pL/min after overnight stabilization of the1 nL/min flow rate. The timing was relaxed to 15 min at eachflow rate with steps of 0.5, 1, and 10 nL/min at low,intermediate, and large flow rates, respectively (see Table 1).

Mass Spectrometry

Mass spectra were acquired on an orthogonal time-of-flightMS instrument (G1969A LC/MSD TOF; Agilent Technol-ogies, Santa Clara, CA, USA) modified with a dual ionfunnel interface [44] and multi-inlet capillary interfaceheated to 120 °C. Electrospray emitters with internaldiameters of 2, 5, and 10 μm were fabricated by chemicallyetching sections of 150-μm-o.d. fused silica capillary tubing(Polymicro Technologies) as described previously [33]. Theelectrospray voltage was applied at the stainless steel unionconnecting the emitter to the transfer line.

Data Analysis

MS intensity data spanning approximately 2.5m/z units (100data points) preceding the analyte peaks of interest were used tocalculate average background levels, N, and standard devia-tions, σN. Signal-to-noise ratios were then calculated asS=N ¼ I − N

� �=3σN , where I stands for the intensity of the

analyte peak.

Results and DiscussionOperating an electrospray ion source at flow rates below thosenormally achieved by nanoESI was expected to lead to furthergains in sensitivity; such gains in combination with new samplehandling techniques will be crucial for extending ESI-MS tosmaller biological samples than can be analyzed at present.Heated and pulled capillary emitters lack the process control toreproducibly operate below 10 nL/min, and self-fedelectrosprays exhibit an interdependence between flow rateand applied voltage, making flow rate determination difficult.In contrast, the highly reproducible chemical etching processcombined with pump-driven flow employed in this workenable the sub-nanoflow regime to be systematically charac-terized for the first time. Figure 1 shows a photograph of a

chemically etched emitter with a 2 μm i.d. used to achieve thelowest flow rates in this study.

Several precautions were taken to ensure that the flow ratedelivered to the electrospray emitter was accurate. Theseincluded always operating the syringe/pump combination atflow rates at least a factor of 10 higher than the manufacturer’sminimum recommendations, stabilizing the flow over-night at 1 nL/min, and testing for hysteresis in the MS signalwhile increasing and decreasing flow rates in the rangeof 0.3–1 nL/min. The responsiveness of the MS signal tochanges in pump settings, the high degree of reproducibility,and the lack of hysteresis served to validate the approach.

Signal stability was measured at different flow rates todetermine the lowest practical flows achievable with the 2-μm-i.d. emitters. Figure 2 shows ion traces for 1 μM doublycharged fibrinopeptide (m/z = 768.85) at various flow rates.

Figure 1. Photo of a 2 μm i.d. chemically etched fused silicaelectrospray emitter similar to those used for MSmeasurementsat low and ultralow (300 pL/min–5 nL/min) liquid flow rates

0 3 6 9 12 150

20

40

60

80nL/min (± RSD)

Inte

nsi

ty (

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. x 1

03 )

Time (min)

0.3 (± 39.7%)

3 (± 8.3%)

30 (± 7.2%)

300 (± 4.5%)

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1000 (± 9.3%)

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400 (± 14.7%)

300 (± 39.7%)

Inte

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ty (

a.u

. x 1

03 )

Time (min)

(a)

(b)

Figure 2. Stability of fibrinopeptide signal (m/z = 768.85) forelectrospray operation at 0.3–300 nL/min (a) and 300–1,000 pL/min (b)

32 I. Marginean et al.: Picoelectrospray Ionization Mass Spectrometry

When the flow rate decreased from 300 nL/min, the signalstability decreased monotonically but maintained an acceptablelevel until 300 pL/min, where the signal relative standarddeviation (RSD) increased dramatically to 40 %. As such,400 pL/min was considered to be the lowest flow rate achievablefor these emitters. The corresponding signal intensity decreasedonly by a factor of ~25 despite a reduction of three orders ofmagnitude in the amount of analyte delivered to the emitter. Thehigher ion utilization efficiency associated with low flowelectrosprays is discussed below.

Depending on the application, ESI-MS analyses can beeither concentration- or mass-limited. To investigate concen-tration-limited performance of the ultralow flow regime andcompare it with more conventional analyses, an equimolarmixture of peptides was measured across a broad range of flowrates (0.3–400 nL/min) and concentrations (1–1,000 nM).Figure 3a shows the 2-min-averaged signal intensity form/z = 530.79 (bradykinin 2+ peak) measured for the 10 nMpeptide mix. Emitters with 2–10 μm i.d. were selected for thedifferent, overlapping flow ranges (see Table 1). The resultsshow intensity optima for each emitter in the correspondingflow rate range (e.g., ~ 1, ~20, and ~200 nL/min for the 2, 5,and 10 μm emitters, respectively), which may be related toefficient and reproducible charged droplet formation. Gradualloss of electrospray stability and less efficient liquid dispersioncan contribute to the drop in signal at flow rates lower and higherthan optimum, respectively. The data points corresponding tosuboptimal performance were eliminated from the subsequentplots for clarity.

