Improved proteome coverage by using iTRAQ labelling and peptide OFFGEL fractionation
© Ernoult et al; licensee BioMed Central Ltd. 2008
Received: 01 August 2008
Accepted: 13 October 2008
Published: 13 October 2008
The development of mass spectrometric techniques and fractionation methods now allows the investigation of very complex protein mixtures ranging from subcellular structures to tissues. Nevertheless, this work is particularly difficult due to the wide dynamic range of protein concentration in eukaryotic tissues. In this paper, we present a shotgun method whereby the peptides are fractionated using OFFGEL electrophoresis after iTRAQ labelling.
We demonstrated that iTRAQ peptide labelling enhances MALDI ionisation and that the OFFGEL fractionation of the labelled peptides introduces a supplementary criterion (pI) useful for validation and identification of proteins. We showed that iTRAQ samples allowed lower-concentrated proteins identification in comparison with free-labelled samples.
The combined use of iTRAQ labelling and OFFGEL fractionation allows a considerable increase in proteome coverage of very complex samples prepared from total cell extracts and supports the low-concentrated protein identification.
The iTRAQ-reagent is well known for relative and absolute quantitation of proteins [1–3]. The interest of this multiplexing reagent is that 4 or 8 analysis samples  can be quantified simultaneously.
In this technique, the introduction of stable isotopes using iTRAQ reagents occurs on the level of proteolytic peptides. The iTRAQ technology uses an NHS ester derivative to modify primary amino groups by linking a mass balance group (carbonyl group) and a reporter group (based on N-methylpiperazine) to proteolytic peptides via the formation of an amide bond. Due to the isobaric mass design of the iTRAQ reagents, differentially-labelled peptides appear as a single peak in MS scans, reducing the probability of peak overlapping. When iTRAQ-tagged peptides are subjected to MS/MS analysis, the mass balancing carbonyl moiety is released as a neutral fragment, liberating the isotope-encoded reporter ions which provides relative quantitative information on proteins.
An inherent drawback of the reported iTRAQ technology is due to the enzymatic digestion of proteins prior to labelling, which artificially increases sample complexity. Since it has been shown that a reliable determination of protein dynamics requires quantitative evaluation of an adequate set of proteolytic peptides derived from each protein, the iTRAQ approach needs a powerful, multi-dimensional fractionation method of peptides before MS identification.
Reported peptide separation methods include strong cation exchange (SCX) chromatography and reverse-phase chromatography . Recently, isoelectric focusing (IEF), a high-resolution electrophoresis technique for separation and concentration of amphoteric biomolecules at their isoelectric point (pI), has been used in shotgun proteomic experiments . IEF runs in a buffer-free solution containing carrier ampholytes or in Immobilized pH gradient (IPG) gels. Recently, the use of IPG-IEF for the separation of complex peptide mixtures has been applied to the analysis of plasma and amniotic fluid [7, 8] as well as to bacterial material . However, a major limitation of this method is the tedious post-IEF sample processing. The IPG gel strip is divided into small sections for extraction and cleaning up of the peptides. A new concept called OFFGEL electrophoresis was recently introduced with the primary aim of purifying proteins and peptides . This technique recovers the sample from the liquid phase and was demonstrated to be of great interest in shotgun proteomics . IEF is not only a high resolution and high capacity separation method for peptides, it also provides additional physicochemical information like their isoelectric point [12, 13]. The pI value provided is used as an independent validating and filtering tool during database search for MS/MS peptide sequence identification .
Recently, the compatibility of iTRAQ isotope labelling and OFFGEL-IEF for relative quantification and validation of sequence matches from database searching was shown from a BSA tryptic digest sample and complex eukaryotic samples [15, 16] but surprisingly, no attempts was done to undertake comprehensive analysis of influence of iTRAQ labelling on the proteome coverage ratio.
In our work, we combined free-labelled peptides or iTRAQ labelled peptides and OFFGEL fractionation for the proteomic study of a very complex sample like the human neuroblastoma SH-SY5Y cell line [17–19] in a wide pI-range (pH 3–10) and compared the proteome coverage between free-samples and iTRAQ-samples.
Results and discussion
The influence of iTRAQ-reagent tagging
The list of experiments and the number of identified proteins
Proteins with 2 peptides (at least)
SCX + OFFGEL-IEF
Increase in peptide mass following iTRAQ labelling
The presence of iTRAQ label on the NH2 terminal adds a mass of 145 to the peptide m/z. If the peptide had a lysine residue at the Cterm position, two iTRAQ labels are added (290 Da). Looking at the list of peptides identified in the iTRAQ-310 label experiment, 61 unique peptides without lysine in their sequence had a m/z between 800 and 945, and 55 other peptides with a lysine at the Cterm extremity had a mass between 800 and 1090. These 116 peptides were not visible without iTRAQ labelling; nevertheless, they were found in the sequence of 60 proteins already identified by at least 2 peptides. Only 3 proteins were identified by a second peptide. The mass increase due to iTRAQ-labelling cannot be the unique explanation of the higher number of proteins identified nor the higher number of peptides contributing to the identification for each protein.
iTRAQ increases ionisation of peptides containing lysines
The influence of the quantity of iTRAQ-labelled peptides
From only 400 μg of reduced, blocked, digested cellular lysate labelled using iTRAQ, we were able after SCX separation to identify, 492 proteins, of which 429 with at least 2 peptides, giving an identification rate of 87% (iTRAQ-429). By doubling the amount of biological material to start with, identification rate for proteins with at least 2 peptides raised by 39% (Table 1).
