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520 Biochemical Society Transactions (2004) Volume 32, part 3 The use of isotope-coded affinity tags (ICAT) to study organelle proteomes in Arabidopsis thaliana T.P.J. Dunkley*, P. Dupree*1 , R.B. Watson† and K.S. Lilley* *Department of Biochemistry, Cambridge Centre for Proteomics, University of Cambridge, Building O, Downing Site, Cambridge CB2 1QW, U.K., and †Applied Biosystems, Lingley House, 120 Birchwood Boulevard, Birchwood Point, Warrington, Cheshire WA3 7QH, U.K. Abstract Organelle proteomics is the analysis of the protein contents of a subcellular compartment. Proteins identified in subcellular proteomic studies can only be assigned to an organelle if there are no contaminants present in the sample preparation. As a result, the majority of plant organelle proteomic studies have focused on the chloroplast and mitochondria, which can be isolated relatively easily. However, the isolation of components of the endomembrane system is far more difficult due to their similar sizes and densities. For this reason, quantitative proteomics methods are being developed to enable the assignment of proteins to a specific component of the endomembrane system without the need to obtain pure organelles. Introduction Organelle proteomics is the study of the protein complement of a specific subcellular compartment and involves subcellular fractionation followed by protein identification, typically by MS. Within the eukaryotic cell, proteins are spatially organized according to their function, so the assignment of an uncharacterized protein to an organelle provides the biologist with an insight into its possible role in the cell [1]. In addition, the enrichment of an organelle before proteomic analysis enables the identification of low-abundance proteins that would not be detected in unfractionated samples. The biggest challenge in organelle proteomics is the production of pure organelle preparations, which are important since the presence of contaminating proteins reduces the confidence with which novel proteins can be assigned to an organelle of interest. For the plant endomembrane system, it is technically impossible to obtain absolutely pure preparations of its component organelles. Therefore to assign specific locations to integral membrane proteins within the plant endomembrane system, novel methods that are not dependent on the production of pure organelle preparations are required. Such a method will be introduced later in this paper. Organelle proteomics In plants, the majority of subcellular proteomic studies have focused on the mitochondria and chloroplast. This is due to the relative ease with which highly enriched preparations of these organelles can be obtained. Peltier Key words: Golgi, isotope-coded affinity tag (ICAT), membrane protein, organelle, quantitative proteomics. Abbreviations used: 2D, two-dimensional; ER, endoplasmic reticulum; ICAT, isotope-coded affinity tagging; LeuD3, leucine containing three deuteriums; SILAC, stable isotope-labelling with amino acids in cell culture. 1 To whom correspondence should be addressed (email [email protected]). C 2004 Biochemical Society et al. [2], for example, prepared thylakoids from Arabidopsis leaves and subsequently separated the soluble lumenal proteins by two-dimensional PAGE. A total of 81 different proteins were identified by a combination of peptide mass fingerprinting and peptide sequencing by both MS/MS (tandem MS) and Edman degradation (see [3] for a review of MS in proteomics). Of these, 30 lumenal proteins were identified, as well as 32 proteins that are associated with the thylakoid membrane on the stromal side. Also, 12 proteins that were of unknown location were identified, as well as six non-chloroplast contaminants from the ER (endoplasmic reticulum), mitochondria and cytosol. These results demonstrate that highly enriched chloroplasts can be obtained. However, as the isolation of a pure thylakoid lumen fraction was not achieved, the 12 identified proteins with no known location could not be localized to the thylakoid lumen with confidence. Two-dimensional PAGE has also been used to study the plant mitochondrial proteome. Kruft et al. [4] and Millar et al. [5] both purified mitochondria from Arabidopsis darkgrown suspension cell culture. Both projects reported no, or very little, contamination from other organelles. However, mitochondria prepared from Arabidopsis leaves and stems did contain chloroplast proteins, indicating that the success of a subcellular proteomics experiment is dependent on the choice of starting material [4]. These studies resulted in the identification of soluble or membrane-associated proteins, but very few integral membrane proteins. Integral membrane proteins are underrepresented on two-dimensional gels as strong ionic detergents, which are required for membrane protein solubilization, are incompatible with protein separation by isoelectric focusing [6]. Therefore the identification of integral membrane proteins requires the use of non-two-dimensional gel-based protein separation. Ferro et al. [7] prepared chloroplast Proteomics of Plant Proteins envelopes from spinach leaves and utilized the solubility of hydrophobic proteins in chloroform/methanol mixtures to enrich for integral membrane proteins. These membrane proteins were then solubilized with SDS and separated using conventional one-dimensional SDS/PAGE. Bands were cut from the gel, their protein contents digested with trypsin and the resulting peptides were sequenced by MS/MS. Using this approach, 54 proteins were identified including 21 with at least four predicted transmembrane spans. Ferro et al. [7] also