Improved protein identification: Off-line multidimensional LC/MS as aneffective tool for proteomics research
Application Note
Ralf MoritzMartin Vollmer
Edgar Nägele
Abstract
Compared to fully automated on-line two-dimensional LC/MS of digested
proteome samples, an off-line two-dimensional approach greatly
increases chromatographic resolution resulting in more significant
protein identification results. In this Application Note we present
off-line 2D LC/MS as an alternative technique to on-line 2D LC/MS for
the analysis of complex proteome samples. The first dimension involves
off-line fraction collection with the Agilent 1100 Series micro fraction
collection system. The second dimension uses the Agilent Nanoflow
Proteomics Solution together with the Agilent Spectrum Mill MS
Proteomics Workbench for LC/MS/MS analysis and subsequent protein
identification.
Introduction
Today, automated on-line 2D LC/MS/MS of digested proteinsis generally applied for proteomecharacterization1. Usually on-line2D LC/MS/MS is performed usinga strong cation exchange (SCX)column in series with a reversedphase (RP) column. In the courseof analysis tryptic peptides areeluted stepwise by injecting saltplugs of increasing ionic strengthfrom the SCX column in the firstdimension. In the second dimensionthese peptides are first trapped on areversed phase (RP) enrichmentcolumn and finally separated onan analytical RP column2. It hasbeen shown that this methodology,especially in nano scale, is capableof resolving large proteomes3 aswell as identifying a subset of proteins expressed under specialconditions4 in differential pro-teomics research.
In contrast to this well establishedon-line methodology off-line
2D LC/MS/MS significantly increaseschromatographic resolution ofhighly complex proteome samples.This improvement is attributed to continuous gradient SCX-chromatography in the firstdimension without intermittingRP-chromatography intervals. As a consequence a higher peakcapacity and therefore a highernumber of identified proteins is associated with the off-line 2D LC/MS/MS approach.
In this Application Note off-line2D LC/MS is presented as an alternative technique for analysisand identification of complex proteome samples. The Agilent 1100Series micro fraction collectionsystem5 was used for fraction collection in the first dimension(SCX). In the second dimension (RP)the Agilent Nanoflow ProteomicsSolution6 in combination with the Agilent Spectrum Mill MS Proteomics Workbench7 was usedfor reliable protein identification.The results obtained with the on-
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line and the off-line approachesare compared and discussed.
Experimental
Equipment
Agilent 1100 Series micro
fraction collection system:
Capillary pump with micro vacuum degasser, thermostattedwell-plate autosampler, ther-mostatted column compartmentwith micro 2-position/6-port microvalve, DAD with 500 nL cell and thermostatted micro fraction collector (figure 1 A).
Agilent 1100 Series Nanoflow
Proteomics Solution:
Nanoflow pump with micro vacuumdegasser, thermostatted microwell-plate autosampler, 2-position/6-port micro switchingvalve box with holder, quaternarypump, LC/MSD Trap XCT withorthogonal nanospray ion source(figure 1B).
Solvent tray
Degasser
Nanoflowpump
Cooler
µ-well-plate sampler
MSD ion trap XCT
Orthogonal nanospray sourceand nanocolumn
Solvent trayDegasser
Capillary pump
µ-well-plate sampler
Cooler Cooler
µ Fraction collector
DAD
TCC + 6-port µ-valve
A
B
Solvent tray
2ndpump
Micro switchingvalve and holder
Figure 1 Off-line 2D LC/MS system. A: Micro fraction collection system. B: Agilent Nanoflow Proteomics Solution
Software:
Agilent ChemStation rev. A.10.01and Spectrum Mill MS ProteomicsWorkbench software.
The typical workflow for an off-line2D LC proteome analysis is shownin figure 2. The first dimensionstarts with the elution of trypticpeptides from a SCX column witha linear salt gradient and the sub-sequent collection of fractions inthe micro liter range with the microfraction collector (figure 2 A). Thewell-plate, containing all collectedfractions was then transferreddirectly to the second dimension – the Agilent Nanoflow ProteomicsSolution (figure 2 B). After reinjection the peptides from eachfraction are concentrated on ashort C18 enrichment columnlocated between the ports of themicro valve. The enrichment column was then switched into thenanoflow path, the concentratedpeptides were eluted, further separated by a gradient withincreasing organic solvent on ananalytical nanobore column andsprayed directly into the MS.