The influence of the fractioning method on free-labelled peptides
The quality of OFFGEL separation of iTRAQ-labelled peptides
As shown in Figure 3, 78% of the identified peptides are found in only one fraction and about 90% are found in one or two fractions, confirming the distribution of free-labelled peptides in our work and the results from earlier studies [16, 21].
The distribution of peptides per fraction is shown in Figure 4. The pI value for each identified peptide was calculated by using Bjellqvist's algorithm without taking into account the iTRAQ groups in N-term position and/or on the lateral lysine chain. Using these data, average pI values with standard deviations were calculated for all peptides identified in each fraction (Figure 4A). The average experimental pI value deviated from the theoretical pI value by an average error of +/- 0.43. In the pH range of 3 to 8, the average pI value of the labelled peptides fits very well with the average value of the non-labelled peptides. Major deviations were observed at the basic pH range (8.3–10.0) for fractions 18–24. By splitting OFFGEL fractionation into 2 zones, we noticed that the mean error in the pH 3–8 range was +/- 0.34 and was rising to +/- 0.64 in the more alkaline range (pH 8–10); this can probably be explained by the fact that the software used for calculating the pI value does not take into account the presence of the iTRAQ label. As for the free-labelled peptides, we introduced the pI iTRAQ-labelled peptides property as a filter to verify the identification of the set of peptides. For the fractions F1 to F17 (pH 3.3–8.0 range), all peptides with an unused ProteinPilot score above 1.3 and with an experimental pI difference larger than 0.4 pI unit were excluded. For the fractions F18-F24 (pH 8.3–10.0), we excluded all peptides with experimental pI difference greater than 0.7 pI unit. All iTRAQ-labelled peptides presented in this work were filtered using this protocol.
Combining results of all experiments, we end up with 947 proteins (Additional file1) identified with at least 2 peptides and 6119 unique peptides. 380 detected proteins have at least 5 or more unique peptides identified.
By comparing MW profiles of the proteins identified in the iTRAQ-947 and iTRAQ-739 experiments, we can see that the profiles are comparable (Figure 7), strengthening the hypothesis that the iTRAQ-739 experiment correctly reflects proteome coverage of the cell line SH-SY5Y.
Relative abundance of proteins
iTRAQ is at evidence a very powerful tool, recognised for its ability to relatively quantify proteins. In this work, we showed that the iTRAQ reagent improves MALDI ionisation, especially for peptides containing lysine. A direct consequence of this property is the better chance of identifying low abundance proteins in complex biological materials. We were also able to demonstrate that an OFFGEL fractionation step, has a positive influence on the number of proteins identified compared to SCX method.
From standard clinical protein quantities (400 μg), we proposed a methodology allowing the identification of more than 800 proteins. Having established a protein database of SH-SY5Y cells, we can now use it as a reference for further research in this domain.
Materials and methods
Cell culture and protein extraction
The human neuroblastoma cell line SH-SY5Y was a gift from Dr F. Vallette (INSERM U601, Nantes, France). Cells were grown in RPMI-1640 medium (Lonza) supplemented with 10% (v/v) fetal bovine serum (Lonza) without antibiotics in a, 5% humid CO2 atmosphere at 37°C. Cells were scraped and washed 3 times with PBS (300 × g, 5 min.). Cell pellets were then lysed by a solution containing 7M urea, 2M thiourea, 4% (w/v) CHAPS at 4°C for 1 h using a rotary shaker. Lysis was achieved by sonication on ice (3 × 5s pulses), and the lysates were clarified by centrifugation at 14,000 × g at 4°C for 15 min.
Protein digestion and peptide labelling with iTRAQ reagents
Protein samples were cleaned up by precipitation with 6 volumes of cold acetone at -20°C overnight followed by resuspension of pellets in 0.5M triethylammonium bicarbonate (TEAB) pH 8.5 (Sigma-Aldrich) and final centrifugation step at 14,000 × g at 4°C for 15 min. We quantified proteins from supernatant with the 2-D Quant Kit (GE Healthcare, München, Germany) before diluting the protein samples up to 5 mg/ml with TEAB buffer. We took 50 or 100 μg of proteins for further reduction, alkylation, digestion and iTRAQ labelling using iTRAQ Reagents Multiplex Kit (Applied Biosystems) according to manufacturer's protocol. Briefly, protein samples were reduced with 5 mM tris-(2-carboxyethyl)phosphine (TCEP) at 60°C for 1 h. and the cysteine-groups were blocked using a 10 mM methyl methanethiosulfonate (MMTS) solution at room temperature for 10 min. . The proteins were then digested by 10 μg of trypsin at 37°C for 16 h. Each peptide solution was labelled at room temperature for 1 h with one iTRAQ reagent vial (mass tag 114, 115, 116 or 117) previously reconstituted with 70 μl of ethanol. Samples of the same protein content, and labelled respectively with 114, 115, 116 and 117 iTRAQ reagents, were combined and labelling reaction stopped by evaporation in a Speed Vac to obtain a brown pellet.
