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Cryo-slicing Blue Native-Mass Spectrometry (csBN-MS), a Novel Technology for High Resolution Complexome Profiling

2015; Elsevier BV; Volume: 15; Issue: 2 Linguagem: Inglês

10.1074/mcp.m115.054080

1535-9484

Catrin Swantje Müller, Wolfgang Bildl, Alexander Haupt, Lars Ellenrieder, Thomas Becker, Carola Hunte, Bernd Fakler, Uwe Schulte,

Mass Spectrometry Techniques and Applications

Blue native (BN) gel electrophoresis is a powerful method for protein separation. Combined with liquid chromatography-tandem mass spectrometry (LC-MS/MS), it enables large scale identification of protein complexes and their subunits. Current BN-MS approaches, however, are limited in size resolution, comprehensiveness, and quantification. Here, we present a new methodology combining defined sub-millimeter slicing of BN gels by a cryo-microtome with high performance LC-MS/MS and label-free quantification of protein amounts. Application of this cryo-slicing BN-MS approach to mitochondria from rat brain demonstrated a high degree of comprehensiveness, accuracy, and size resolution. The technique provided abundance-mass profiles for 774 mitochondrial proteins, including all canonical subunits of the oxidative respiratory chain assembled into 13 distinct (super-)complexes. Moreover, the data revealed COX7R as a constitutive subunit of distinct super-complexes and identified novel assemblies of voltage-dependent anion channels/porins and TOM proteins. Together, cryo-slicing BN-MS enables quantitative profiling of complexomes with resolution close to the limits of native gel electrophoresis. Blue native (BN) gel electrophoresis is a powerful method for protein separation. Combined with liquid chromatography-tandem mass spectrometry (LC-MS/MS), it enables large scale identification of protein complexes and their subunits. Current BN-MS approaches, however, are limited in size resolution, comprehensiveness, and quantification. Here, we present a new methodology combining defined sub-millimeter slicing of BN gels by a cryo-microtome with high performance LC-MS/MS and label-free quantification of protein amounts. Application of this cryo-slicing BN-MS approach to mitochondria from rat brain demonstrated a high degree of comprehensiveness, accuracy, and size resolution. The technique provided abundance-mass profiles for 774 mitochondrial proteins, including all canonical subunits of the oxidative respiratory chain assembled into 13 distinct (super-)complexes. Moreover, the data revealed COX7R as a constitutive subunit of distinct super-complexes and identified novel assemblies of voltage-dependent anion channels/porins and TOM proteins. Together, cryo-slicing BN-MS enables quantitative profiling of complexomes with resolution close to the limits of native gel electrophoresis. Blue native (BN) 1The abbreviations used are:BNblue native2Dtwo-dimensionalBisTris2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)-1,3-propanediolcsBN-MScryo(-microtome) slicing BN-MSdddouble-deionized (Milli-Q)FTFourier transformFWHMfull width at half-maximumHA(influenza) hemagglutininIBisolation bufferOXPHOSoxidative phosphorylationPVpeak volumeTEMEDtetramethylethylenediamineTOMtranslocase of the mitochondrial outer membraneVDACvoltage-dependent anion channel.-PAGE and its colorless variant, colorless native PAGE, were originally developed by Schägger and co-workers as end point separation methods for characterization of solubilized mitochondrial membrane protein (super-)complexes under close-to-native conditions (1.Schägger H. von Jagow G. Blue native electrophoresis for isolation of membrane protein complexes in enzymatically active form.Anal. Biochem. 1991; 199: 223-231Crossref PubMed Scopus (1906) Google Scholar, 2.Schägger H. Pfeiffer K. Supercomplexes in the respiratory chains of yeast and mammalian mitochondria.EMBO J. 2000; 19: 1777-1783Crossref PubMed Scopus (1037) Google Scholar, 3.Wittig I. Schägger H. Native electrophoretic techniques to identify protein-protein interactions.Proteomics. 2009; 9: 5214-5223Crossref PubMed Scopus (76) Google Scholar). Subsequently, native gel electrophoresis became the method of choice for first dimension separation followed by second dimension SDS-PAGE in two-dimensional gel-based proteomic analyses (2D-BN) of membrane protein complexes. After staining of the gel-separated proteins, protein spots are individually analyzed by different mass spectrometric methods, and the identified proteins were assigned to complexes based on their co-migration pattern (2D-BN-MS (4.Reisinger V. Eichacker L.A. How to analyze protein complexes by 2D blue native SDS-PAGE.Proteomics. 