Improved Electrospray Ionization Efficiency Compensates for Diminished Chromatographic Resolution...

Improved Electrospray Ionization EfficiencyCompensates for Diminished ChromatographicResolution and Enables Proteomics Analysis ofTyrosine Signaling in Embryonic Stem Cells

Scott B. Ficarro,†,‡,§ Yi Zhang,†,‡ Yu Lu,†,§ Ahmadali R. Moghimi,† Manor Askenazi,†,‡,§,|

Elzbieta Hyatt,⊥ Eric D. Smith,†,‡ Leah Boyer,# Thorsten M. Schlaeger,# C. John Luckey,⊥ andJarrod A. Marto*,†,‡,§

Department of Cancer Biology and Blais Proteomics Center, Dana-Farber Cancer Institute, 44 Binney Street, Smith1158A, Boston, Massachusetts 02115-6084, Department of Biological Chemistry and Molecular Pharmacology, andDepartment of Pathology, Brigham and Women’s Hospital, Harvard Medical School, 77 Avenue Louis Pasteur,Boston, Massachusetts 02115, Stem Cell Program and Division of Hematology/Oncology, Children’s Hospital Boston,Boston, Massachusetts 02115, and Department of Biological Chemistry, The Hebrew University of Jerusalem, Israel

Characterization of signaling pathways in embryonic stemcells is a prerequisite for future application of these cellsto treat human disease and other disorders. Identificationof tyrosine signaling cascades is of particular interest butis complicated by the relatively low levels of tyrosinephosphorylation in embryonic stem cells. These hurdlescorrelate with the primary limitations of mass spectrometry-based proteomics; namely, poor detection limit anddynamic range. To overcome these obstacles, we fabri-cated miniaturized LC-electrospray assemblies that pro-vided ∼15-fold improvement in LC-MS performance.Significantly, our characterization data demonstrate thatelectrospray ionization efficiency compensates for dimin-ished chromatographic performance at effluent flow ratesbelow Van Deemter minima. Use of these assembliesfacilitated quantitative proteomics-based analysis of ty-rosine signaling cascades in embryonic stem cells. Ourresults suggest that a renewed focus on miniaturized LCcoupled to ultralow flow electrospray will provide a viablepath for proteomic analysis of primary cells and rare post-translational modifications.

To date, pluripotent cells have been isolated from the innercell mass of blastocysts (ESC), 1 cultured postimplantationepiblasts, 2,3 and reprogrammed differentiated fibroblasts (iPS). 4-8

Each of these pluripotent cell populations can in principle undergo

programmed differentiation to generate nearly any cell type. Thusfor human pluripotent cells, there is growing excitement that thesecells may form the basis of new therapies to fight disease.

The majority of large-scale data for ESC are based on genome-wide transcriptional profiles and interrogation of transcriptionfactor binding sites. 9 By comparison a relatively small numberof protein signaling pathways have been functionally linked to self-renewal and differentiation in ESC. For example, leukemiainhibitory factor (LIF) maintains self-renewal in murine ESC(mESC) via signaling through the receptor g130, resulting inSTAT3 phosphorylation and activation,10 while self-renewal inhuman ESC (hESC) is dependent upon basic fibroblast growthfactor (bFGF) signaling.11,12 Similarly, activity of selected Src-family kinases,13,14 glycogen synthethase kinase 3 beta (GSK),and phospho-inositol 3- kinase (PI3K) have been implicated in bothself-renewal and differentiation.15-17 Collectively, these results

* To whom correspondence should be addressed. E-mail: [email protected]. Phone: (617) 632-3150. Fax: (617) 582-7737.

† Department of Cancer Biology, Dana-Farber Cancer Institute.‡ Blais Proteomics Center, Dana-Farber Cancer Institute.§ Department of Biological Chemistry and Molecular Pharmacology, Harvard

Medical School.| The Hebrew University of Jerusalem.⊥ Department of Pathology, Brigham and Women’s Hospital, Harvard Medical

School.# Children’s Hospital Boston.

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10.1021/ac802720e CCC: $40.75 2009 American Chemical Society3440 Analytical Chemistry, Vol. 81, No. 9, May 1, 2009Published on Web 03/30/2009

illustrate that multiple and interdependent signaling cascades laythe molecular groundwork for irreversible cell fate decisions.Hence, methods that facilitate analysis of signaling events in ESCwill provide valuable information in conjunction with studies aimedat directed differentiation for therapeutic or tissue engineeringapplications.

