Agilent 3100 OFFGELFractionatorpI-based fractionation of proteins and peptides with liquid-phase recovery

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Achieve unprecedented sensitivity in LC/MS-based proteomics experiments

Sample complexity and large dynamic range of protein and peptideconcentrations are major hurdles inthe analysis of low abundance proteins.Today, for almost any proteomicanalysis, robust and reproduciblefractionation techniques are key to ensure that MS/MS can identify the maximum number of proteins. At the same time, to avoid inadvertentloss or alteration of critical proteins, it is important that fractionation steps minimize the number of samplemanipulations.

OFFGEL electrophoresis is a noveltechnique jointly developed by AgilentTechnologies and Diagnoswiss* that separates proteins or peptidesaccording to their isoelectric points,whereby the separated componentsare recovered in liquid phase. Up- or downstream sample processingsteps such as immunodepletion, proteindigestion and liquid chromatographycan be easily interfaced with thistechnique for multi-dimensionalseparations of complex samples.

*Technology and product developed and licensed by DiagnoSwiss S.A., Monthey, Switzerland. Technology based on scientific research done at the Swiss Federal Institute of Technology, Lausanne, Switzerland.

“The instrument is highly user-friendly and simple to use. In our group, the scientists can operate the

OFFGEL system after just an hour of training” Hookeun Lee, Senior Scientist at ETH, Zurich

“We had the opportunity to test the system prior to market release and have found that high resolution pI prefractionation allowed

us to increase our sensitivity in analysis of complex samples”

Edward Dratz, Montana State University

Easy integration into anyprotein analysis workflow

Whether your task is biomarkerdiscovery, protein identification orpurification of functional proteins or peptides, the Agilent 3100 OFFGELFractionator fits into any proteinanalysis workflow. Integration of theOFFGEL system is easy and ultimatelyincreases sensitivity of subsequentMS detection.

Benefits of the Agilent 3100 OFFGEL Fractionator

• pI-based OFFGEL fractionation withliquid-phase recovery for easytransfer to downstream techniquessuch as LC/MS

• Reproducible fractionation withresolution as low as 0.1 pH for maximum MS sensitivity

• Compatibility with up- ordownstream techniques such as immunodepletion, LC/MS or gel-based analysis for maximumflexibility

• pI values obtained act as additionalvalidation parameters of MS hits andcan be used to search for peptideswith charged posttranslationalmodifications (PTMs)

• All additives can easily be removedafter fractionation to avoidinterference with nano-electrosprayand MS detection

• Fractionation of up to 16 samples inparallel on two trays (eight sampleseach) for highest throughput

• Broad loading capacity from 50 µg to 5 mg of sample for analyticalapplications or maximum enrichmentof low abundance proteins

• OFFGEL mode as well asconventional isoelectric focusing(IEF) based on immobilized pHgradient (IPG) gels

HPLC-Chip/MS analysis of E.colilysate, showing total number of non-redundant proteins and peptidesidentified with and without OFFGELfractionation (pH 4-7, 24 fractions).

Adding OFFGEL fractionation into the workflow prior to digestion significantlyincreases the number of proteins and peptides identified by subsequent HPLC-Chip/MS analysis.

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Example workflow

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OFFGEL electrophoresis – high resolution pI fractionation based on proven IPG technology

The Agilent 3100 OFFGEL Fractionatorperforms isoelectric focusing ofproteins or peptides in immobilized pH gradient (IPG) gel strips. Thetechnique applied by the 3100 OFFGELFractionator differs from conventionalgel electrophoresis in that the sample components are recoveredfrom the liquid phase. This makes the technology directly compatible with liquid-phase workflows, forexample LC-MS.

a) The IPG gel strip is rehydrated andseals tightly against the well frame

b) The diluted sample is equallydistributed into all wells in theframe, and a cover seal is placed on top of the frame to preventsample evaporation

c) A high voltage is applied to theends of the gel strip and the proteinor peptide molecules migratethrough the gel until they reach a position where the pH equals the pI of the molecule

d) After fractionation, the separatedproteins or peptides remain in the buffer solution and can beeasily removed using a pipette forsubsequent downstream processing

Open design of fractionatorfor easy access to trays

Anode connectors

Fixed electrode (anode)

Well frames

Moveable electrode (cathode)

Tray

Cover seals

Cathode connectors

Cooling platform

Snap-in electrodes and wellframes facilitate easy set up of a fractionation

Fractionation principle

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1. Place a dry IPG gel strip in the tray.

2. Place a well frame over the IPG gel strip, pipette20 µl rehydration solutioninto each well and allowthe IPG gel to swell.

3. Pipette 150 µl of the diluted sample into each well and close the framewith a cover seal.

4. Attach the electrodes to the tray.

5. Place the loaded tray into the fractionator and press “Start”.

Five easy steps to set up and run a fractionation

Typical current pattern on tray I when every second lane is running an OFFGEL fractionation.