studied the Arabidopsis chloroplast envelope proteome using a combination of chloroform/methanol, high pH and high salt extraction techniques to enrich for membrane proteins. The extracted proteins were digested with trypsin and the resulting peptides were separated and analysed by reverse-phase capillary LC-MS/MS. Using this approach, 112 proteins were identified, of which 79% were envelope proteins, 15% were stromal or thylakoid and 6% were contaminants from other organelles [8]. Millar and Heazlewood [9] also identified hydrophobic integral membrane proteins from mitochondria by using a high pH wash to remove membraneassociated and soluble proteins, followed by SDS/PAGE and MS/MS. To date, no comprehensive study of any component of the plant endomembrane system has been reported, reflecting the difficulty in purifying the component organelles which have similar sizes and densities. However, proteomic studies of a component of the animal endomembrane system, the Golgi apparatus, have been conducted. Taylor et al. [10,11] prepared intact Golgi stacks from rat liver and separated their protein contents by two-dimensional PAGE. Spots were cut from the gel and after tryptic digestion their protein contents were analysed by MS/MS. Of the 55 proteins identified with known subcellular locations, approx. 50% were Golgi localized. As a result of this, the proteins of unknown location could not be assigned to the Golgi apparatus based on this study. In addition, the 2D (twodimensional) PAGE approach used in this study was not suitable for the identification of the highly hydrophobic integral membrane components of the Golgi. Bell et al. [13] addressed this problem by enriching Golgi membrane proteins, prepared from rat liver, by Triton X-114 phase partitioning. These membrane proteins were then solubilized with SDS and separated by SDS/PAGE [12,13]. Using MS and Edman sequencing, 17 Golgi integral membrane proteins were identified as well as 24 contaminating proteins, most of them from the ER and mitochondria. The large number of non-Golgi proteins identified in the two Golgi studies discussed highlights the difficulty in isolating a specific component from the endomembrane system. The components of the endomembrane system have similar sizes and densities and are therefore difficult to separate by ultracentrifugation. In addition, the endomembrane system is dynamic, with membranes and proteins continuously trafficking between different organelles. Contaminating proteins are even more of a problem for the identification of organelle proteins that are present at very low copy numbers. The identification of low-abundance proteins requires either development of higher sensitivity MS techniques or the use of larger amounts of starting material. Either approach will lead to the identification of low levels of contaminants as well as the proteins of interest, unless the organelle preparations are completely pure [14]. Quantitative proteomics for assigning proteins to organelles Quantitative proteomics can be used to circumvent the problem of contamination in assigning proteins to subcellular compartments. Several techniques have been developed to compare proteomes quantitatively; these include difference gel electrophoresis (DIGE) [15,16], ICAT (isotope-coded affinity tagging) [17] and SILAC (stable isotope-labelling with amino acids in cell culture) [18]. Of these, difference gel electrophoresis is a 2D-PAGE-based technique and thus is not suitable for the quantitative comparison of membrane proteins. ICAT and SILAC, on the other hand, are not based on 2D-PAGE and rely on MS for protein quantification. Therefore ICAT and SILAC can be used to compare membrane proteomes. Foster et al. [19] used SILAC to identify lipid-raft-localized proteins in HeLa cells, despite the presence of contaminating proteins in the lipid raft preparation. SILAC is a quantitative proteomics technique that involves feeding one set of cultured cells with the essential amino acid leucine containing three deuteriums (LeuD3) in place of three hydrogens and a second set of cells with nondeuterated leucine (Leu). After several generations of growth on LeuD3-containing media, cells contain LeuD3 in place of Leu in all of their proteins. As a consequence of this, leucine-containing peptides from the LeuD3 grown cells are 3 Da heavier than the equivalent peptide from the Leu cells. MS can then be used to distinguish peptides derived from the LeuD3 and Leu cells and their relative abundance can be calculated, enabling the quantitative comparison of protein levels in the two samples. Foster et al. [19] treated the Leu grown cells with a drug to disrupt lipid rafts. The drug-treated Leu cells were then pooled with untreated LeuD3 cells and lipid rafts were prepared. The proteins in the lipid raft fraction were then solubilized, digested and analysed by LC-MS/MS. Peptides with higher abundance in the untreated LeuD3 cells were assigned as lipid raft proteins because the drug treatment disrupts rafts and therefore should result in lower levels of lipid raft proteins and consequently peptides in the lipid raft enriched fraction. Out of a total of 392 quantifiable proteins identified in the lipid raft fraction, 241 proteins were found to be genuinely lipid-raft localized. The use of a quantitative proteomics technique thus enabled the localization of proteins to a subcellular domain despite the presence of contaminating proteins. We have applied a quantitative proteomics approach to study the plant endomembrane system. First, membrane fractions enriched in ER and Golgi were prepared from Arabidopsis callus. Soluble and membrane associated proteins were then removed from