Mass spectrometry-based proteomics analysis of pluripotentembryonic stem cells (ESC) has focused primarily on globalprotein profiling, 18,19 with identification of only a few specific sitesof phosphorylation. 20 The latter result is not unexpected giventhat detection of phosphorylation events in primary or otherkaryotypically normal cells may be confounded by the rarity ofthe cells themselves, intrinsically low signaling activity, or somecombination thereof. Moreover, and independent of absolutesignaling levels, it is typically the case that biological samplesenriched by cell type, protein class, or specific post-translationalmodification, contain a concentration dynamic range that exceedsthe analytical capabilities of current LC-MS instruments.

In principle, improvements to the sample introduction interfacefor mass spectrometry can provide gains in detection limit anddynamic range. Recent efforts to advance proteomics sampleintroduction for LC-MS focused on either the use of small columnsand particles for so-called fast separations21-26 or platforms withrobotically controlled nozzle arrays to introduce LC effluent;27

arguably these approaches emphasize reproducibility and robust-ness over optimum chromatographic and electrospray perfor-mance. Interestingly, the respective advantages of small innerdiameter (I.D.), capillary-based liquid chromatography for im-proved separation performance28-31 and very low flow electrosprayfor improved ionization efficiency32-36 were independently de-scribed in multiple publications over a decade ago. However, thedifficulty inherent to fabrication of the small and robust emittertips required to support stable electrospray at increasingly low

effluent flow rates34,35,37 has exacerbated efforts to decipher therelative performance compromise between otherwise divergenttrends in chromatographic resolution and electrospray ionizationefficiencies expected in low flow regimes. Anecdotal evidencesuggests that researchers struggle to find a suitable convergencebetween key metrics that will facilitate exploration of small-scale,low flow LC-MS assemblies. Despite these obstacles we anticipatedthat fabrication of miniaturized LC-ESI assemblies would facilitateexploration of experimental parameters heretofore inaccessibleand ultimately provide a viable path for proteomics-based analysisof low-level tyrosine signaling in ESC or other primary cells.

EXPERIMENTAL SECTION

Construction of Fused Silica Analytical Columns withIntegrated Emitter Tips. Analytical columns (AC) were con-structed from 360 µm O.D. × 50 µm I.D. and 360 µm O.D. × 25µm I.D. fused silica capillary tubing (O.D., outer diameter; I.D.,inner diameter). Figure 1a depicts the major features of thesecolumns, with images that illustrate discrete steps during fabrica-tion (b-f). Silicate-based frits were cast in situ based on thefollowing protocol. A 2.5 cm section of polyimide was removedapproximately 3 cm from one end of the fused silica tubing. Asilicate solution was allowed to migrate via capillary action to themidpoint of the exposed window (Figure 1b). Next, polymerizationwas induced using a soldering iron as described above, with caretaken to form frits of 1-2 mm in length (Figure 1c). After ejectionof excess silicate solution the frits were reheated with thesoldering iron at 400 °C for several seconds. Reversed phase bedswere packed as described above, with 5 µm diameter beads usedfor the 50 µm I.D. AC and 3 µm diameter beads used in the 25µm I.D. AC. For both column sizes bed lengths of 12 cm wereattained in 10 min or less at pressures of 500-1500 psi. Next, thecolumns were dried with pressurized helium for 5 min (Figure1d). Panel (e) shows a cross-section image of a fritted 25 µm I.D.capillary, taken at the frit midpoint. Finally, an integrated emittertip of 0.75-1.5 µm diameter (Figure 1f) was formed 2-4 mmbeyond the frit using a laser-based pipet puller (P-2000, SutterInstruments, Novato, CA). A Nikon I.C.-66 microscope (MicroOptics, Fresh Meadows, NY) was used at 1000 × or 1500 ×magnification for visual confirmation of tip diameter. Prior to firstuse the end of the column (opposite the tip) was cleaved to within<1 cm of the reversed phase bed to minimize dead volume.

