Key words: multidimensional liquid chromatography (MDLC) • reversed-phase chromatography (RPC) • LC-MS/MS • DeCyder MS • differential analysis
Ettan™ MDLC was used in conjunction with tandem mass spectrometry (MS/MS) to separate and identify allergenic proteins extracted from the pollen of the olive tree, Olea europaea. DeCyder™ MS Differential Analysis Software (DeCyder MS) was used to quantitate differentially expressed proteins in two different samples, Ole_e1_"mer" and Ole_e1_"lite."
Ole e 1 is the most frequent sensitizing agent affecting more than 70% of the patients suffering from olive (Olea europaea) pollinosis. The protein consists of a single polypeptide chain of 145 amino acids (MW 16.3 kDa) and has been suggested to be involved in the hydration of pollen during germination (1).
Ole e 1 exists in several isoforms, displaying microheterogeneities, that are difficult to resolve by gel-based methods. Therefore, we used an LC-MS/MS protein separation and identification strategy.
DeCyder MS Differential Analysis Software (DeCyder MS) is a tool for visualization, detection, comparison, and label-free relative quantitation of LC-MS/MS data (2). In brief, the software displays LC-MS analyses as two-dimensional intensity maps, resulting in a much better overview of spectral quality and data compared with the traditional way to present LC-MS data. Statistical analysis is carried out on all peptide spots within minutes.
The DeCyder MS algorithm is based on accurate mass and reproducible retention times. Ettan MDLC is ideally suited to deliver reproducible retention times (3), and should be used to generate the data that will be analyzed by DeCyder MS.
In this application note, relative quantitation of allergenic proteins in two different samples, Ole_e1_"mer" and Ole_e1_"lite," was determined using DeCyder MS.
DeCyder MS Differential 11-0013-32
(including PC and single concurrent network user license)
Ettan MDLC 18-1176-44, 11-0008-41
Ettan LC System 18-5050-50
Fraction Collector Frac- 950 18-6083-00
Superdex™ 75 10/300 GL 17-5174-01
NAP™ 10 Columns 17-0854-02
Trypsin, sequencing grade 17-6002-75
PlusOne™ Dithiothreitol (DTT) 17-1318-01
PlusOne Tris 17-1321-01
PlusOne Urea 17-1319-01
PlusOne Iodoacetamide RPN6302
Finnigan™ LTQ™ mass spectrometer (Thermo Electron)
TurboSEQUEST™ protein identification software (Thermo Electron)
Zorbax™ 300-SB C18 trap column, 300 µm i.d. x 5 mm, 3 µm (Agilent)
Zorbax 300-SB C18 analytical column, 75 µm i.d. x 150 mm, 3 µm (Agilent)
Formic acid, ultrapure (Fluka)
Acetonitrile, HPLC grade
Water, HP LC grade
Protein samples were obtained from the pollen of the olive tree, Olea europaea, by aqueous extraction at low pH. The two samples used in this study, Ole_e1_"mer" and Ole_e1_"lite," refer to different batches of collected pollen containing varying amounts of the allergen.
Two mg each of Ole_e1_"mer" and Ole_e1_"lite" was partially purified using an Ettan LC System. The chromatographic separation was performed on a Superdex 75 10/300 GL gel filtration column at 0.5 ml/min in 80 mM sodium acetate, pH 4.5, 50 mM NaCl, and 0.5-ml fractions were collected using Fraction Collector Frac-950. Fractions were examined using MALDI-ToF MS. Those containing proteins with molecular weights 14–20 kDa were pooled and dried in vacuo.
Protein reduction, alkylation, and trypsin digestion
The pooled, dried fractions from the gel filtration step were dissolved in 0.5 ml of 9 M urea containing 50 mM DTT and incubated at ambient temperature for 60 min. An equal volume of a solution consisting of 8 M urea, 250 mM Tris-HCl, pH 8.8, and 125 mM iodoacetamide was added, and incubation was continued for another 60 min in darkness. The samples were desalted against 20 mM ammonium bicarbonate on NAP-10 Columns, and then digested with 5 µg trypsin for 2 h at 37 °C. The digestions were stopped by acidification with formic acid.