the membranes using a high pH carbonate wash [20]. To assess the degree of cross-contamination C 2004 Biochemical Society 521 522 Biochemical Society Transactions (2004) Volume 32, part 3 Figure 1 Standard cleavable ICAT workflow used for the quantitative comparison of membrane protein levels in an ER-rich Figure 2 Membrane proteins from an ER-rich and a Golgi-rich fraction were labelled with light and heavy ICAT respectively and and a Golgi-rich fraction prepared from Arabidopsis callus subjected to the standard cleavable ICAT procedure as shown in Figure 1 MS spectra for peptides derived from two ER proteins and a Golgi protein are shown. The relative abundance of each protein in the two fractions can be determined by comparing the intensity of the ICAT light (ER)- and ICAT heavy (Golgi)-labelled peptides. between the two organelles, proteomic analysis of the Golgirich fraction was performed. Golgi membrane proteins were identified, as well as numerous uncharacterized proteins. However, the presence of contaminating ER proteins in the Golgi-rich fraction prevented the assignment of these uncharacterized proteins to the Golgi membrane. To address this problem, a quantitative comparison of an ERrich fraction and a Golgi-rich fraction was performed to discriminate between proteins resident in the two organelles, since Golgi proteins should be more abundant in the Golgirich fraction and vice versa. The quantitative technique applied centred around the use of the cleavable ICAT reagents from Applied Biosystems (Warrington, Cheshire, U.K.). Cleavable ICAT reagents consist of an iodoacetamide group, which reacts specifically with cysteine thiol functional groups, connected to biotin by a linker that contains either nine 13 C or nine 12 C. The mass difference between these two tags is therefore 9 Da. The biotin group enables ICATlabelled peptides to be avidin affinity-purified to remove unlabelled peptides and hence simplify the peptide mixture. The cleavable ICAT reagent also contains an acid-cleavable group, so that the biotin can then be removed by treating the labelled peptides with trifluoroacetic acid, resulting in higher quality MS/MS data. In this study, the ER-rich fraction was labelled with the light version of the cleavable ICAT reagent and the Golgi-rich fraction with the heavy 13 C version. The two samples were then pooled and digested. The resulting peptides were fractionated by cation-exchange chromatography and the ICAT-labelled cysteine-containing peptides were isolated by running each fraction through an avidin affinity column. After removal of the biotin group C 2004 Biochemical Society with trifluoroacetic acid, the peptides were analysed by LCMS/MS (see Figure 1). Peptides derived from the ER-rich and Golgi-rich fractions were separated by 9 Da due to the mass difference between the heavy and light tags. The relative abundance of a protein in the two fractions could thus be determined by comparing the intensities of the ICAT lightand ICAT heavy-labelled versions of each peptide. Spectra obtained for peptides from two known ER proteins and a Golgi protein are shown in Figure 2. The peptides shown are doubly charged, so the light- and heavy-labelled versions Proteomics of Plant Proteins of each peptide are separated by m/z 4.5. Each peptide is represented by a series of peaks due to the natural abundance of 13 C. As shown in Figure 2, the ER proteins calnexin and cytochrome b5 and the Golgi protein gtl-6 are present in both the ER-rich and Golgi-rich fractions, due to the cross-contamination already discussed. However, the use of ICAT labelling enables us to compare the amount of each protein in the two fractions. Calnexin and cytochrome b5 are clearly more abundant in the ER-rich fraction and gtl-6 is more abundant in the Golgi-rich fraction, thus enabling us to distinguish between Golgi-localized and ER-localized proteins without the need to obtain pure Golgi and ER preparations. A comprehensive analysis of the membrane proteins present in the ER-rich and Golgi-rich membrane fractions, and the quantification of their relative abundances, is currently in progress. This approach should enable the localization of previously uncharacterized endomembrane proteins by comparing their relative abundances with those of proteins of known locations. Conclusions Subcellular proteomic analysis has typically involved the cataloguing of proteins present in a single organelle-enriched fraction. This approach has been reasonably successful for easily purified organelles, such as the mitochondria and chloroplast. However, the plant endomembrane system has so far proved refractory to subcellular proteomic analysis, due to the difficulty in isolating its component organelles. The use of quantitative proteomics to analyse the relative levels of proteins in different organelle-enriched fractions provides a solution to this problem and enables us to distinguish between proteins from different subcellular compartments without the need to obtain pure organelles. We thank the Biotechnology and Biological Sciences Research Council and Applied Biosystems for financial support and also Applied Biosystems for the gift of the ICAT reagents. We also thank the members of Cambridge Centre for Proteomics for their technical assistance and Zhinong Zhang for maintaining the Arabidopsis callus. References 1 Dreger, M. (2003) Mass Spectrom. Rev. 22, 27–56 2 Peltier, J.B., Emanuelsson, O., Kalume, D.E., Ytterberg, J., Friso, G., Rudella, A., Liberles, D.A., Soderberg, L., Roepstorff, P., von Heijne, G. et al. 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Cell Biol. 93, 97–102 Received 25 November 2003 C 2004 Biochemical Society 523