To date we have used this strategy to construct frits incapillaries ranging from 200 to 10 µm I.D.. Furthermore the useof a controlled, point heat source facilitates polymerization of fritse2 mm in length, and thus ensures good flow rate across thecolumn and compatibility with standard, low pressure (e5000 psi)HPLC systems. The high porosity frit structure (Figure 1e)exhibits very good mechanical stability as evidenced by our useof emitter tips e1.5 µm in diameter over multiple, automated LC-MS analyses spanning several days of continuous operation.Although our typical HPLC operating conditions are <5000 psi,we have tested these frits in 25, 50, and 75 µm I.D. capillaries atpressures up to 9500 psi, indicating that these columns are alsocompatible with UHPLC systems.

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For convenient handling and storage the columns are sheathedin a 1.5 cm length of Teflon tubing (0.062′′ O.D. × 0.0115′′ I.D.)that is counter-bored (0.038′′ I.D.) at one end using a pin visedrill to provide protective cover for the tip when not in use(Supporting Information, Figure S1). Note that filling the reservoirwith solvent eliminates clogging that otherwise occurs due to driedresidue at the tip. For extended storage the column may be placedinto a conical style tube (Teflon sleeve oriented downward)containing 5 mL of 0.1% acetic acid.

Imaging of Column Frits and Electrospray Emitter Tips.Capillary cross-section images were acquired on a Nikon E600upright microscope with a 10× objective and Insight 4MS Mosaic

color CCD camera. Digital images were processed using SPOTimage software. Electron micrographs of emitter tips wereobtained on a Zeiss Leo 982 scanning electron microscope at theHarvard Center for Nanoscale Systems. The off-axis perspectivewas imaged with the following parameters: 20,000× magnification,3 mm working distance; 1.90kV. The on-axis image was acquiredat 30,000× magnification, 2 mm working distance, 1.90kV.

All other experimental parameters including tissue culture andLC-MS are included as Supporting Information.

Safety Considerations. Safety glasses should be worn at alltimes during construction and use of fused silica based capillaries.In addition, a laboratory coat and gloves should be worn whenmanipulating cell cultures in biosafety cabinets or when handlingneat TFA and other volatile organics in a chemical fume hood.

RESULTS AND DISCUSSIONESC Display Reduced Levels of Tyrosine Phosphorylation.

Despite the success reported for proteomics-based analysis oftyrosine signaling in cancer models, our initial results in mESCwere inconsistent and generally yielded modest numbers ofphosphorylation sites (data not shown). We speculated thattyrosine signaling in self-renewing ESC may be diminished relativeto that in immortalized cell lines that express constitutively activereceptor tyrosine kinases. To address this question we usedantiphosphotyrosine antibodies to probe lysates from murine andhuman ESC, and from immortalized cells expressing oncogenicFlt-3 and Bcr-abl. Figure 2 shows that tyrosine signaling in mESCwas significantly reduced as compared to typical tumor cell lines.Given the comparably low levels of tyrosine phosphorylation inmESC, and hence the corresponding challenges in large-scale,quantitative analysis of tyrosine signaling in these cells, we nextexplored potential improvements in chromatographic separationand electrospray ionization performance beyond that available withthe 75 and 100 µm I.D. fused silica capillary columns typicallyused in mass spectrometry-based proteomics.

Electrospray Ionization Efficiency Compensates for Di-minished Chromatographic Performance at Effluent FlowRates below Van Deemter Minima. In previous work designedto optimize enrichment of phosphopeptides from stable, tran-formed human-derived myeloid cells expressing constitutivelyactivated Bcr-Abl, we reached a point of diminishing return in

Figure 1. Discrete steps for analytical column construction, shownfor 360 µm OD × 50 µm ID fused silica (b-d) and 360 µm OD × 25µm ID fused silica (e,f). (a) Schematic of complete analytical column(b) Frits are polymerized in situ by first drawing silicate solution tothe midpoint of a 1 cm length bare fused silica window via capillaryaction. (c) Slow heating at 300 °C of the region immediately adjacent(to the left in this image) to the silicate meniscus provides uniformand controlled polymerization, resulting in a frit approximately 2 mmin length (d) and with mesoporous fine structure (e). Next, HPLCresin is packed behind the frit and a ∼1 µm diameter emitter tip (f) ispositioned approximately 2-3 mm past the frit.

Figure 2. Silver stain (a) reveals that mESC and hESC expresssimilar total protein levels as compared to murine (Baf3) or human(K562) transformed cell lines expressing constitutively active Flt-3(Baf3) or Bcr-abl (K562) oncogenic kinases. However, Western blotwith anti-pY antibody (b) shows that mESC and hESC exhibitsignificantly less tyrosine phosphorylation as compared to murine andhuman transformed cells.