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Chromatographic methodOff-line approach:1st dimension (SCX chromatography): Agilent 1100 Series micro fraction collection system Solvents: A: 5 % AcN +0,03 % formic acid,
B: 500 mM NaCl, + 5 % AcN + 0.03 % formic acid.Gradient: 0 min 0 % B, 5 min 0 % B, 35 min 20 % B, 38 min 100 % B,
41 min 100 % B.Flow: 5 µL/min.Column: Agilent BioSCX Series II, 0.30 x 35 mm, 3.5 µm particlesAutosampler: 5 mL sample injection volumeTime-based fraction collection mode: 1st fraction 0 - 5 min, 2nd to 13th fraction each 3 min, liquid
contact control mode, fraction size 15 µL, cooling 4 °C .
2nd dimension (RP Chromatography): Agilent 1100 Series Nanoflow Proteomics SolutionNanoflow pump:Solvents: A = water + 0.1% formic acid,
B = AcN + 0.1% formic acidGradient: 0 min 5 % B, 5 min 5 % B, 12 min 15 % B, 72 min 55 % B,
74 min 75 % B, 75 min 75 % B, 75.01 min 5 % B. Stop time: 90 min. Post time: 10 min.Flow: 300 nL/minColumns: Zorbax 300SB C18, 75 µm x 150 mm, 3.5 µm particles
Zorbax 300SB C18, 0.3 x 5 mm, 5 µm particlesMicro valve: Enrichment column switch: 5 minAutosampler: 15 µL sample injection volumeQuaternary pump:Solvent: water + 3% AcN + 0.1 % formic acid, flow: 20 µL/min
On-line approach2:Agilent 1100 Series Nanoflow Proteomics Solution
Nanoflow pump:Solvents: A = water + 0.1 % FA, B = AcN + 0.1 % FA.Flow: 300 nL/min.Gradient: 0 min 5 %B, 5 min 5 %B, 12 min 15 %B, 72 min 55 %B,
74 min 75 %B, 75 min 75 %B, 75.01 min 5 %B. Stop time: 90 min.Post time: 10 min.Columns: Agilent BioSCX Series II, 0.30 x 35 mm, 3.5 µm particles
Zorbax 300SB C18, 75 µm x 150 mm, 3.5 µm particlesZorbax 300SB C18, 0.3 x 5 mm, 5 µm particles
Micro valve:Enrichment column switch: 0 min in-line with SCX; 5 min in-line with nanocolumn;
85 min in-line with SCXAutosampler:Injection volume: 5 µL sample injection volume, 20 µL salt solution injection
volumeSalt steps injected on SCX (NaCl): 0, 15, 30, 45, 60, 75, 90, 105, 120, 135, 150, 300, 500 mM
MS conditions for both experiments:Source: positive orthogonal nanospray sourceDrying gas flow: 3 L/min Drying gas temp: 225 °CSkim 1: 40 VCapillary exit: 135 V Trap drive: 80 VICC: on
Target: 40,000Max. accu. time: 150 msAverages: 4
Automatic MS/MS: Number or precursors: 3Isolation width: 1.15 VFragmentation amplitude: 4Preferred charge state: +2SmartFrag: On, 30-200%
Sample preparation
Lyophilized yeast cells (S. cerevisiae), resuspended incooled 50 mM NH4HCO3 containing8 M urea were disrupted in a beadbeater (0.5-mm glass beads). After
4
162
Waste
1
6
54
32
15
4
6
Cooled µ well-plate sampler
Cooled µ well-plate sampler
Waste
Capillary pump
1
6543
Plug
Micro fraction collector
2nd pump
6543
2Plug
75 µm x 150 mm, 3.5 µmanalytical column
Waste
Nanoflow pump3
2
6
4
MS detection
B. Inject fractions to trap column, wash and analyze
A. Continous salt gradient
Transfer well-plate to autosampler
SCX column
C18 enrichment column
Figure 2Workflow for proteome identification by off-line 2D LC/MS
centrifugation to remove cell debrisproteins in the supernatant werereduced with 1 mM DTT at 37 °Cfor 1h, alkylated in the dark with10 mM iodoacetamide for 30 minat RT, ultrafiltrated for bufferexchange and tryptically digested
with TPCK trypsin at 37 °C for 16 h.Finally, the sample was lyophilizedin a SpeedVac and dissolved in 5 %AcN, 0.03 % formic acid prior toanalysis.
