Advantages of using Intelligent Sample Loading with the HPLC-Chip ...

Advantages of using Intelligent SampleLoading with the HPLC-Chip for automatedsample enrichmentApplication NoteMihaela GhitunEric BonneilLinda CôtéGeorges L. GauthierPierre ThibaultAbstractThe Agilent HPLC-Chip integrates enrichment column, analytical column,associated connection capillaries and a nanospray emitter directly ona single, small, reusable microfluidic chip and the HPLC-Chip and companionHPLC-Chip Cube interface provide an easy to use, robust andreliable nanospray LC/MS platform when compared to conventionalnanocolumn LC/MS technology. When using an enrichment column,appropriate selection of valve timetable events is critical for properloading and transfer of the sample to the analytical nanocolumn andrequires re-adjustment each time the injection volume is changed.Optimization of this process is often time consuming and improper settingscan significantly degrade the efficiency of the enrichment process.This Application Note will demonstrate how Intelligent Sample Loadingautomates and optimizes sample enrichment on the HPLC-Chip. Directcomparison with the timetable approach will also show how IntelligentSample Loading increases the number of peptides detected in a proteindigest test mixture.

IntroductionMicrofluidic HPLC-Chip/MS technologyis rapidly establishing itselfas a robust, reliable easy to usealternative to conventionalnanocolumn LC-MS system 1 .Integration of the enrichmentcolumn, analytical columns,connecting capillaries andnanospray emitter directly on tothe micro-fluidic HPLC-Chip hasdemonstrated improved sensitivityand enhanced chromatographicperformance leading to improvedpeptide and protein identification2-3 . HPLC-Chips and theHPLC-Chip Cube interface havebeen used with Ion-Trap and TOFmass spectrometers. Recent applicationsusing HPLC-Chip/MStechnology include biomarkerdiscovery 4 , investigation of thenucleolar proteome 5, phophoproteomeanalysis 6 and oligosaccharideseparations in mucins andhuman milk 7 . When usingnanocolumn LC/MS, long injectiontimes and very small or very dilutesamples requires the use of anenrichment column. This enrichmentcolumn is used to concentratethe sample and also allowsloading the sample at higher flowrates reducing the overall injectioncycle time. In addition, biologicalsamples can be desalted onthe enrichment column to minimizethe introduction of salt in the MSsource. Flow switching is requiredfor sample loading on to theenrichment column and subsequenttransfer to the analyticalnanocolumn. With the HPLC-Chip/MS system, this is achievedusing a novel microvalve systemcontained in the HPLC-Chip Cubeinterface that ensures seamlesszero dead volume leak tight connectionswith the HPLC-Chip.Automation and optimization ofthe flow switching process is criticalfor proper loading of the sampleon to the enrichment columnand transfer to the analyticalnanocolumn. For conventionalnanocolumn LC/MS system, valvecontrol is typically performedusing a valve event timetable.However, this approach can createunnecessarily large data files andrequires re-optimization of thevalve event timetable each timethe sample injection volume ismodified. This process is quiteoften time consuming and errorprone and improper settings cansignificantly degrade the efficiencyof the enrichment process.This will negatively impact boththe amount and number of compoundsretained on the enrichmentcolumn. In this ApplicationNote, the features and performanceof Intelligent SampleLoading will be discussed indetail. Direct comparison with thetime table approach will showhow Intelligent Sample Loadingincreases the number of peptidesdetected in a protein digest testmixture and reduces data file size.Equipment, sample and dataprocessingThe