Abstract
Detailed knowledge of the composition of protein complexes is crucial for the understanding of their structure and function; however, appropriate techniques for compositional analyses of complexes largely rely on elaborate tagging, immunoprecipitation, cross-linking and purification strategies. The proteasome is a prototypical protein complex and therefore an excellent model to assess new methods for protein complex characterisation. Here we evaluated the applicability of Blue Native (BN) PAGE in combination with label-free protein quantification and protein correlation profiling (PCP) for the investigation of proteasome complexes directly from biological samples. Using the purified human 20S proteasome we showed that the approach can accurately detect members of a complex by clustering their gel migration profiles. We applied the approach to address proteasome composition in the schizont stage of the malaria parasite Plasmodium falciparum. The analysis, performed in the background of the whole protein extract, revealed that all subunits comigrated and formed a tight cluster with a single maximum, demonstrating presence of a single form of the 20S proteasome. This study shows that BN PAGE in combination with label-free quantification and PCP is applicable to the analysis of multiprotein complexes directly from complex protein mixtures.
This is a preview of subscription content, log in via an institution to check access.
Similar content being viewed by others
Abbreviations
- BN PAGE:
-
Blue native polyacrylamide gel electrophoresis
- MS:
-
Mass spectrometry
- PCP:
-
Protein correlation profiling
- PAI:
-
Protein abundance index
- AUC:
-
Area under the curve
- iBAQ:
-
Intensity-based absolute quantification
References
Andersen JS, Wilkinson CJ, Mayor T, Mortensen P, Nigg EA, Mann M (2003) Proteomic characterization of the human centrosome by protein correlation profiling. Nature 426(6966):570–574. doi:10.1038/nature02166
Aurrecoechea C, Brestelli J, Brunk BP, Dommer J, Fischer S, Gajria B, Gao X, Gingle A, Grant G, Harb OS, Heiges M, Innamorato F, Iodice J, Kissinger JC, Kraemer E, Li W, Miller JA, Nayak V, Pennington C, Pinney DF, Roos DS, Ross C, Stoeckert CJ Jr., Treatman C, Wang H (2009) Plasmodb: a functional genomic database for malaria parasites. Nucleic Acids Res 37 (Database issue):D539–543. doi:10.1093/nar/gkn814
Binh VQ, Luty AJ, Kremsner PG (1997) Differential effects of human serum and cells on the growth of Plasmodium falciparum adapted to serum-free in vitro culture conditions. Am J Trop Med Hyg 57(5):594–600
Bochtler M, Ditzel L, Groll M, Hartmann C, Huber R (1999) The proteasome. Annu Rev Biophys Biomol Struct 28:295–317. doi:10.1146/annurev.biophys.28.1.295
Borchert N, Dieterich C, Krug K, Schütz W, Jung S, Nordheim A, Sommer RJ, Macek B (2010) Proteogenomics of Pristionchus pacificus reveals distinct proteome structure of nematode models. Genome Res 20(6):837–846. doi:10.1101/gr.103119.109
Camacho-Carvajal MM, Wollscheid B, Aebersold R, Steimle V, Schamel WW (2004) Two-dimensional blue native/SDS gel electrophoresis of multi-protein complexes from whole cellular lysates: a proteomics approach. Mol Cell Proteomics 3(2):176–182. doi:10.1074/mcp.T300010-MCP200
Cox J, Mann M (2008) Maxquant enables high peptide identification rates, individualized p.p.b.-range mass accuracies and proteome-wide protein quantification. Nat Biotechnol 26(12):1367–1372. doi:10.1038/nbt.1511
Cox J, Matic I, Hilger M, Nagaraj N, Selbach M, Olsen JV, Mann M (2009) A practical guide to the maxquant computational platform for SILAC-based quantitative proteomics. Nat Protoc 4(5):698–705. doi:10.1038/nprot.2009.36
Elias JE, Gygi SP (2007) Target‐decoy search strategy for increased confidence in large-scale protein identifications by mass spectrometry. Nat Methods 4(3):207–214.doi:10.1038/nmeth1019
Eubel H, Jänsch L, Braun HP (2003) New insights into the respiratory chain of plant mitochondria. Supercomplexes and a unique composition of complex ii. Plant Physiol 133(1):274–286