Dual reversed-phase nanoscale liquid chromatography
Ettan MDLC was used in the high-throughput configuration using two reversed-phase (RPC) trap columns 300 µm i.d. x 5 mm (Zorbax 300-SB C18) for online desalting and sample cleanup, followed by two nanoscale RPC analytical columns 75 µm i.d. x 150 mm (Zorbax 300-SB C18) for high-resolution separation. One set of RPC trap/analytical columns was equilibrated while the second set separated the sample. The mobile phases were A: HPLC-grade water with 0.1% formic acid, and B: 84% HPLC-grade acetonitrile with 0.1% formic acid. Approximately 0.50 µg of material was loaded onto the columns and separation was performed at a flow rate of 200 nl/min by applying a linear gradient of 0–60% B for 50 min. The digested samples were run in triplicate.
A Finnigan LTQ linear ion trap mass spectrometer equipped with a nanospray interface was used as the detector for the peptides that eluted from the RPC column. Full scan mass spectra were collected in profile mode and MS/MS spectra in centroid mode.
Detection of significantly varying peptides using DeCyder MS
Detection, profile comparison, background subtraction, and quantitation were done on the full scan precursor mass spectra in fully automatic mode using DeCyder MS (1).
The PepDetect module of DeCyder MS was used for automated peptide detection, charge-state assignments, deconvolution, and quantitation based on MS signal intensities of individual LC-MS analyses.
The PepMatch module was used to match peptides in different intensity maps from the different runs, which resulted in a quantitative comparison including statistical evaluation. The background intensity was used for normalization (no internal standards were added to the samples). Peptides were identified using intact masses by exporting the MS/MS files into TurboSEQUEST protein identification software and subsequently importing TurboSEQUEST search results into the PepMatch module. The peptide matches were filtered based on cross-correlation scores (Xcorr) of 1.5, 2, and 2.5 for charge states 1+, 2+, and 3+, respectively.
Results and discussion
Protein prefractionation of allergen samples
The two allergen samples, Ole_e1_"lite" and Ole_e1_"me r," were subjected to a protein prefractionation step using Ettan LC System and gel filtration on Superdex 75 to reduce the complexity of the samples. Fractions of 0.5 ml were collected throughout the analyses. The overlaid chromatograms from the gel filtration analyses are shown in Figure 1.
Fractions A4–A6 were pooled because they contained proteins varying in size from 14 to 20 kDa, thus covering the expected molecular weight of Ole e 1 according to analyses by MALDI-ToF MS (data not shown). As can be seen from the chromatograms, Ole_e1_"mer" contained about 3.5 times more material in the collected fractions in comparison with Ole_e1_"lite," as judged by integration of the UV traces of the collected fractions.
Nanoscale LC-MS/MS of tryptic allergen peptides
Fractions A4–A6 from the gel filtration analyses of Ole_e1_"mer" and Ole_e1_"lite," respectively, were pooled, digested with trypsin, and analyzed by LC-MS/MS using the Ettan MDLC coupled to a Finnigan LTQ linear ion trap mass spectrometer. The resulting base peak ion chromatogram of Ole_e1_"lite" is shown in Figure 2.
The full scan MS data was acquired in profile mode, which is required in order to get enough data points for evaluation by DeCyder MS, while MS/MS data (not used for matching) was collected in centroid mode. The other batch, Ole_e1_"mer," displayed the same base peak chromatogram profile (data not shown). The generated MS and MS/MS files were transferred to TurboSequest software, which resulted in identification of about 40 proteins with at least two peptide hits for each protein from each of the examined samples. For each batch the highest ranked protein candidate was identified as the main olive allergen.
Detection of olive allergens using DeCyder MS
The individual intensity maps created from the Ettan MDLC-MS/MS analyzed fractions of Ole_e1_"mer" and Ole_e1_"lite" were examined using the PepDetect module of DeCyder MS, where features are detected and quantitated, background intensities are subtracted, and charge states are assigned. Furthermore, the LC-MS data is presented as two-dimensional intensity maps with m/z on the y axis and retention time (RT) on the x axis, as shown in Figure 3. The density of the grayscale pattern confined within the blue boxes is proportional to the signal intensity of a peptide peak at a certain m/z, RT, and charge state, and thus corresponds to full-scan MS data. The red crosses indicate positions (scan numbers) where MS/MS data is available that can be used for identification.
The intensity map in Figure 3 is derived from the same LC-MS data set that generated the base peak chromatogram shown in Figure 2. By comparing the two figures it is clear that the presentation of MS data in the form of an intensity map gives a much better overview of the LC-MS part of the analysis. For example, the signal intensity map clearly shows isotopic distribution of detected peptides, exact retention times, overlapped peptides, and the presence of MS/MS data not directly visible in a base peak ion chromatogram. This type of visualization thus results in a quick overview of the data and can be used to optimize chromatographic and MS conditions, observe unique patterns within the data, or recognize contaminants. At least 1000 ions were detected in each LC-MS analysis of the two allergen samples, which after applying the PepDetect module of DeCyder MS resulted in a final list consisting of ~ 500 deconvoluted and identified peptides.