Results and discussion
Collection principleIn order to collect small volumesin the lower µL range reliably aunique collection principle wasdeveloped which is illustrated infigure 3. During the entire collectionprocess, the fraction collectorensures that the droplet formingat the capillary tip is in constantcontact with the surface of thedeposited liquid. At the start offraction collection, the dropletbeing formed is precisely depositedat the bottom of the well. As thewell is filled the capillary tip movescontinuously upwards, keepingconstant contact with the solventsurface. When fraction collectionis finished the capillary tip movesquickly upwards, ensuring that thedroplet currently being formed atthe capillary tip is delivered to thewell. The well filling velocity duringfraction collection is not only calculated from the current flowrate but also from the geometry ofthe actual well type. Such a uniquecollection principle guarantees airbubble-free, precise deposition oflow volume fractions in a broadvariety of well plate types and ves-sels from different manufacturers.In addition, cross contaminationbetween collected fractions iseffectively prevented.
Data evaluationTrypsin-digested Saccharomyces
cerevisiae proteome was subjectedto off-line and on-line 2D LC-MS/MSanalysis. For both approaches thesame conditions were chosen forsolvents, sample and columns. Forthe on-line version the individualsalt step concentrations were
5
1 2 3
4 5 6
Figure 3Collection principle for small volumes in the lower µL range. During the collection process the capillary stays in constant contact with the solvent. This approach leads to air bubble free accurately collected fractions without cross contamination.
min0 5 10 15 20 25 30 35 40
mAU
0
250
500
750
1000
1250
1500
1750
01 02 03 04 05 06 07 08 09 10 11 12 13
100mM NaCl
500mM NaCl
2000
Figure 4Time-based collected fractions from the first dimension (SCX) and the applied salt gradient.
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adjusted such that they practicallycorresponded to the fractions collected in the off-line approach.After evaluation of the MS/MSdata with the Agilent SpectrumMill software using the identicalstringent parameters for bothapproaches the following resultswere obtained.
For the nano on-line 2D LC-MS/MSmethodology, which comprisesinjected salt steps within the 1st
dimension and a nano LC RP separation in the 2nd dimension101 proteins were identified. Forthe off-line methodology that wasperformed by applying continuousgradient elution during the 1st
dimension and a nano LC RP separation in the 2nd dimension,144 proteins were identified. Theseresults were confirmed in threerepeated analyses. Proteins withhighest scores for both analysesindicate that basically the samehigh abundance proteins wereidentified in both experiments(table 1). However, the values ofthe scores are different and in general higher in the off-line than inthe on-line approach. This clearlydemonstrates that the higher chromatographic resolutionobtained with a continuous linearsalt gradient leads to well separatedpeaks, and finally to a higher number of detected peptides inthe MS.
On-line 2D LC/MS analysis of the yeast proteomeScore Peptides Spectra Coverage Protein name
150,46 10 45 36 Phosphopyruvate hydratase123.75 8 52 25 Phosphoglycerate kinase114.54 8 21 24 Pyruvate decarboxylase98.57 7 76 23 Translation elongation factor eEF-181,53 6 16 21 Pyruvate kinase 65.13 4 16 13 Hexokinase A62.63 5 39 16 Alcohol dehydrogenase I55.54 4 16 23 Phosphoglycerate mutase 148.01 4 6 18 Triosephosphate isomerase47.39 3 7 22 Eukaryotic translation initiation factor 5A-2 33.54 3 3 5 Heat-shock Protein HSC8232.65 2 15 14 BMH2 Protein31.18 2 2 13 Ribosomal protein S629.24 2 7 6 Phosphogluconate dehydrogenase24.70 2 2 27 Superoxide dismutase23.30 2 2 20 Cytochrome C oxidase18.24 1 1 10 Malate dehydrogenase17.65 1 3 6 40S Ribosomal protein S516.97 1 3 3 Citrate synthase 16.79 1 2 5 Glycerol-3-phosphate dehydrogenase16.33 1 2 4 Glucose kinase
Off-line 2D LC/MS analysis of the yeast proteomeScore Peptides Spectra Coverage Protein name
161.50 12 27 42 Phosphopyruvate hydratase150.45 10 32 32 Phosphoglycerate kinase112.92 8 14 14 Pyruvate decarboxylase97.77 6 8 19 Glucose kinase93.12 7 17 23 Translation elongation factor eEF-173.11 5 7 13 Citrate synthase 71.66 5 8 19 Pyruvate kinase 64.03 4 6 14 Hexokinase A63.04 4 19 21 Glyceraldehyde-3-phosphate
dehydrogenase56.07 4 5 23 Eukaryotic translation initiation factor 5A-255.24 4 8 13 Phosphoglycerate mutase 154.04 4 5 20 60S Ribosomal protein L4-A43.59 3 4 8 Phosphogluconate dehydrogenase43.35 3 5 18 BMH1 Protein42.49 3 9 15 Alcohol dehydrogenase41.15 3 5 11 Glycerol-3-phosphate dehydrogenase39.39 3 4 6 Aconitate hydratase37.57 3 3 15 Ribosomal protein S436.99 3 6 11 Ketol-acid reductoisomerase33.77 3 3 19 Guanine nucleotide-binding protein30.14 2 3 27 Superoxide dismutase
Table 1Proteins with highest scores for on-line and off-line analysis of the yeast cell lysate.