Fandino AS, Rais I, Vollmer M, Elgass H, Schägger H, Karas M (2005) LC-nanospray-MS/MS analysis of hydrophobic proteins from membrane protein complexes isolated by blue-native electrophoresis. J Mass Spectrom 40(9):1223–1231. doi:10.1002/jms.903
Foster LJ, de Hoog CL, Zhang Y, Xie X, Mootha VK, Mann M (2006) A mammalian organelle map by protein correlation profiling. Cell 125(1):187–199. doi:10.1016/j.cell.2006.03.022
Fry M, Beesley JE (1991) Mitochondria of mammalian Plasmodium spp. Parasitology 102(Pt 1):17–26
Gardner MJ, Hall N, Fung E, White O, Berriman M, Hyman RW, Carlton JM, Pain A, Nelson KE, Bowman S, Paulsen IT, James K, Eisen JA, Rutherford K, Salzberg SL, Craig A, Kyes S, Chan MS, Nene V, Shallom SJ, Suh B, Peterson J, Angiuoli S, Pertea M, Allen J, Selengut J, Haft D, Mather MW, Vaidya AB, Martin DM, Fairlamb AH, Fraunholz MJ, Roos DS, Ralph SA, McFadden GI, Cummings LM, Subramanian GM, Mungall C, Venter JC, Carucci DJ, Hoffman SL, Newbold C, Davis RW, Fraser CM, Barrell B (2002) Genome sequence of the human malaria parasite Plasmodium falciparum. Nature 419(6906):498–511. doi:10.1038/nature01097
Gavin AC, Bosche M, Krause R, Grandi P, Marzioch M, Bauer A, Schultz J, Rick JM, Michon AM, Cruciat CM, Remor M, Hofert C, Schelder M, Brajenovic M, Ruffner H, Merino A, Klein K, Hudak M, Dickson D, Rudi T, Gnau V, Bauch A, Bastuck S, Huhse B, Leutwein C, Heurtier MA, Copley RR, Edelmann A, Querfurth E, Rybin V, Drewes G, Raida M, Bouwmeester T, Bork P, Seraphin B, Kuster B, Neubauer G, Superti-Furga G (2002) Functional organization of the yeast proteome by systematic analysis of protein complexes. Nature 415(6868):141–147. doi:10.1038/415141a
Gille C, Goede A, Schloetelburg C, Preissner R, Kloetzel PM, Gobel UB, Frommel C (2003) A comprehensive view on proteasomal sequences: Implications for the evolution of the proteasome. J Mol Biol 326 (5):1437–1448. pii:S0022283602014705
Gingras AC, Gstaiger M, Raught B, Aebersold R (2007) Analysis of protein complexes using mass spectrometry. Nat Rev Mol Cell Biol 8(8):645–654. doi:10.1038/nrm2208
Glickman MH, Ciechanover A (2002) The ubiquitin-proteasome proteolytic pathway: destruction for the sake of construction. Physiol Rev 82(2):373–428. doi:10.1152/physrev.00027.2001
Groll M, Ditzel L, Lowe J, Stock D, Bochtler M, Bartunik HD, Huber R (1997) Structure of 20S proteasome from yeast at 2.4 A resolution. Nature 386(6624):463–471. doi:10.1038/386463a0
Hershko A, Ciechanover A (1992) The ubiquitin system for protein degradation. Annu Rev Biochem 61:761–807. doi:10.1146/annurev.bi.61.070192.003553
Ho Y, Gruhler A, Heilbut A, Bader GD, Moore L, Adams SL, Millar A, Taylor P, Bennett K, Boutilier K, Yang L, Wolting C, Donaldson I, Schandorff S, Shewnarane J, Vo M, Taggart J, Goudreault M, Muskat B, Alfarano C, Dewar D, Lin Z, Michalickova K, Willems AR, Sassi H, Nielsen PA, Rasmussen KJ, Andersen JR, Johansen LE, Hansen LH, Jespersen H, Podtelejnikov A, Nielsen E, Crawford J, Poulsen V, Sorensen BD, Matthiesen J, Hendrickson RC, Gleeson F, Pawson T, Moran MF, Durocher D, Mann M, Hogue CW, Figeys D, Tyers M (2002) Systematic identification of protein complexes in Saccharomyces cerevisiae by mass spectrometry. Nature 415(6868):180–183. doi:10.1038/415180a
Hochuli E, Bannwarth W, Dobeli H, Gentz R, Stuber D (1988) Genetic approach to facilitate purification of recombinant proteins with a novel metal chelate adsorbent. Nat Biotech 6(11):1321–1325
Kreidenweiss A, Kremsner PG, Mordmüller B (2008) Comprehensive study of proteasome inhibitors against Plasmodium falciparum laboratory strains and field isolates from gabon. Malar J 7:187. doi:10.1186/1475-2875-7-187
Leitner A, Walzthoeni T, Kahraman A, Herzog F, Rinner O, Beck M, Aebersold R (2010) Probing native protein structures by chemical cross-linking, mass spectrometry, and bioinformatics. Mol Cell Proteomics 9(8):1634–1649. doi:10.1074/mcp.R000001-MCP201