Agilent 1200 HPLC-Chip/MSsystem includes the followingcomponents:• Agilent 1200 Series nanoflow LCsystem consisting of thenanoflow pump, micro vacuumdegasser, and thermostattedmicro well-plate autosampler(µ-WPS)• Agilent 1200 Series capillarypump for sample loading anddesalting• Agilent 1200 HPLC-Chip CubeMS interface• Agilent LC/MSD Trap XCT Ultramass spectrometers• Agilent Protein ID chip• Spectrum Mill Protein IDsofwareChromatographic conditions:HPLC-Chip/MS:Enrichment column: ZORBAX 300SB-C18,40 nL, 5 µmAnalytical column: ZORBAX 300SB-C18, 75 µm x 43 mm, 5 µmLoading flow rate:4 µL/minLoading mobile phase: 3 % acetonitrile, 0.2 % formic acidInjection flush volume: 6 µL for IFV method and 0 µL for TT method respectivelyInjection volume: 5 µLValve positions (for TT method): 0 min enrichment, 6 min analysis, 69 min enrichmentFlow rate:Mobile phase:Gradient:TT method:Time (min)% B0 86 863 4067 6068 6069 8Stoptime: 75Post time: 5 min300 nL/minA = 0.2 % formic acid in waterB = 0.2 % formic acid in acetonitrileIFV method:Time (min)% B0 857 4061 6062 6063 8Stop time: 64Post time: 10 minNeedle flush solvent: 20 % methanol + 0.2 % formic acid in waterTable 1HPLC-Chip chromatographic conditions.2

For peptide detection and clustering,a script was developed forconversion of the Agilent Ion-Trapdata files into text files containinginformation on all m/z, intensityand retention time values above auser specified threshold. Theresulting text file is then processedusing proprietary software thatenables data reduction, peptidedetection and alignment of peptideion maps (m/z, retention time,intensity) 8 . Protein and peptideidentification was performed usingSpectrum Mill. The sample usedfor this evaluation was a peptidemixture from the tryptic digest of8 different proteins (bovine serumalbumin, rabbit aldolase, yeastalcohol dehydrogenase, bovinecatalase, bovine glutamate dehydrogenase,E.Coli glycerokinase,human lactotransferrin, bovinelactoperoxidase, MichromBioresources, Auburn, CA). Thedigests were dissolved in waterwith 5 % acetonitrile and 0.2 %formic acid, and mixed togetherto reach the concentration of40 fmol/µL each. The final injectionvolume contained 80 ng ofthe mixture (ProMix). Chromatographicand Ion Trap MS conditionsfor the experiments aredescribed in tables 1 and 2.Chromatographic conditions:Ionization mode:HPLC-Chip/MS interfaceDrying gas flow:3.5 L/minDrying gas temperature: 300 °CCapillary voltage:1900 VSkimmer 1:30 VCapillary exit:75 VTrap drive: 85Averages: 1ICC: OnMaximum accumulation time: 150 msSmart target: 500000MS scan range: 400-1600Automatic MS/MS:Peptide scan mode (standard-enhanced for MSand ultra-scan for MS/MS)Number of precursors: 3Averages: 1Fragmentation amplitude: 1.3 VActive exclusion:On, 2 spectra, 1 minPrefer +2:OnMS/MS scan range: 100-2200ICC target: 500000Table 2Ion Trap MS conditions.Nano LC columnHV ESI contactRF tagEnrichment column,capillaries, fittings, fritsNanospray emitter, emitterassembly and fittingsWorkflow and description of theHPLC-ChipThe chip fabrication process andthe chip interface for MS havebeen previously described 3,9 andfor this work, the Protein ID (p/nG4240-62001) chip was used. Thischip includes a 40-nL enrichmentcolumn and a 75-µm x 43-mmanalytical column which are bothpacked with 5-µm 300A ZORBAXHPLC-Chip in chip holderfor use with the HPLC-Chip interfaceFigure 1Design of the Protein ID chip. The different elements necessary for nanoflow LC/MS areintegrated on the HPLC-Chip.SB-C18. Figure 1 shows thecomponents integrated ontothis microfluidic HPLC-Chip.Components and workflows integratedon to the HPLC-Chip areoutlined in figure 2 and clearlyillustrate the requirement for flowswitching so that the sample can3