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0 10 20 30 40 50 60 70 80Time [min]
0
1
2
37x10
Intens.
0
1
2
37x10
Intens.Fraction 5: BPC 200 -1800 ±All MS
0 10 20 30 40 50 60 70 80Time [min]
0 10 20 30 40 50 60 70 80Time [min]
0 10 20 30 40 50 60 70 80Time [min]
Fraction 6: BPC 200 -1800 ±All MS
Fraction 8: BPC 200 -1800 ±All MS
0
1
2
3
4
Intens. Fraction 7: BPC 200 -1800 ±All MS7x10
0
1
2
3
4
Intens.7x10
Figure 5Base peak chromatograms from SCX fractions fractions after RP separation
Figure 6Peptide from Orothate phosphoribosyltransferase. Protein number 140 in the off-line experiemnt, Score 9.28, Peptides 1, Spectra 1, Sequence coverage 8 %, MW 24.6 kDa, pI 5.80.
The chromatogram obtained forthe first dimension with themarked fractionations and theapplied gradient is shown in figure4. This chromatogram clearlyshows that the majority of thepeptides is eluted for a salt con-centration up to 100 mM NaClfrom the SCX column and collect-ed in fractions 2-9. The MS basepeak chromatograms corresponding to fraction 5-8 areshown in figure 5. As a consequenceof the better resolution the detectionof more peptides per single proteinleads to higher sequence coverageand therefore to a more significantprotein identification. Furthermore,a better resolution leads to thedetection of additional lowerabundant proteins for the off-lineapproach. Due to a higher
experiment and not found in theon-line experiment. The MS/MSfragmentation pattern of the identified peptide gives a reliableidentification of this particularprotein (figure 6).
The Agilent Spectrum Mill software provides an excellent
chromatographic resolution in the off-line method peptides and corresponding proteins which fall below significance level in the on-line procedure wereidentified. This is demonstratedwith the identification of Orothatephosphoribosyltransferase, whichis protein number 140 in the off-line
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tool to compare complex proteomeanalyses from multidimensionalseparations. Results from individualsalt step injections and single fractions as well as distribution ofpeptides in different fractions canbe displayed and reviewed easily.This is shown for the distributionof the tryptic peptides over the
Sequence m/z MH+ FRAC1-3 FRAC4 FRAC5 FRAC6 FRAC7 FRAC8 FRAC9 FRAC10 FRAC11 FRAC12-13Measured Matched Score Score Score Score Score Score Score Score Score Score
(K)AAQDSFAAGWGVMVSHR(S) 597.49 1789.84 10.6014.9010.83
(K)AVDDFLISLDGTANK(S) 790.13 1578.80 13.71 14.259.79
14.19(R)GNPTVEVELTTEK(G) 708.94 1416.72 12.75(R)IEEELGDNAVFAGENFHHGDKL(-) 814.69 2441.14 18.84(K)IGLDCASSEFFK(D) 687.51 1373.64 12.43
12.34(K)NVNDVIAPAFVK(A) 644.07 1286.71 13.21
11.83(K)NVPLYKHLADLSK(S) 749.95 1497.84 9.09 12.49(R)SGETEDTFIADLVVGLR(T) 911.52 1821.92 10.00
15.50 13.19(R)SIVPSGASTGVHEALEMR(D) 921.50 1840.92 12.74 16.50
9.62 12.3013.90
(K)TAGIQIVADDLTVTNPK(R) 878.37 1755.95 18.3516.79
(K)VNQIGTLSESIK(A) 645.09 1288.71 12.20(K)WLTGPQLADLYHSLMK(R) 625.09 1872.97 9.23
Table 2Distribution of the tryptic peptides over the collected fractions from the protein Phosphopyruvate hydratase identified in the off-line experiment.
shows the detailed results afterdatabase search of all identifiedpeptides from the protein. Theproteins’ identity, as well as itssequence, the sequence coverage,the species and the databaseaccession number is displayed.
