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.

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“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 releaseand have found that high resolution pI prefractionation allowed us

to increase our sensitivity in analysis of complex samples” Edward Dratz, Montana State University

OFFGEL electrophoresis separatesproteins or peptides according totheir isoelectric points, whereby theseparated components are recoveredin liquid phase. Up- or downstreamsample processing steps such asimmunodepletion, protein digestionand liquid chromatography can beeasily interfaced with this techniquefor multi-dimensional separations of

complex samples. With its newstate-of-the-art web-based userinterface OFFGEL electrophoresishas just become even moreintuitive to use. Directly connect itto the supplied Netbook orconnect to your local area networkto operate from any PC* or iPhone.

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 samplecomponents are recovered from liquidphase. This makes the technologydirectly compatible with liquid-phaseworkflows, for example 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 the endsof the gel strip and the protein orpeptide molecules migrate throughthe gel until they reach a positionwhere the pH equals the pI of themolecule.

d) After fractionation, the separatedproteins or peptides remain in thebuffer solution and can be easilyremoved 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, pipette40 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 1 displayed onlineduring OFFGEL fractionation.

Individual sample diagnosticsfor a quick and easy check offractionation quality

With its state-of-the-art user interfacethe 3100 OFFGEL Fractionator measuresand records voltage, power and currentduring fractionation in OFFGEL andtraditional IEF mode.

Currents of each sample are measuredand displayed online individually.Current and voltage information can bevisualized using the online plot or byexporting the data to a spreadsheetapplication, providing direct diagnosticsfor the quality of each individual samplefractionation.

Features of the Agilent3100 OFFGEL Fractionator

• Easy setup with preinstalled methods forOFFGEL and conventional IPG-based IEF modes

• State-of-the-art user interface for intuitiveinstrument setup and control and onlinemonitoring of all key parameters that affectfractionation quality

• Independent high-voltage power supplies allowtwo different methods or sample sets to berun simultaneously

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

• Monitoring of all operations and error events inself-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 OFFGEL electrophoresis(24 fractions, pH 3-10). The overlay ofelectropherograms from five inde-pendent fractionations shows excellentreproducibility (UM - upper marker,bCA - bovine carbonic anhydrase II,BSA - bovine serum albumine, 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 2010 Agilent Technologies Published May 1, 2010Publication Number 5990-5596EN

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Type Specification Comments Weight 1 4 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

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

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