e loaded using higher flow ratesonto the enrichment column,flushed onto the analytical column,and then analyzed by nanoflowLC/MS.LoadingpumpHPLC-ChipWasteIntelligent sample loadingThe HPLC-Chip/MS system includeda novel Intelligent Sample Loading(ISL) feature that decouples thesample loading process from theLC/MS analysis. ISL will allow thesystem to automatically calculatethe required time to effectivelyload the sample. At the end ofthis time, the HPLC-Chip Cubemicrovalve will automaticallyswitch from enrichment to analysismode and the pump gradient andMS data acquisition will begin.This novel feature eliminates theneed to adjust the method whenchanging the injection volume andsaves time by using the shortestpossible injection cycle. Furthermore,data file size is also reducedsince the MS data acquisitioncoincides with the switchingfrom enrichment to analysis modeinstead of starting at the beginningof the autosampler sample loadingcycle.Intelligent Sample Loading parametersAutomatic calculation of the optimumsample loading time requiresthat the HPLC-Chip/MS system hasthe ability to measure actual flowrates from the sample loadingpump and prior knowledge ofthe capillary tubing delay volumebetween the autosampler seatand the enrichment column onthe HPLC-Chip. This delay volumeplus a flush factor are defined asthe injection flush volume (IFV)and is calculated as follows:IFV = (VSeatCap +VTransferCap)x factorwhere:InjectorFigure 2Workflow diagram for the HPLC-Chip/MS system.VSeatCap = delay volume of theµ-WPS seat capillaryVTransferCap = delay volume ofthe capillary from µ-WPS toChip Cubefactor: number of time to flushthe delay volume.Typical values are:VSeatCap: 75 µm x 150 mm (blue)= 0.663 µLVSeatCap: 100 µm x 150 mm (black)= 1.178 µLVTransferCap: 25 µm x 1050 mm(yellow) = 0.515 µLfactor = 3 (typical flush factorsare in the range of 2 to 6)EnrichmentcolumnC18NanocolumnNanoflowLC pumpOnlineMS/MSFor this experiment, the HPLC-Chip Cube was used with 100-µmseat capillary and the IFV is:(1.178 µL + 0.515 µL) x 3,5 = 5.926 µL.Measurement of the actual flowrate from the capillary loadingpump is achieved by linking theloading pump to the HPLC-ChipCube during the initial system setup.Linkage of the capillary loadingpump to the Chip Cube willallow the Chip Cube to monitorreal time flow from the capillaryloading pump and automaticallyadjust the sample loading timewhen the injection volume ischanged. Sample loading time isautomatically calculated by thesystem as follows:Sample loading time = IFV +injection volume / Actualcapillary loading pump flow4

Thus, given an IFV of 5.926 µL,an injection volume of 1 µL anda flow rate of 4 µL/min at thecapillary loading pump:Sample loading time:(5.926 µL + 1 µL) / 4 µL/min = 1.73 minFigure 3 illustrates the dialogbox from the Chemstation pumpconfiguration pull-down menurequired to link the capillary loadingpump and the nanoflow pumpto the HPLC-Chip Cube. In additionto allowing real time measurementof flows, linking both pumps providean additional safety functionby allowing them to react to thestatus of the Chip Cube and turnthe flow off when the chip is notin Operate (spraying) position.This ensures that no solvent isspilled into the Chip Cube whilethe chip tip is retracted or whenthe chip is unloaded and the valvestator ports are open.perform real time measurementof the capillary loading pump flowensures continued optimized sampleloading time throughout thelifetime of the HPLC-Chip. Thisensures that changing backpressures(and flows) will not affectthe sample transfer.Smaller data file sizeDecoupling the sample loadingprocess from the LC/MS data acquisitionreduces the data file sizesince data acquisition is started atthe same time the sample is transferredto the analytical nanocolumninstead of at the beginning of thesample injection cycle. Figure 