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Figure 3. Comparison of chromatographic and electrospray ionization performance characteristics for 360 µm O.D. × 50 µm I.D. (red) and360 µm O.D. × 25 µm I.D. (blue) analytical columns. (a,b) Box plots showing median peak widths (white lines) and interquartile ranges (IQRs;colored boxes) for (a) reconstructed ion chromatograms (RICs) of 198 peptide sequences identified in common across eleven runs, or (b) highconfidence peptide identifications where the (a) flow rate (nL/min) or (b) organic gradient slope was systematically varied for each column set.(c) High confidence (FDR ∼ 1.5%) peptide identifications from experiments shown in (b). Box plot (d) showing median apex peak intensity(white lines) and IQRs (colored boxes) for 198 peptide sequences identified in common across eleven runs shown in (a). High confidence (e)peptide identifications (FDR ∼ 1.5%) derived from experiments shown in (d). Colored dots above histogram bars indicate those data used in (h)to assess reproducibility. Bottom labels for x-axis show calculated separation peak capacity for each acquisition, based on 198 peptides identifiedacross all eleven LC-MS analyses. Box plot (f) showing median peak widths and IQRs for all high confidence peptides detected in each columnsize and flow rate combination. Histogram of peptide identifications (g) observed using the small column (blue) and large column (red) assemblies,binned according to precursor intensity. Venn diagram (g, inset) illustrates overlap of peptide identifications observed at the two extremesshown in (e). Reproducibility (h) based on overlap of peptide sequence identifications obtained for (left, red) 50 µm I.D. × 12 cm and (right,blue) 25 µm I.D. × 12 cm columns operated at flow rates of 12.5, 2.5, <1 nL/min and 10.5, 4.9, <1 nL/min, respectively.


our ability to increase overall phosphopeptide identification(pSer, pThr, pTyr) by extension of the organic gradient orreduction of the effluent flow rate.38 We were particularlyintrigued by the latter option because of the potential forimproved ionization efficiency, 32-36,39,40 but unfortunately itappeared that longitudinal diffusion broadened chromato-graphic profiles at low effluent flow rates in our capillarycolumns (data not shown). Moreover, our protocol for fabrica-tion of fused silica columns with integrated emitter tips reliedon particle-based frits and in our hands was less robust whenused with capillaries <50 µm I.D. 41 These limitations, combinedwith our desire to probe tyrosine phosphorylation in ESC,motivated our quest for improved chromatographic and elec-trospray performance. We speculated that elucidation of theinterplay between predicted divergence in the performance ofelectrospray ionization and chromatographic separation atincreasingly lower effluent flow rates would provide valuableclues for improved LC-MS performance. Toward this end wedeveloped a column fabrication strategy based on in situpolymerization of liquid silicates42 to form frits of high me-chanical stability and porosity that were also compatible withincorporation of integrated electrospray emitter tips (seeExperimental Section and Figure 1).

We next sought to elucidate the respective contributions ofchromatographic separation and electrospray ionization efficien-cies to overall performance in LC-MS proteomics studies. Forgeneral characterization we prepared a working solution of trypticpeptides from human-derived, myeloid cell lysate. Figure 3 showsa comparison of chromatographic and electrospray ionizationperformance characteristics for 50 µm I.D. × 12 cm (red) and 25µm I.D. × 12 cm (blue) capillary columns fabricated as describedabove. Figure 3a shows the interquartile range for reconstructedion chromatogram (RIC) peak widths plotted for 198 peptidesequences identified in common across eleven runs in which theeffluent flow rate was systematically varied across each columnset. These data show the expected Van Deemter behavior, withthe smaller columns exhibiting narrower elution profiles acrossall flow rates examined, and with both column sets showingbroadened peaks at flow rates at or below 1 nL/min.