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Figure 7MS/MS spectrum of one identified peptide from Phosphopyruvate hydratase. The total sequence coverage is shown below the spectrum.
collected fractions from the protein Phosphopyruvate hydrataseidentified in the off-line experiment(table 2). This peptide distribution-also demonstrates the perfor-mance of the SCX separtion. Thequality of the linear gradient separa-tion is indicated by the fact thatmost of the peptides are onlyfound in one fraction. SingleMS/MS
spectra can be retrieved in a fastmanner for manual inspection ofthe automated peptide and proteinvalidation settings. As an examplethe single peptide MS/MS spectrumof Phosphopyruvate hydratasethat was identified by 2D off-lineis shown in figure 7. All identifiedfragments of the y- and b-seriesare indicated. The lower panel
longer analysis time. Of course,using less stringent parameters inthe database search will alsoincrease the number of identifiedproteins. However, these hits arepresumably false positive identifiedproteins and their correspondingpeptide fragmentations patternneeds a careful, time consumingmanual inspection to decide theirvalidity.
Furthermore, the off-line methodology adds more fexibilityand a high degree of automationto a multidimensional chromato-graphic approach. Besides SCXand RP other additional separa-tion or prefractionation tech-niques can be combined. Further-more, after one dimension the col-lected samples might be stored forlater repeated analysis or subjectedto chemical or enzymatic modifi-cation before they are transferredto the next dimension.
A comparison of the applicabilityof both approaches indicates thatthe off-line approach is best suitedfor highly complex samples orwhen a separation step for furtherprocessing is necessary. In such acase the higher investment andmore complex handling for an off-line system is justified.
Conclusion
Off-line 2D LC-MS/MS represents a powerful alternative to on-linemethodologies for protein identification from complex proteomes. In such an approachthe trypsin-digested peptides areeluted with a continuous salt gradient from a SCX column andcollected as small fractions. These fractions are subsequentlyseparated on a RP column, analyzed by ESI-MS/MS and identified with the Agilent SpectrumMill MS Proteomics Workbench. In contrast, the conventional on-line technique elutes in the firstdimension with discrete salt steps.In the second dimension the elutedpeptides are directly separated on a RP column, analyzed by ESI-MS/MS and finally identified.
In this study we were able todemonstrate that in the off-lineapproach about 40 % more pro-teins were identified than in acomparable on-line approach. Fur-thermore, we could show that thescores for the identified proteinsby both approaches are higherwhen applying the off-line tech-nique. This means that the proba-bility for false positive proteinidentification decreases whenapplying the off-line technique andtherefore makes analyses morereliable. The total number of iden-tified proteins in an off-line analy-sis can be increased further by per-forming the SCX dimension with alonger shallow gradient. This,however, requires the collection ofadditional fractions and significant
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References
1.Davis MT, Beierle J, Bures ET,McGinley et.al. “Automated LC-LC—MS-MS platform usingbinary ion-exchange and gradientreversed-phase chromatographyfor improved proteomic analysis”.J. Chromatogr. B 752: 281-291,2001.
2. Vollmer M, Nägele E, Hörth P.“Two-dimensional nano LC/MS forcomplex proteomics application”.Agilent Technolgies Application
Note, Publication number
5988-9287EN, 2003.
3. Washburn MP, Wolters D, Yates JR.“Large-scale analysis of the yeastproteome by multidimensionalprotein identification technology”.Nat. Biotechnol. 19: 242-247,
2001.
4. Nägele E, Vollmer M, Hörth P.“Proteome profiling in E. coli:Effect of heat-shock conditions onprotein expression pattern”. Agilent Technolgies Application
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5988-8629EN, 2003.
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Product Brochure, Publication
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Copyright © 2003 Agilent TechnologiesAll Rights Reserved. Reproduction, adaptationor translation without prior written permissionis prohibited, except as allowed under thecopyright laws.
Published August 1, 2003Publication Number 5988-9913EN
www.agilent.com/chem/proteomics
Ralf Moritz is Application
Chemist, Martin Vollmer is R&D
Biochemist and Edgar Nägele is
Application Chemist, all at
Agilent Technologies GmbH,
Waldbronn, Germany.
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