4highlights the difference in runtime achieved when using ISL comparedto a microvalve time tablemethod for the ProMix tryptic peptidesample. In this example, dataacquisition time could be reducedby 16 % (from 51 to 43 min) anddata file size by 11 %. Reducing theacquisition time will increasethroughput. Smaller data files willaccelerate data processing timeand save valuable disk space onthe host computer system.Identify more peptides with ISLFlow switching timing is criticalfor optimum loading of the sampleon to the enrichment column andtransfer to the analytical nanocolumn.Unfortunately, the processof selecting flow switching parameterscan be time consuming andrequire multiple experiments toselect and optimize all parameters.Because of this, sample loadingtimes are often set arbitrarilyfollowing a limited set of optimizationexperiments. Improper settingsof the sample loading timewill significantly degrade theefficiency of the enrichmentprocess and negatively impactboth the amount and number ofcompounds retained on theenrichment column.1082.01.51.00.5Start of injection cycle and data acquisitionSample transfer to analytical nanocolumn, gradient startTT methodEnd of dataacquisitionFigure 3Pump configuration dialog box.Changing the injection volumeA change to the injection volumespecified in the method orautosampler sequence table willautomatically trigger a recalculationof the sample loading time.No other operator input isrequired. Over time, the enrichmentcolumn flow characteristicswill change and the ability to10 82.01.51.00.5Sample transfer to analytical nanocolumn,gradient and data acquisition startISL methodEnd of dataacquisition0 5 10 15 20 25 30 35 40 45 Time [min]Figure 4ProMix tryptic digest peptide sample using TimeTable (upper, TT) and ISL (lower, ISL).Sample analysis time was decreased by 18 %. Data file size was reduced by 11 %.5

Figures 5 and 6 clearly demonstratethis problem. In figure 5, thebase peak chromatogram (BPC) ofProMix is compared using both amanual timetable (TT) and ISLapproach. For the most part, theBPC’s of both the TT and ISL runsappear to be identical. However,significant differences are noticeablein the initial part of the BPCwith several more peaks observedin the sample analysed using ISL(figure 5). This is further highlightedwith the comparison of the peptideion maps generated from boththe TT and ISL runs (figure 6).Zooming in on the initial segmentof the peptide ion map of eachrun, we can clearly observe theadditional peptides detected whenusing the ISL approach. For thisexample with the 8 proteinProMix tryptic digest sample, wewere able to detect 6.1 % morepeptides using ISL than with theTT approach. Table 3 highlights alist of additional peptides detected.Because of the overall chromatographicperformanceobserved for the TT approach(figure 6, left), the fact that thesample loading process was notfully optimized could have passedunnoticed and resulted in significantloss of information from thesample. This demonstrates the10 8 2.0TT method1.51.00.510 82.01.51.00.5ISL method0 5 10 15 20 25 30 35 40 45 Time [min]Figure 5BPC of ProMix with TT (top) and ISL (bottom) with scale expansion for first part of the BPC.Additional peaks are clearly visible in the ISL chromatogram.Figure 6Peptide ion map of ProMix with TT (left) and ISL (right). 6.1 % more peptides were detected usingISL. Sample loading on to the analytical nanocolumn occurred at t = 6 min for the TT approachand t = 0 for the ISL.m/z measured MH + MH +(Da) Matched (Da) Error (Da) Sequence RT (min) Intensity Protein1 667.83 1334.67 0.02 R.EFRPGIETTER.N 6.19 6.37e+008 Glycerol kinase2 503.34 1004.67 0.15 R.ETTIVWEK.E 9.00 0.90e+007 Glycerol kinase3 625.35 1249.62 0.09 R.FKDLGEEHFK.G 6.67 3.01e+008 BSA4 722.94 1443.87 0.25 K.YICDNQDTISSK.L 4.4 0.83e+007 BSA5 745.90 1490.71 0.08 R.LQSIGTENTEENR.R 2.65 2.51e+007 Aldolase6 481.82 962.57 0.07 K.DAQLFZIKK.A 8.94 7.56e+007 Catalase7 656.43 1310.85 0.17 K.RLCENIAGHLK.D 9.2 1.2e+007 Catalase8 740.36 1479.68 0.03 R.FNSANDDNVTQV.R 4.58 2.03e+008 Catalase9 575.90 1149.79 0.17 R.THYYAVAVVK.K 8 1.1e+007 Lactotransferrin10 498.43 994.85 0.32 R.RFTMELAK.K 9.1 0.70e+007 Glutamate dehydrogenase11 522.86 1043.71 0.14 R.QLLLTADDR.V 9 3.90e+007 AldolaseTable 3Additional ProMix peptides detected using Intelligent Sample Loading.6