Next, equal aliquots of the same peptide mixture were analyzedat constant flow rate with systematic variation of organic gradientslope across both column sets. Analysis of RICs for all high-confidence peptide sequences identified under each condition(Figure 3b) shows that the smaller dimension columns exhibitednarrower peak widths, as gauged both by the median and by theinterquartile range. Furthermore, the smaller column set consis-tently provided for a greater number of identified peptidesequences (Figure 3c). These data demonstrate important advan-tages inherent to the use of smaller scale LC assemblies for massspectrometry-based proteomics studies; for example, the dashed

line in Figure 3c illustrates that the 25 µm I.D. × 12 cm columnsprovided for high-confidence identification of some 1500 peptidesequences in approximately one-half the time (60 min versus 120min) required for an equivalent LC-MS analysis performed with50 µm I.D. × 12 cm columns.

Although the above data are consistent with previous reportsdescribing the advantages of microscale chromatography, 28-31,43

we believe that the key open question is whether predictedincreases in electrospray ionization efficiency will override thebroadening of chromatographic peaks expected at effluent flowrates below Van Deemter minima. These data are critical deter-minants for continued efforts designed to improve detection anddynamic range in LC-MS studies. Hence, we returned to the datain Figure 3a and extracted RIC apex intensities for each of the198 commonly detected peptides; Figure 3d shows that peptideintensities vary inversely with both effluent flow rate and columnsize, with the median intensity increasing nearly 15-fold acrossthe conditions analyzed. Surprisingly, the median apex intensityfor these 198 common peptides continued to increase for bothcolumn sets even at flow rates e1 nL/min, or below the clearonset of chromatographic peak broadening due to longitudinaldiffusion (Figure 3a). This observation supports the notion thatincreased electrospray ionization efficiency at progressively lowerflow rate can compensate for chromatographic peak broadeningdue to longitudinal diffusion. To understand the extent of thisphenomenon, we next plotted (Figure 3e) the total number ofhigh-confidence peptide identifications for each column size andflow rate combination. Although the numbers generally increaseinversely with both column size and effluent flow rate, there is aclear decrease for the 50 µm I.D. × 12 cm columns operated atthe lowest flow rate. To reconcile the apparent disagreementbetween the continued increase in signal intensity observed forthe 198 commonly detected peptides (Figure 3d) and the drop intotal number of peptide identifications observed for the largecolumn set operated at e1 nL/min (Figure 3e), we plotted theinterquartile range of RIC peak widths (Figure 3f) for all highconfidence peptides detected in each column size and flow ratecombination. As in Figure 3a, these data exhibit typical VanDeemter behavior; however, it is clear that the 50 µm I.D. × 12cm columns suffer more severe band broadening, as comparedto that for the 25 µm I.D. × 12 cm columns, when operated ateffluent flow rates of e1 nL/min. We speculate that diminishedchromatographic resolution eventually leads to a higher degreeof peptide co-elution, and possibly ion suppression effects, bothof which result in fewer peptide identifications in toto. In contrast,the smaller column set shows a consistent and inverse correlationbetween effluent flow rate and number of high-confidence peptideidentifications, reaching nearly 2500 sequence IDs at the lowestflow rate. This interpretation was further corroborated by calcula-tion of separation peak capacity based on the 198 commonlydetected peptides (Figure 3e). For each column geometry, thecalculated peak capacity mirrors the trend in peptide identification,except for the lowest flow rate, where the separation peak capacitydiverges significantly for the 50 µm I.D. × 12 cm, and 25 µm I.D.× 12 cm columns, respectively.

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To elucidate potential improvements in dynamic range realizedfrom the various column size and flow rate combinations weconstructed a Venn Diagram (Figure 3g, inset) for the twoextremes represented in Figure 3e. Note that the 25 µm I.D. × 12cm columns operated at effluent flow rates of e1 nL/min identifyfully 89% (570 out of 639) of the high-confidence peptides foundin the large column set (operated at 299 nL/min), plus anadditional 1726 peptides (e.g., not found with the large columnset). More importantly, the majority of peptides unique to the smallcolumn, low flow rate configuration (Figure 3g, blue bars) haveRIC apex intensities well below those for peptides identified incommon between the large and small columns sets (Figure 3g,red bars); comparison of mean intensity values (Figure 3g, dashedvertical lines) or the full width of each intensity distribution(Figure 3g, blue and red horizontal arrows) demonstrates aneffective dynamic range improvement of between 4- and 15-foldrespectively, for the 25 µm I.D. × 12 cm columns operated ateffluent flow rates of e1 nL/min.