Individual sample diagnosticsfor a quick and easy check offractionation quality

The 3100 OFFGEL Fractionator measuresand records voltage, power and currentduring fractionation in OFFGEL andtraditional IEF mode. The current foreach sample is measured individually.The current/voltage information canbe visualized using the controller'sonline plot or by exporting the data toa spreadsheet application, and providesa diagnostic indicator for the quality of each individual sample fractionation.

Features of the Agilent3100 OFFGEL Fractionator

• Easy setup with preinstalledmethods for OFFGEL andconventional IPG-based IEF modes

• Independent high-voltage powersupplies allow two differentmethods or sample sets to be run simultaneously

• Online display of all importantfractionation parameters and onlinecurrent measurement of eachindividual sample for monitoring of key parameters that affectfractionation quality

• Automated storage of up to 1200 data files and easy export of run parameters

• Monitoring of all operations anderror events in self-updating logbook

• Cover design allows fractionation of light-sensitive protein samples

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Protein Fractionation

Fractionation of E.coli into 12 fractions (pH 3-10). Analysis of fractions 3-10 by 1D electrophoresis from three independent fractionations.

Fractionation of E.coli into 24 fractions (pH 4-7). Analysis of fractions 9 and 12 by 2D electrophoresis. The theoretical resolution of 0.13 pH units is indicated by the red bars.

Obtain greater reproducibility

Fractionation by OFFGEL electrophoresisis highly reproducible. Fractions from several runs may be pooled forenrichment of low abundance proteinsor peptides.

Achieve highest resolution

The Agilent 3100 OFFGEL Fractionatorprovides pI-based fractionation ofpeptides or proteins with exceptionalresolution. Samples can be fractionatedinto 12 or 24 fractions for standard or highest resolution.

Analysis of a protein IEF marker mixwith the Agilent 2100 bioanalyzerbefore and after OFFGELelectrophoresis (24 fractions, pH 3-10).The overlay of electropherograms fromfive independent fractionations showsexcellent reproducibility (UM - uppermarker, bCA - bovine carbonicanhydrase II, BSA - bovine serumalbumine, ConA - conalbumine).

Fluo

resc

ence

uni

ts

15 28 46 63 95 150 240 kDa

Fluo

resc

ence

uni

ts

Unfractionated IEF marker mix

15 28 46 63 95 150 240 kDa

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Peptide Fractionation

Gain sensitivity

Efficient reduction of sample complexityincreases sensitivity of downstreamanalysis. This example compares theefficiency of fractionation of the trypticpeptides of immunodepleted plasma by strong cation exchange (SCX) withOFFGEL electrophoresis.

Increase efficiency

Whereas proteins are oftenrepresented by isoforms with differentpI values, peptides have distinct,individual pI values. In this example, 90 % of all individual tryptic peptidesfocused efficiently into one or twoOFFGEL fractions.

Peptide fractionation by OFFGEL electrophoresis (pH 3-10, 24 fractions) is more efficient than separation by SCX (50 fractions) as indicated by the 3-foldhigher number of peptides and 2-fold higher number of proteins identified with HPLC-Chip/MS.

Number of OFFGEL fractions containing each individual peptide (absolute numbers of peptides in parenthesis).

75.3% (2603)

1 Fraction

1.2% (42)

0.6% (20)

0.3% (12)

2.6% (90)

5.5% (192)

15.5% (536)

2 Fractions

3 Fractions

4 Fractions

5 Fractions

6 Fractions

7 Fractions

© Copyright 2006 Agilent Technologies Published March 1, 2007Publication Number 5989-5700EN

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Type Specification Comments Weight 14 kg (31 lbs)

Dimensions(height × width × depth)

157 × 355 × 427 mm(6.2 × 14 × 16.8 inches)

Line voltage 100 – 240 VAC, ±10% Wide ranging capability

Line frequency 50 – 60 Hz, ±5%

Power consumption 140 VA

Ambient operating temperature 5 – 40 ºC (41 – 104 °F)

Humidity < 80% at 40° C (104 °F) Non-condensing

Operating altitude Up to 2000 m (6500 ft)

Safety standards IEC, CSA, UL Installation Category II, Pollution Degree 2

Flash memory card minimum 512 MB storage capacity

Platform temperature 10 ºC – 35 ºC Maximum 10 degrees belowambient, non-condensing

Power supplies Two independent high voltagepower supplies Voltage-Range: 0.5 – 10 kVCurrent-Range: 0 – 150 µA/stripPower: 0 – 1 W/stripModes:Constant voltageConstant currentContant powerTimetable programable

Individual currentmeasurement for eachsample

Fractionation capacity 2 × 8 samples (12 or 24 fractions) in OFFGELmode and IPG-IEF (in-gel) on 2 individual trays

Instrument specifications

Agilent 3100 OFFGEL Fractionator