Luber CA, Cox J, Lauterbach H, Fancke B, Selbach M, Tschopp J, Akira S, Wiegand M, Hochrein H, O’Keeffe M, Mann M (2010) Quantitative proteomics reveals subset-specific viral recognition in dendritic cells. Immunity 32(2):279–289. doi:10.1016/j.immuni.2010.01.013
Maiolica A, Cittaro D, Borsotti D, Sennels L, Ciferri C, Tarricone C, Musacchio A, Rappsilber J (2007) Structural analysis of multiprotein complexes by cross-linking, mass spectrometry, and database searching. Mol Cell Proteomics 6(12):2200–2211. doi:10.1074/mcp.M700274-MCP200
Monti S, Tamayo P, Mesirov J, Golub T (2003) Consensus clustering: a resampling-based method for class discovery and visualization of gene expression microarray data. Mach Learn 52(1–2):91–118
Mordmüller B, Kremsner PG (2006) Malarial parasites versus antimalarials: never-ending rumble in the jungle. Curr Mol Med 6(2):247–251
Mordmüller B, Fendel R, Kreidenweiss A, Gille C, Hurwitz R, Metzger WG, Kun JF, Lamkemeyer T, Nordheim A, Kremsner PG (2006) Plasmodia express two threonine-peptidase complexes during asexual development. Mol Biochem Parasitol 148(1):79–85. doi:10.1016/j.molbiopara.2006.03.001
Olsen JV, de Godoy LM, Li G, Macek B, Mortensen P, Pesch R, Makarov A, Lange O, Horning S, Mann M (2005) Parts per million mass accuracy on an orbitrap mass spectrometer via lock mass injection into a c-trap. Mol Cell Proteomics 4(12):2010–2021. doi:10.1074/mcp.T500030-MCP200
Perkins DN, Pappin DJ, Creasy DM, Cottrell JS (1999) Probability-based protein identification by searching sequence databases using mass spectrometry data. Electrophoresis 20(18):3551–3567. doi:10.1002/(SICI)1522-2683(19991201)20:18<3551:AID-ELPS3551>3.0.CO;2-2
Petrotchenko EV, Borchers CH (2010) Crosslinking combined with mass spectrometry for structural proteomics. Mass Spectrom Rev 29(6):862–876. doi:10.1002/mas.20293
R Development Core Team (2011) R: A language and environment for statistical computing. R Foundation for Statistical Computing, Vienna
Rappsilber J, Ryder U, Lamond AI, Mann M (2002) Large-scale proteomic analysis of the human spliceosome. Genome Res 12(8):1231–1245. doi:10.1101/gr.473902
Rappsilber J, Mann M, Ishihama Y (2007) Protocol for micro-purification, enrichment, pre-fractionation and storage of peptides for proteomics using stagetips. Nat Protoc 2(8):1896–1906. doi:10.1038/nprot.2007.261
Sanders PR, Cantin GT, Greenbaum DC, Gilson PR, Nebl T, Moritz RL, Yates JR 3rd, Hodder AN, Crabb BS (2007) Identification of protein complexes in detergent-resistant membranes of Plasmodium falciparum Schizonts. Mol Biochem Parasitol 154(2):148–157. doi:10.1016/j.molbiopara.2007.04.013
Schägger H, von Jagow G (1991) Blue native electrophoresis for isolation of membrane protein complexes in enzymatically active form. Anal Biochem 199(2):223–231
Schwanhäusser B, Busse D, Li N, Dittmar G, Schuchhardt J, Wolf J, Chen W, Selbach M (2011) Global quantification of mammalian gene expression control. Nature 473(7347):337–342. doi:10.1038/nature10098
Tschan S, Mordmüller B, Kun JF (2011) Threonine peptidases as drug targets against malaria. Expert Opin Ther Targets. doi:10.1517/14728222.2011.555399
Wessels HJ, Vogel RO, van den Heuvel L, Smeitink JA, Rodenburg RJ, Nijtmans LG, Farhoud MH (2009) LC-MS/MS as an alternative for SDS-PAGE in blue native analysis of protein complexes. Proteomics 9(17):4221–4228. doi:10.1002/pmic.200900157
Wittig I, Schägger H (2008) Features and applications of blue-native and clear-native electrophoresis. Proteomics 8(19):3974–3990. doi:10.1002/pmic.200800017
Wittig I, Braun HP, Schägger H (2006) Blue native page. Nat Protoc 1(1):418–428. doi:10.1038/nprot.2006.62
Acknowledgments
The authors wish to thank Dr. Tobias Lamkemeyer and Dr. Rolf Fendel for helpful discussions in the initial stages of the project. This study was supported by the Landesstiftung BW (Juniorprofessoren-Programm), DFG and PRIME-XS (to Boris Macek).