Finally, we asked whether the performance increase affordedby the small column and low flow rate combination would translateinto improved reproducibility. To address this question, wecompared the overlap in unique peptide identifications obtainedfor 50 µm I.D. × 12 cm and 25 µm I.D. × 12 cm columns operatedat flow rates of 12.5, 2.5, e1 nL/min and 10.5, 4.9, e1 nL/min,respectively. Figure 3h shows that reproducibility across thiseffluent flow regime is substantially higher for the 25 µm I.D. ×12 cm columns.

As an orthogonal readout of dynamic range and sensitivity weassembled a 25 µm I.D. × 12 cm analytical column in a “ventedcolumn” configuration (Supporting Information, Figure S2) tofacilitate automated and rapid sample loading and rinsing, followedby efficient gradient elution of peptides to the analytical columnand mass spectrometer with minimal intercolumn band broaden-ing.44 Consistent with standardization efforts within the proteomicscommunity we characterized our automated system with readilyavailable and well-defined protein standards. Figure 4a showscalibration curves for commonly detected peptides from injectedquantities spanning a range of 10 amol to 1 fmol, acquired at 25(red) and 5 (blue) nL/min., respectively. In addition to overalllinear response, we achieved a 2-fold increase in sensitivity at areduced flow rate, again highlighting the importance of electro-spray ionization efficiency. Next we explored maximum dynamicrange by automated injection of peptides across a total range of25 amol to 250 fmol. Figure 4b shows that we readily achievelinear dynamic range of 10,000:1 under these conditions, andmoreover that our 25 µm I.D. × 12 cm columns are robust andwell-suited for use in automated experiments.

Significantly, our data above provide the first in-depth dem-onstration that electrospray ionization efficiency effectively com-pensates for diminished chromatographic performance at effluentflow rates below predicted Van Deemter minima; furthermore,this phenomenon is enhanced for smaller scale LC columns. Ourfabrication protocol provides for high-performance columns thatare very robust and well suited to high-throughput studies. Webelieve these data provide compelling evidence that a renewedfocus on miniaturized LC assemblies coupled to ultralow flow

electrospray will provide significant improvements in dynamicrange and detection limit for proteomics-based analysis of complexbiological mixtures.

Improved Detection of Tyrosine Phosphorylation in mESC.We next sought to elucidate early signaling mediators of dif-ferentiation in mESC; cells were established in standard cultureconditions and subjected to enforced differentiation by LIFwithdrawal. This key cytokine regulates self-renewal via signalingthrough the receptor gp130, with subsequent activation of theSTAT3 and MAP-Kinase pathways.10 Our use of iTRAQ stableisotope reagents45 for quantification in these experiments allowedus to verify the performance of our miniaturized LC-ESI as-semblies on a different mass spectrometry platform (see Support-ing Information, Supplementary Methods). Figure 5 shows that,consistent with our performance characterization data above, theuse of 25 µm I.D. × 12 cm columns provided improved results foranalysis of tyrosine signaling in mESC. For example, we observedan approximate 5-fold increase in phosphotyrosine peptide precur-

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Figure 4. Calibration curve (a) for commonly detected peptides frominjected quantities spanning 10 amol to 1 fmol, acquired at 25 (red)and 5 (blue) nL/min., respectively (see Supporting Information,Supplementary Methods). Observed sensitivity improves approxi-mately 2-fold at the lower effluent flow rate. Analysis of peptides (b)from commercial protein digest standard (see Supporting Information,Supplementary Methods) demonstrates a linear dynamic range of10,000:1 (25 amol to 250 fmol).


sor intensity (Figure 5c) and doubled the number of high-confidence peptide sequence identifications (Figure 5a and b),as compared to a large column, high flow rate combination. Wealso observed higher intensities for the iTRAQ reporter ionsassociated with commonly detected peptides (Figure 5c). In totalwe identified 158 unique tyrosine phosphorylated peptides (5%FDR) and detected modulation of tyrosine phosphorylation onproteins previously reported to be downstream of gp130, includingSTAT3 (Figure 5d) and MAPK1. Supporting Information, TablesS1 and S2 list all peptides and source proteins identified in ourstudies.