Peptide OFFGEL fractionation
For pI-based peptide separation, we used the 3100 OFFGEL Fractionator (Agilent Technologies, Böblingen, Germany) with a 24-well set-up. Prior to electrofocusing, samples were desalted onto a Sep-Pak C18 cartridge (Waters). For 24-well set-up, peptide samples were diluted to a final volume of respectively 3.6 mL using OFFGEL peptide sample solution. To start, the IPG gel strips of 24 cm-long (GE Healthcare, München, Germany) with a 3–10 linear pH range were rehydrated with the Peptide IPG Strip Rehydradation Solution according to the protocol of the manufacturer for 15 min. Then, 150 μL of sample was loaded in each well. Electrofocusing of the peptides is performed at 20°C and 50 μA until the 50 kVh level was reached. After focusing, the 24 peptide fractions were withdrawn and the wells rinsed with 200 μL of a solution of water/methanol/formic acid (49/50/1) after 15 min, the rinsing solutions were pooled with their corresponding peptide fraction. All fractions were evaporated by centrifugation under vacuum and maintained at -20°C. Just prior nano-LC, the fractions were resuspended in 20 μL of H2O with 0.1% (v/v) TFA.
Capillary LC separation
The samples were separated on an Ultimate 3,000 nano-LC system (Dionex, Sunnyvale, USA) using a C18 column (PepMap100, 3 μm, 100A, 75 μm id × 15 cm, Dionex) at a flow rate of 300 nL/min. . Buffer A was 2% ACN in water with 0.05% TFA and buffer B was 80% ACN in water with 0.04% TFA.
Peptides were desalted for 3 min. using only buffer A on the precolumn, followed by a separation for 60 min. using the following gradient: 0 to 20% B in 10 min., 20% to 55% B in 45 min. and 55% to 100% B in 5 min. Chromatograms were recorded at the wavelength of 214 nm. Peptide fractions were collected using a Probot microfraction collector (Dionex).
For the SCX fractionation, we used a salt gradient steps: 20-μl injections of 5 mM, 10 mM, 25 nM, 50 mM, 75 mM,100 mM, 125 mM, 150 mM, 200 mM, 300 mM, 500 mM, 1000 mM NaCl
We used CHCA (LaserBioLabs, Sophia-Antipolis, France) as MALDI matrix. The matrix (concentration of 2 mg/mL in 70% ACN in water with 0.1% TFA) was continuously added to the column effluent via a micro "T" mixing piece at 1.2 μL/min flow rate. After 14 min run, a start signal was sent to the Probot to initiate fractionation. Fractions were collected for 10s and spotted on a MALDI sample plate (1,664 spots per plate, Applied Biosystems, Foster City, CA).
MS and MS/MS analyses of off-line spotted peptide samples were performed using the 4800 MALDI-TOF/TOF Analyser (Applied Biosystems). After screening of all LC-MALDI sample positions in MS-positive reflector mode using 1500 laser shots, the fragmentation of automatically-selected precursors was performed at collision energy of 1 kV using air as collision gas (pressure of ~2 × 10-6 Torr). MS spectra were acquired between m/z 800 and 4000. For internal calibration, we used the parent ion of Glu1-fibrinopeptide at m/z 1570.677 diluted in the matrix (3 femtomoles per spot). Up to 12 of the most intense ion signals per spot position having a S/N > 12 were selected as precursors for MS/MS acquisition. Peptide and protein identification were performed by the ProteinPilot™ Software V 2.0 (Applied Biosystems) using the Paragon algorithm . Each MS/MS spectrum was searched for Homo sapiens specie against the Uniprot/swissprot database (release 51 of October 2006). The searches were run using with the fixed modification of methylmethanethiosulfate labelled cysteine parameter enabled. Other parameters such as tryptic cleavage specificity, precursor ion mass accuracy and fragment ion mass accuracy are MALDI 4800 built-in functions of ProteinPilot software.
The ProteinPilot software calculates a confidence percentage (the unused score) which reflects the probability that the hit is a false positive, meaning that at 95% confidence level, there is a false positive identification chance of about 5%.
While this software automatically accepts all peptides having an identification confidence level >1%, only proteins having at least one peptide above 95% confidence were initially recorded. The low confidence peptides cannot give a positive protein identification by themselves, but may support the presence of a protein identified using other peptides with higher confidence. Performing the search against a concatenated database containing both forward and reversed sequences allowed estimation of the false discovery rate below1%.
strong cation exchange
Immobilized pH gradient
protein abundance indice
This work was supported by grants from the "Ligue Nationale Contre le Cancer" (program équipe labellisée).
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