critical aspect of optimizing sampleloading times and the advantagesof using the automated ISLfeature of the HPLC-Chip/MSsystem.ConclusionsIntelligent Sample Loading optimizessample loading on to theenrichment column and transferto the analytical nanocolumnwhen using the HPLC-Chip.Automation of the entire processensures that the operator canchange injection volumes withoutthe need to re-optimize methodparameters for sample loading.By automatically selecting thebest parameters, ISL shortensthe analysis time and reduces thedata file size, saving disk storagespace and accelerating thedata processing of the sample.Compared to a time table method,ISL automatically selects the mostefficient sample loading parametersallowing for the detection ofadditional peptides missed whenusing the timetable method.References1.Gauthier, G-L., Grimm, R.,“Miniaturisation: Chip-basedLiquid Chromatography andProteomics.” Drug DiscoveryToday, DDTEC., 3(1), 59-66,2006.2.“Performance of the Agilent 1100HPLC-Chip/MS System.” AgilentApplication Note, publicationnumber 5989-4148, 2005.3.“Comparison of HPLC-Chip/MSwith conventional nanoflowLC/MS for proteomic analyses.”Agilent Application Note, publicationnumber 5989-3538, 2005.4.Fortier, M-H, Bonneil, E., Goodley,P., and Thibault, P., “IntegratedMicrofluidic Device for MassSpectrometry-Based Proteomicsand Its Application to BiomarkerDiscovery Programs,” Anal.Chem., 77(6), 1631-1640, 2005.5.Vollmer, M., Hörth, P., Rozing, G.,Couté, Y., Grimm, R., Hochstrasser,D., and Sanchez, J-C.,“Multi-dimensional HPLC/MS ofthe nucleolar proteome usingHPLC-chip/MS.” J. Sep. Sci.,29(4) ,499-509, 2006.6.Ghitun, M., Bonneil, E., Fortier,M-H, Yin, H., Killeen, K. andThibault, P., “Integrated MicrofluidicDevices with EnhancedSeparation Performances;Application to PhosphoproteomeAnalyses of Differentiated CellModel Systems.” J. Sep. Sci.,in press 2006.7.Niñonuevo, M., An, H., Yin, H.,Killeen, K., Grimm, R., Ward, R.,German, B., and Lebrilla, C.,“Nanoliquid chromatography-massspectrometry of oligosaccharidesemploying graphitized carbonchromatography on microchipwith a high-accuracy mass analyzer”Electrophoresis, 26, 3641–3649,2005.8.Kearney, P.; Thibault, P., “Bioinformaticsmeets proteomics –Bridging the gap between massspectrometry data analysis andcell biology” J. Bioinf. Comput.Biol., 1 (1), 183-200, 2003.9.Yin H., Killeen K., Brennen R.,Sobek D., Werlich M., and van deGoor T., “Microfluidic Chip forPeptide Analysis with anIntegrated HPLC Column, SampleEnrichment Column, andNanoelectrospray Tip,” Anal.Chem., 77 (2), 527-533, 2005.7

Mihaela Ghitun and PierreThibault – Institute for Researchin Immunology and Cancer,Université de Montréal, Montréal,Canada and Department ofChemistry, Université deMontréal, Montréal, Canada;Eric Bonneil – Institute forResearch in Immunology andCancer, Université de Montréal,Montréal, Canada; Georges L.Gauthier – Agilent Technologies,Waldbronn, Germany; LindaCoté – Agilent Technologies,Montréal, Canadawww.agilent.com/chem/hplc-chipCopyright © 2006 Agilent TechnologiesAll Rights Reserved. Reproduction, adaptation ortranslation without prior written permission isprohibited, except as allowed under the copyrightlaws.Published June 1, 2006Publication Number 5989-5222EN