Figure 3b includes similar results for the 1, 10, 100, and1,000 nM peptide mix solutions. The signal was found todecrease monotonically with the flow rate for the highestconcentration mixtures while a maximum in signal intensity isobserved at ~1 nL/min for lower concentrations. Both trendshave been observed previously [10–24], but the dependence onconcentration had not been identified. The optimum seems to berelated to striking the right balance between the flow rateensuring the most favorable ionization/desolvation conditions(lower flow rates) and providing sufficient analyte for analysis(larger flow rates). This optimum may change somewhat whenusing liquids exhibiting different physical properties (volatility,surface tension, viscosity, etc.). Importantly, for the lowestconcentration studied (1 nM), the only detectable signal wasobserved around 1 nL/min, whereas higher S/N are obtained atincreased flow rates at larger concentrations (Figure 3c). Therelative ion utilization efficiency (also included in Figure 3c for10 nM bradykinin) was calculated by normalizing the S/N withthe flow rate and shows an exponential growth with decreasingflow rate. The results in Figure 3 imply that for analyses that areconcentration-limited but not limited in terms of available mass,typical “nanospray” flow rates are often sufficient and the greaterefforts to operate at ultralow flow rates may not be justified.

In the case of mass-limited studies, the use of ultralowflow rates are expected to become increasingly important.Mass-limited analyses are common for biological systemsand can arise from the scarcity of material (e.g., circulating

tumor cells or cancer stem cells) or the heterogeneity of thesystem that would be unresolved if a larger sample wererequired (providing motivation for, e.g., laser-capture mi-

0.1 1 10 100

100

200

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400

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.)

Flow Rate (nL/min)

5 m 10 m2 m

0.1 1 10 100

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1000 nM 100 nM 10 nM 1 nM

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5 m 10 m2 m

0.1 1 10 1000

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Flow rate (nL/min)

0.01

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100

Rel

ativ

e io

n u

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atio

n e

ffic

ien

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a.u

)

(a)

(c)

(b)

Figure 3. Effect of liquid flow rate and analyte concentra-tion on analyte signal (a), (b), and signal-to-noise ratio (c), forbradykinin (m/z = 530.78). The ion utilization efficiency [cyanplot in panel (c)] grows exponentially with decreasing flowrate

I. Marginean et al.: Picoelectrospray Ionization Mass Spectrometry 33

crodissection and single cell analyses). Besides optimizingthe flow rate, a mass-limited sample could potentially bedelivered to the emitter as a low volume, concentrated plug

(e.g., from an on-line CZE separation), or as a more dilute,larger volume allowing for proportionately more signalaveraging. To determine the conditions providing the highestsignal-to-noise with a given mass, we adjusted the number ofaveraged spectra at different flow rates and concentrations tomaintain constant the amount of sample consumed. Figure 4acompares the m/z = 521–527 region of the MS spectracorresponding to consumption of 5 amol angiotensin II as afunction of analyte concentration. For example, three spectrawere averaged for the 100 nM sample delivered at 1 nL/min,resulting in a S/N of 14. Similarly, 30 and 300 spectra wereaveraged for the 10 and 1 nM “diluted” samples, resulting in S/N ratios of 11 and 5, respectively. The trend of decreasing S/Nratio with decreasing concentration arises in part because muchof the background is chemical in nature, arising for examplefrom the solvent, which does not decrease substantially withadditional averaging, and suggesting that sample dilutionshould be avoided for such modestly dilute samples.

Figure 4b compiles the S/N ratios for the analysis of 5 amoltotal amount of angiotensin II at several flow rates andconcentrations. Each data point is the average of three S/Nratios calculated as described above. The S/N generallyimproves with increasing concentration and shows an optimumat flow rates between 1 and 1.5 nL/min. These results suggestthat operating at ~1 nL/min while avoiding unnecessary sampledilution is optimum for this case. Pushing to even smalleramounts of consumed analyte, a mass spectrum correspondingto just ~500zmol of each peptide (an average of seven spectrameasured for the 3 nM peptide mix solution delivered at 1.5 nL/min) is presented in Figure 4c. Most peptides in the mix weredetected with signal between the detection level (S/N = 3) andthe quantitation level (S/N = 10), whereas Substance P, mellitin,and angiotensinogen were not detected. It is important to notethat this comparative study to determine optimum conditionsused an older instrument and was not designed to provide the

(AII 5 amol)

(AII 5 amol)

522 524 5260

20

40

60

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100

Inte

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.)