We next used a Fisher’s Exact Test to identify enrichment ofKEGG pathways46 based on all tyrosine phosphorylated proteinsidentified in our mESC data (see Supporting Information, Supple-mentary Methods). Figure 6a shows a histogram of KEGGpathways ranked by p-value. Several pathways previously de-scribed in the context of ESC self-renewal and differentiation,including ErbB, insulin signaling, MAPK, and JAK-STAT, 47 werewell represented in our data. Interestingly, these results suggestthat cell adhesion, junction, and matrix related proteins mayrepresent underexplored molecular pathways relevant for main-tenance of ESC self-renewal and execution of normal commitmentprograms. Finally we considered pathway representation as afunction of column geometry. Figure 6b shows that the 25 µmI.D. × 12 cm columns provided greater evidence in terms of uniqueprotein identifications for the majority of significant KEGGpathways. Collectively these results demonstrate that the improvedanalytical performance realized with our miniaturized columns and

lower effluent flow rates translates to higher quality biologicalannotation for the associated proteomics data.

CONCLUSIONOur analytical results have significant implications for the

application of mass spectrometry-based proteomics to questionsin biomedical research. Characterization data for our miniaturizedLC assemblies provides compelling evidence that electrosprayionization efficiency dictates overall LC-MS performance, even ateffluent flow rates below Van Deemter minima. Improved pro-ductivity (e.g., more peptides identified per unit time and over awider concentration dynamic range) is particularly relevant giventhe growing interest in label-free quantification,48 and the prolif-eration of large scale efforts to discover biomarkers of disease inclinical tissues, 49,50 large networks of protein interactions,51-54

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Figure 5. Comparison of 75 µm I.D. × 12 cm and 25 µm I.D. × 12 cm column assemblies for quantification of tyrosine phosphorylated peptidesin mESC as a function of LIF withdrawal. The number of unique pY peptide sequence identifications (a, b) is approximately 2-fold higher acrossvarious false discovery thresholds when using the smaller column assemblies. In addition (c) increased intensities for both peptide precursorand iTRAQ reporter ions are observed for analyses performed with the 25 µm I.D. × 12 cm columns. (d) MS/MS spectrum that shows modulation(inset) of STAT3 tyrosine phosphorylation (pY705) in response to LIF withdrawal. Red and blue dots indicate detection of b- and y-type ions,respectively.


comprehensive organelle proteomes,55-59 and complete proteincatalogues for eukaryotes.60 Moreover, our fabrication protocolprovides the practical advantage that columns can be made in less

than 30 min and for a raw materials cost of approximately $3 each.This is a truly portable technology, immediately accessible to alllaboratories, that provides more than an order of magnitudeimprovement in LC-MS performance. Extrapolation of our analyti-cal findings suggests that open tubular technology 61,62 may bean ideal format for continued exploration of lower effluent flowrates (e1 nL/min.) in smaller I.D. (e5 µm) columns.

We leveraged these analytical improvements to probe low-abundance tyrosine signaling events in ESC. Despite recentsuccess in large scale transcriptional profiling,9 the phosphory-lation cascades in these cells remain largely undefined. Our dataprovide intriguing new avenues for future studies that may provideimportant molecular links between intracellular signaling47 cas-cades and the pathways driven by cell-cell and cell-matrixinteractions 63-66 in the surrounding microenvironment thatcollectively regulate the balance between ESC self-renewal anddifferentiation.

ACKNOWLEDGMENTThe first four authors contributed equally to this work. The

authors thank David Lange at the Harvard Center for NanoscaleSystems for assistance with acquisition of SEM images. Generousfinancial support for this work was provided by the Dana-FarberCancer Institute. J.A.M. acknowledges financial support from theW.M. Keck Foundation.

SUPPORTING INFORMATION AVAILABLEFurther details on methods and data as noted in the text. This

materialisavailablefreeofchargeviatheInternetathttp://pubs.acs.org.

Received for review December 22, 2008. AcceptedFebruary 28, 2009.

AC802720E

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Figure 6. Proteomics analysis of tyrosine phosphorylation in mESC.(a) Significant (p e 0.01, Bonferroni corrected, see SupportingInformation, Supplementary Methods) KEGG pathways, classifiedunder “Environmental Information Processing” and “Cellular Pro-cesses”, based on tyrosine phosphorylated proteins identified inmESC subjected to LIF withdrawal. (b) Protein evidence for KEGGpathways in (a) based on tyrosine phosphorylated proteins identifiedthrough use of a 25 µm I.D. × 12 cm column (y-axis) or a 75 µm I.D.× 12 cm column (x-axis). Dashed line represents equal contribution.


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