Peptides OFFGEL electrophoresis: a suitable pre-analytical step for complex eukaryotic samples fractionation compatible with quantitative iTRAQ labeling
© Chenau et al; licensee BioMed Central Ltd. 2008
Received: 01 December 2007
Accepted: 26 February 2008
Published: 26 February 2008
The proteomes of mammalian biological fluids, cells and tissues are complex and composed of proteins with a wide dynamic range. The effective way to overcome the complexity of these proteomes is to combine several fractionation steps. OFFGEL fractionation, recently developed by Agilent Technologies, provides the ability to pre-fractionate peptides into discrete liquid fractions and demonstrated high efficiency and repeatability necessary for the analysis of such complex proteomes.
We evaluated OFFGEL fractionator technology to separate peptides from two complex proteomes, human secretome and human plasma, using a 24-wells device encompassing the pH range 3–10. In combination with reverse phase liquid chromatography, peptides from these two samples were separated and identified by MALDI TOF-TOF. The repartition profiles of the peptides in the different fractions were analyzed and explained by their content in charged amino acids using an algorithmic model based on the possible combinations of amino acids. We also demonstrated for the first time the compatibility of OFFGEL separation technology with the quantitative proteomic labeling technique iTRAQ allowing inclusion of this technique in complex samples comparative proteomic workflow.
The reported data showed that OFFGEL system provides a highly valuable tool to fractionate peptides from complex eukaryotic proteomes (plasma and secretome) and is compatible with iTRAQ labeling quantitative studies. We therefore consider peptides OFFGEL fractionation as an effective addition to our strategy and an important system for quantitative proteomics studies.
The proteomes of mammalian cells, tissues and body fluids are complex and display a wide dynamic range of proteins concentration. In order to overcome the human proteome complexity and determine the proteome content, it is necessary to use sample fractionation steps.
The recent introduction of commercially available OFFGEL fractionator system by Agilent Technologies, provides an efficient and reproducible separation technique [1, 2]. This separation is based on immobilized pH gradient (IPG) strips and permits to separate peptides and proteins according to their isoelectric point (pI), but is realized in solution [1, 3]. Therefore, its micropreparative scale provides fraction volumes large enough to perform subsequent analyses as reverse phase (RP) – liquid chromatography (LC) – MALDI MS/MS.
The secretome, first introduced by Tjalsma et al in 2000, describes the global study of secreted proteins by a cell, tissue or organism at any given time or under certain conditions [4, 5]. Secretome is the origin of circulating proteins in the body and a very promising source for discovery of new biomarkers candidates. Human plasma is the most complex body fluid and contains a large number of proteins with a dynamic range of at least 9–10 orders of magnitude . This complexity is a problem for proteomic analysis and it is necessary to develop efficient separation techniques to determine its precise protein composition.
In this study, we applied OFFGEL fractionation in combination with RP LC-MALDI MS/MS analysis to separate peptides of complex samples as secretome and plasma and we analyzed and explained the specific repartition profiles obtained. We also evaluated the efficiency of OFFGEL fractionation with peptides previously labeled by the isobaric amine-specific tags used in recently developed iTRAQ™ (isobaric tags for relative and absolute quantification) technology . The validation of OFFGEL electrophoresis on separation of eukaryote tryptic digests will permit to include it in proteomic workflow of complex samples.
Results and discussion
The expected pH ranges per fraction were calculated according to the IPG strip supplier data and the OFFGEL well dimensions and compared with experimental pI obtained for plasma and secretome samples.
Expected pH range
Peptide pI Average
Peptide pI Average
iTRAQ labeling influence on peptides repartition for OFFGEL fractions 2, 11 and 24 for secretome sample.
Peptide pI Average
Peptide pI Average
% of Coverage
Sample fractionation is an essential step for proteomics analysis. The data reported here show that OFFGEL system provides a highly valuable tool to fractionate peptides from complex eukaryotic samples like plasma or secretome. We also demonstrated the compatibility of OFFGEL fractionation with the iTRAQ labeling reagents used for quantitative proteomics. Finally, we consider peptides OFFGEL separation as a highly valuable technique to integrate in a proteomic workflow for complex sample analysis.