Conflict of interest
The authors declare no conflict of interest.
Author information
Authors and Affiliations
Corresponding author
Electronic supplementary material
Below is the link to the electronic supplementary material.
726_2012_1296_MOESM1_ESM.pdf
Online Resource 1 The intensity profiles of the human proteasomal subunits. For the purified 20S proteasome a raw intensities and b adjusted intensities resulted in different profiles. For the 20S proteasome plus five spiked proteins the c raw intensities and d adjusted intensities resulted in profiles that are more similar. The profiles showed that normalisation worked better in a sample with higher complexity (PDF 534 kb)
726_2012_1296_MOESM2_ESM.xls
Online Resource 2 Overview of the identified protein groups in the experiments. The excel file summarises all identified protein groups of the two experiments with human 20S proteasome and the three experiments using the plasmodial proteasome (XLS 1561 kb)
726_2012_1296_MOESM3_ESM.xls
Online Resource 3 Overview of the identified proteins in the analysed slices of the BN gel. The excel file summarises the identification of the proteins according to the analysed BN gel slice. The number of identified peptides per protein and the corresponding intensity values are also included. Thereby, the number of identified peptides per proteins and slice represents redundant peptides (XLS 633 kb)
726_2012_1296_MOESM4_ESM.pdf
Online resource 4 Comparison of the adjusted intensity profiles of the plasmodial 20S proteasome and the contaminating human proteasome in the parasite lysate. In the plasmodial lysate, 13 subunits of the human proteasome were identified with a lower intensity (about 2 orders of magnitude) as the plasmodial ones. Their intensity profiles showed maxima in the same slices as the plasmodial profiles indicating a comigration of the two proteasomal complexes (PDF 251 kb)
726_2012_1296_MOESM5_ESM.pdf
Online Resource 5 Clustering of the adjusted intensities of all 314 quantified P. falciparum proteins in the experiment 2. All 14 proteasomal subunits are contained in cluster 5. The maximum cluster consensus value which represents the robustness of the cluster, was assigned to the same cluster (PDF 267 kb)
726_2012_1296_MOESM6_ESM.xls
Online Resource 6 Overview of the proteins quantified in the experiment 2 and used for the clustering analysis. The proteins in the excel file were classified after their membership to one of the six clusters (XLS 247 kb)
726_2012_1296_MOESM7_ESM.pdf
Online Resource 7 Three additional plasmodial protein complexes were detected in the dataset. The intensity profiles of these complexes showed a good correlation. a shows two members of the low molecular weight rhoptry (RAP) complex, ‘rhoptry-associated protein 1, RAP1′ (PlasmoDB gene ID PF14_0102) and ‘rhoptry-associated protein 2, RAP2′ (PFE0080c), b represents the high molecular weight rhoptry (RhopH) complex with the members ‘Cytoadherence linked asexual protein, 3.2′ (PFC0120w), ‘RhopH3′ (PFI0265c) and ‘High molecular weight rhoptry protein-2′ (PFI1445w) and (c) shows the mitochondrial signal peptide processing complex with the members ‘mitochondrial processing peptidase alpha subunit, putative’ (PFE1155c) and ‘organelle processing peptidase, putative’ (PFI1625c) (PDF 383 kb)
726_2012_1296_MOESM8_ESM.pdf
Online Resource 8 The intensities of the ten quantified subunits of the regulatory particle resulted in different profiles. Most of the profiles had more than one maximum, except (f), and the maxima were located in different slices. Some maxima are located in slice 4, slice 7/8 and/or slice 11/12, like (a), (c) and (h), suggesting that the antibody cross-reactivity is likely related to the regulatory particles. There was no correlation between these proteins visible at all (PDF 480 kb)
726_2012_1296_MOESM9_ESM.pdf
Online Resource 9 Three different label-free quantification methods were used to test the determination of stoichiometry of the plasmodial 20S proteasome. PAI, AUC and two versions of the iBAQ label-free quantification method were applied to Exp. 2 of the analysis of the P. falciparum proteasome to estimate the protein abundance of the subunits. The standard deviation is given in brakes in the legen (PDF 420 kb)
Rights and permissions
About this article
Cite this article
Sessler, N., Krug, K., Nordheim, A. et al. Analysis of the Plasmodium falciparum proteasome using Blue Native PAGE and label-free quantitative mass spectrometry. Amino Acids 43, 1119–1129 (2012). https://doi.org/10.1007/s00726-012-1296-9
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s00726-012-1296-9