The next step in the DeCyder MS software analysis was the matching of peptides, from different intensity maps derived from the fractions of Ole_e1_"lite" and Ole_e1_"mer," using the PepMatch module. In this module features from the two allergen samples were compared resulting in relative quantitation of significantly varying peptides. By assuming th at the peptide content reflects the protein level, a list of significantly and differentially expressed proteins was obtained after which identification of acquired MS/MS data was carried out.
In Figure 4, a screen capture of the PepMatch module of DeCyder MS is shown, covering the peptide LGMYPPNM representing the main olive allergen of these samples. The average ratio of this peptide between Ole_e1_"mer" and Ole_e1_"lite" was 3.8 times, as shown in the peptide table above the 2-D and 3-D signal intensity maps. This is also in agreement with the gel filtration data of the two allergen samples, which showed an average difference of 3.5 times after integration of the UV traces for the collected fractions.
LGMYPPNM was the only peptide that was identified in all Ole e 1 proteins identified in this study, resulting in a total quantitation of the allergens present in the two batches. This finding also reflects the fact that the most intense signals in DeCyder MS for all detected and matched Ole e 1 peptides were obtained for this peptide. From Figure 4 it can also be seen that signal intensities from singly as well as doubly charged forms of LGMYPPNM were used for calculation of the relative abundances of this specific peptide in the two batches of pollen. Also shown in the list above the 2-D and 3-D signal intensity maps are p values, identification details, molecular masses of the compared peptides, number of matched profiles, signal intensities of each matched peptide, etc.
Among other Ole e 1 peptides detected, some were found to be unique for different isoforms of the protein. In Figure 5, another view from PepMatch is shown, demonstrating a four-fold difference in abundance between Ole_e1_"mer" and Ole_e1_"lite" for the peptide DCDEIPIEGWAKPSLK. This peptide was found to be unique for the Ole e 1 protein, with a molecular mass of 16616 Da and accession number CAA73036.1. In total, five isoforms of Ole e 1 could be identified and quantitated by using DeCyder MS.
The graph extracted from the PepMatch module in Figure 6 shows the sequences and signal intensities of the significantly varying peptides that were used for quantitation of different isoforms of Ole e 1. As earlier mentioned, LGMYPPNM is present in all forms of Ole e 1, resulting in the highest signal intensity or most blackened grayscale in the intensity maps created by DeCyder MS. The other peptide pairs in Figure 6 thus represent unique peptides for five different isoforms of the olive allergen.
In Table 1, a list of significantly varying peptides from the two allergen samples is shown, together with Student t-test p values, protein names, pI values, molecular masses, and accession numbers. As can be seen, several of the isoforms were very close in molecular mass and pI value. The smallest deviation in mass was 64 Da between Ole 1 c (16485 Da) and Ole e 1.0103 (16421 Da), while the smallest deviation in pI value was 0.02 units between Ole e 1 (CAA73036.1) and Ole e 1.0103 (CAA73037.1).
DeCyder MS Differential Analysis Software is a tool for the visualization and label-free relative quantitation of complex protein digests. The precise gradient formation and solvent delivery features of Ettan MDLC provided highly reproducible retention times for eluted peptides, resulting in improved matching performance by DeCyder MS.
Quantitation of allergen samples using DeCyder MS revealed a 3.8-fold difference in Ole e 1 content, which is in agreement with other physicochemical quantitation methods. In addition, five differentially regulated isoforms of Ole e 1 could be unambiguously identified and quantitated.
The results indicate that for quantitation of proteins containing microheterogeneities, the LC-MS based proteomics workflow combined with DeCyder MS evaluation could be preferable to the gel- based workflow. This user-friendly and unlabeled approach to relative quantitation of MS data will have wide implications on the LC-MS based proteomics workflow for differential analysis.
Peter Brostedt and Ingrid Holmquist, Pharmacia Diagnostics AB, Uppsala, Sweden, are acknowledged for generously supplying extracted proteins from olive pollen.
1. Huecas, S. et al. J. Biol. Chem. 276, 2795927966 (2001).
2. Application note: Automated identification and quantitation of proteins and peptides using LC-MS/MS, GE Healthcare, 11-0027-39, Edition AA (2005).
3. Application note: Reproducible and robust peptide separations using Ettan MDLC, a multidimensional liquid chromatography system, GE Healthcare, 11-0031-13, Edition AA (2005).
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