For quality and standardization issues, the plasma samples used were IQC samples (Internal Quality Control) provided by the proteomic laboratory of Dijon (Plateform Proteomic IFR- santé-STIC, France) as calibrated and reference samples. IQC was a pool of inactivate plasma packaged in controlled conditions. The samples were received in 50 μL aliquots and stored at -130°C until use. The samples were depleted using PROTEOPREP 20S spin column technology, according to the procedure recommended by Sigma-Aldrich, which remove the 20 highest abundant proteins. After 10 depletion cycles, corresponding to 80 μL of crude plasma, depleted solutions are recovered and then concentrated by ultra filtration using a 5 kDa molecular mass cut-off spin column (Amicon Ultra, Millipore).
H358 human non-small lung adenocarcinoma cell line  were grown in RPMI-1640 medium with glutamine, 10% heat-inactivated fetal bovine serum and Penicillin/Streptomycin 5000 U in 175-cm2 dishes until they reached a confluence state of approximately 60–70 %. They were then gently washed four times with phosphate buffered saline and two times with serum free medium to eliminate serum contaminants and left in serum free medium for 72 hours. The conditioned medium was collected and cooled down on ice. Floating cells and cellular debris were removed by centrifugation (200 × g, 10 min) followed by sterile filtration (pore size: 0.2 μm). Proteins were then concentrated and desalted by ultra-filtration using a 5 kDa molecular mass cut-off spin column (Amicon Ultra, Millipore) according to the manufacturer instructions. The total protein amount was determined using a standard Bradford protein assay (Bio-Rad).
Secretome and plasma samples were treated simultaneously and with the same protocol. 200 μg of each sample were reduced with 45 mM dithiothreitol at 50°C for 35 min and alkylated with 100 mM iodoacetamide at room temperature for 45 min. Trypsin (Promega) was added at an enzyme: protein ratio of 3:100 w/w and incubated overnight at 37°C. The digests were dried by vacuum centrifugation prior to the OFFGEL peptides fractionation.
200 μg of proteins from secretome sample were resuspended, reduced, alkylated, and digested according to the standard protocol supplied by the manufacturer (Applied Biosystems). Then, 100 μg of each digest were labeled either with iTRAQ reagent 114 or iTRAQ reagent 117. After labeling, samples were pooled in a ratio 1:1 (v/v) (for a total of 200 μg of peptide digests) and dried by vacuum centrifugation prior to the OFFGEL peptides fractionation.
OFFGEL peptides fractionation
To perform peptide fractionation according to their pI, the 3100 OFFGEL Fractionator and the OFFGEL Kit 3–10 (both from Agilent Technology) were used following the user protocol. The device was set up for the 24 fractions separation by using 24-cm-long IPG gel strip with a linear pH gradient ranging at 3–10. The peptides are separated in a two-phases system: liquid upper phase (focusing buffer provided by the supplier) separated in wells and lower IPG gel strip phase. The wells are isolated from each others. There is no direct fluidic connection between the wells. The peptides migrate through the IPG gel that plays the role of 'bridge" between each well and are retrieved in the solution at the IPG region where pH is peptides pI. 200 μg of secretome (with or without iTRAQ labelling) or plasma tryptic digests were resuspended with focusing buffer to a final volume of 3.6 mL. 150 μL of this sample was loaded in each of the 24 wells. The sample was focused using the recommended method for OFFGEL peptides 24 wells fractionation with a maximum current of 50 μA. The focusing was stopped after total voltage reaches 50 kVh. During the focusing, oil was added to the electrodes to prevent any evaporation effect. After focusing, 50 to 150 μl of sample was recovered for each well and transferred in individual micro tubes. To recover as much as possible the focusing peptides, 150 μl of methanol was added to each well, incubated for 15 min without voltage . Corresponding peptides fractions were pooled and concentrated by vacuum centrifugation prior to LC-MALDI MS/MS analysis.