1 nMS/N = 5

522 524 526

S/N = 1110 nM

m/z

522 524 526

S/N = 14100 nM

0 1 2 3 4 50

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6

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12

15 1 nM 3 nM 10 nM 30 nM 100 nM

S/N

Flow rate (nL/min)

400 500 600 700 8000

100

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.)

m/z

768.

85 (

2+)

558.

31 (

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432.

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3+)

530.

79 (

2+)

386.

74 (

2+)

523.

77 (

2+)

(a)

(c) (spectrum 500 zmol)

(b)

Figure 4. Mass spectra (a), (c), and signal-to-noise ratios(b), for experiments consuming the same amount of analyte:5 amol (a), (b), and 500 zmol (c). See text for details

300 400 500 600 700 800 900 1000-120

-80

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4+)

300 nL/min

Inte

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%)

m/z

386.

74 (

2+)

523.

77 (

2+)

400 pL/min

Figure 5. Mass spectra measured at 400 pL/min (top) and300 nL/min (bottom) suggesting no trend toward uniformresponse with decreasing flow rate

34 I. Marginean et al.: Picoelectrospray Ionization Mass Spectrometry

greatest absolute sensitivity. Still, the subattomole detectionlimits shown here are among the lowest reported to date.

Besides improved ion utilization efficiency, nanoelectrosprayhas been shown to improve quantitation by reducing ionsuppression and matrix effects. Indeed, several studies [14, 24,35, 36] identified a trend toward uniform response withdecreasing liquid flow rates in which all analytes are ionizedwith nearly 100 % efficiency, and equal concentration analytesmight be expected to display similar signal intensities. It washypothesized that insufficiently low flow rates in the studieswere responsible for deviation from uniform response. Thus, wemight expect to observe a change in relative intensities trendingtoward uniform response with decreasing flow rate, but foundinstead that they were strikingly similar across nearly threeorders of magnitude change in flow rate. Figure 5 comparesspectra measured at 400 pL/min (top) and 300 nL/min (bottom)with the major peaks for each of the nine peptides indicated bymass labels. In both cases, the most intense peak was thatcorresponding to fibrinopeptide (m/z = 768.85), whereas theleast intense peak was that corresponding to mellitin (m/z =712.20). It should be noted that all the analytes considered in thecurrent study were peptides, whereas the previous studies [14,24, 35, 36] demonstrating uniform response at decreased flowrates included analytes from different classes of compounds. Forexample, mixtures of peptides and saccharides have beenemployed to emphasize the advantage of working at low flowrates [14, 36]. At large flow rates, the saccharide moleculesionize poorly because of their hydrophilicity that favorsdistribution inside the charged droplets rather than at the surface[45], displaying less intense analyte peaks than the peptide. Forelectrosprays operated at decreasing flow rates, the saccharideionization efficiency can improve more significantly than that ofpeptides, to the point where uniform response was evident.However, the present results suggest that the ionizationefficiency is analyte-dependent and a trend toward uniformresponse with decreasing flow rate is not universal.

ConclusionsWe explored electrospray ionization in the sub-nanoflow regimeextending to 400 pL/min. In comparison with flow rates as highas 400 nL/min, there was no indication of analyte suppressioneffects or charge competition evident between the selectedpeptide analytes included in this study. The ionization efficiencyof peptides appeared analyte-dependent; uniform response (i.e.,same signal at identical concentration) was not achieved even atthe lowest flow rates. The etched fused-silica emitters couldgenerally handle flow rates much smaller than those suggestedin the literature for similar dimension emitters. By workingunder optimized conditions, S/N ~10 could be obtained for someanalytes with ~500 zmol consumed. Although the flow ratesexplored here are below those currently employed for chemicalseparations (e.g., capillary LC), the benefits of lower flow ratescan be extended to higher rate separations by using electrosprayemitter arrays [46] or for CZE separations in very narrow borecolumns with sheathless interface designs.

AcknowledgmentsThe authors thank William F. Danielson for writing thesyringe pump control software, and Sarah Rausch, AllisonSheen, and Levi Broeske for assistance with data processing.This research was supported by the William R. WileyEnvironmental Molecular Sciences Laboratory (EMSL)intramural program and grants from National Institutes ofHealth: the National Institute of General Medical Sciences(grant 8 P41 GM103493-10) and the National CancerInstitute (1R33CA155252). The EMSL is a national scien-tific user facility sponsored by US DOE’s Office ofBiological and Environmental Research and located at thePacific Northwest National Laboratory (PNNL) in Richland,WA. PNNL is a multiprogram national laboratory operatedby Battelle for the DOE under contract no. DE-AC05-76RLO 1830.

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