Nano Reversed-phased LC-MALDI MS/MS analysis
Peptides were re-dissolved in 20 μl 0.2% trifluoroacetic acid. Peptides separation was performed on an Ultimate nanoHPLC System (Dionex/LC Packings, France) equipped with a PepMapC18 column (Dionex/LC Packings; 3-μm particles, 10 nm pore size, 75-μm i.d.), an autosampler and a Probot microfraction collector. The mobile phase consisted of a gradient of solvents A (0.05% trifluoroacetic acid; 2% acetonitrile in water) and B (0.05% trifluoroacetic acid; 80% acetonitrile in water). Injection was performed with 100% solvent A. The peptides were separated with a linear gradient of solvent B from 0–5% in 5 min, followed by an increase until to 40% of solvent B in 30 min and to 55% in 10 min at a flow rate of 0.3 μL/min. The column was washed and regenerated with 90% solvent B for 10 min and with 100% solvent A. For MALDI MS/MS analysis, column effluent was mixed in a 1:3 ratio with MALDI matrix (2 mg/mL α-cyano-4-hydroxycinnamic acid in 0.1% trifluoroacetic acid/70% acetonitrile) (v/v) and deposed on an Opti-tof LC/MALDI Insert 123 × 81 mm plate (Applied Biosystems) at a frequency of one spot/15s.
MALDI plates were analyzed by MALDI TOF TOF 4800 proteomics Analyzer mass spectrometer (Applied Biosystems) in positive reflector ion mode. MS spectra from m/z 700–3500 were acquired for each spot using 1500 laser shots. The ten most intense peaks in each MS spectrum above an S/N threshold of 100 were selected for MS/MS analysis.
Peptides and proteins identification were performed using the GPS (Global Proteome Server) Explorer software V3.6 (Applied Biosystems) with Mascot (Matrix Science) as the database search engine (V2.0). Each MS/MS spectrum was searched against a database of human protein sequences (Swiss-Prot, downloaded January 2006), resulting in a set of tryptic peptides matches with confidence values. Only the peptides with C.I.% > 85%, for any MS/MS spectrum were retained for further analysis. These peptide identifications were then combined using the MASCOT search engine to yield a set of human protein identifications with confidence values. The MASCOT searches were run using the following parameters: methionine oxidation, cystein carbamidomethylation modifications were selected as variable; 1 missed cleavage allowed; precursor error tolerance at < 50 ppm; MS/MS fragment tolerance set to 0.2 Da and charge set to +1; full trypsin specificity (N- and C-terminal also applied). Only proteins with at least two specific peptides matched were considered positively identified.
The theoretical pI of the peptides, were calculated using "Compute pI/Mw" tool accessible on the Expasy website [13, 17]. The identified peptides with an ion score C.I.% higher than 95% were always kept in the peptide list even if theoretical pI did not match with pI of the relevant individual OFFGEL fraction. For peptide ion score C.I.% between 85% and 95%, peptides were kept if calculated theoretical pI was correlated with pH range ± 2.
Matrix Assisted Laser Desorption Ionization
Isoelectric Point Gel
Internal Quality Control
Human Proteome Organisation.
The medical proteomic platform of the Center for Innovation in Biology (CIB) benefits from financial supports from the Lyon-Auvergne-Rhône-Alpes Canceropole (CLARA), Nanobio project, Grenoble university hospital and the French research minister. This work was supported by a grant from the French research minister to JC. This work was supported by a Grant from INCA (National Institute of Cancer) free projects 2006 PL06_025, "Analysis of secreted and targeted proteins from non-small cell lung cancers for the identification of new seric biomarkers". We would like to thank Patrick Ducoroy (Platform Proteomic IFR-Sante-STIC) for providing Internal Quality Control Plasma samples and Pascal Bertolino for developing specific algorithms.
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