2007; 7: 6-16Crossref PubMed Scopus (57) Google Scholar)). However, these 2D-BN-MS approaches exhibit the following severe shortcomings: (i) they are critically dependent on the staining properties of individual proteins; (ii) the size resolution of protein complexes is low; and (iii) the assignment of identified proteins to spots and complexes may be ambiguous. Therefore, application of 2D-BN-MS has remained largely restricted to the characterization of highly abundant and well defined membrane protein complexes such as complexes I–V of the respiratory chain in mitochondria (5.Devreese B. Vanrobaeys F. Smet J. Van Beeumen J. Van Coster R. Mass spectrometric identification of mitochondrial oxidative phosphorylation subunits separated by two-dimensional blue-native polyacrylamide gel electrophoresis.Electrophoresis. 2002; 23: 2525-2533Crossref PubMed Scopus (62) Google Scholar, 6.Meyer B. Wittig I. Trifilieff E. Karas M. Schägger H. Identification of two proteins associated with mammalian ATP synthase.Mol. Cell. Proteomics. 2007; 6: 1690-1699Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar, 7.Klodmann J. Senkler M. Rode C. Braun H.P. Defining the protein complex proteome of plant mitochondria.Plant Physiol. 2011; 157: 587-598Crossref PubMed Scopus (145) Google Scholar), photosynthetic complexes (8.D'Amici G.M. Rinalducci S. Murgiano L. Italiano F. Zolla L. Oligomeric characterization of the photosynthetic apparatus of Rhodobacter sphaeroides R26.1 by nondenaturing electrophoresis methods.J. Proteome Res. 2010; 9: 192-203Crossref PubMed Scopus (19) Google Scholar, 9.Selao T.T. Branca R. Chae P.S. Lehtiö J. Gellman S.H. Rasmussen S.G. Nordlund S. Norén A. Identification of chromatophore membrane protein complexes formed under different nitrogen availability conditions in Rhodospirillum rubrum.J. Proteome Res. 2011; 10: 2703-2714Crossref PubMed Scopus (10) Google Scholar, 10.Behrens C. Blume C. Senkler M. Eubel H. Peterhänsel C. Braun H.P. The 'protein complex proteome' of chloroplasts in Arabidopsis thaliana.J. Proteomics. 2013; 91: 73-83Crossref PubMed Scopus (20) Google Scholar), or viruses (11.Li Z. Xu L. Li F. Zhou Q. Yang F. Analysis of white spot syndrome virus envelope protein complexome by two-dimensional blue native/SDS PAGE combined with mass spectrometry.Arch. Virol. 2011; 156: 1125-1135Crossref PubMed Scopus (21) Google Scholar). blue native two-dimensional 2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)-1,3-propanediol cryo(-microtome) slicing BN-MS double-deionized (Milli-Q) Fourier transform full width at half-maximum (influenza) hemagglutinin isolation buffer oxidative phosphorylation peak volume tetramethylethylenediamine translocase of the mitochondrial outer membrane voltage-dependent anion channel. In a first attempt to overcome these shortcomings of 2D-BN-MS, Wessels et al. (12.Wessels H.J. Vogel R.O. van den Heuvel L. Smeitink J.A. Rodenburg R.J. Nijtmans L.G. Farhoud M.H. LC-MS/MS as an alternative for SDS-PAGE in blue native analysis of protein complexes.Proteomics. 2009; 9: 4221-4228Crossref PubMed Scopus (72) Google Scholar) coupled BN-PAGE separation more directly to MS analysis by manually cutting the gel lane into 24 slices/sections of about 2 mm width that were separately digested and analyzed by liquid chromatography-tandem mass spectrometry (LC-MS/MS). Their study on HEK cell mitochondria identified 59 of the 90 canonical subunits of the oxidative respiratory chain (OXPHOS) complexes I–V. The respective protein abundance profiles (based on standard label-free quantification) showed clustering of their peak maxima into the expected complexes I–V. Since then, this one-dimensional BN-MS methodology has been gradually improved with respect to quality of the native gel separation, LC-MS/MS sensitivity, and robustness of the quantitative evaluation. Thus, two recent studies on human mitochondrial preparations (each analyzing two BN separations in 60 and 24 slices, respectively) reported identification and hierarchical profile clustering of 464 (13.Heide H. Bleier L. Steger M. Ackermann J. Dröse S. Schwamb B. Zörnig M. Reichert A.S. Koch I. Wittig I. Brandt U. Complexome profiling identifies TMEM126B as a component of the mitochondrial complex I assembly complex.Cell Metab. 2012; 16: 538-549Abstract Full Text Full Text PDF PubMed Scopus (198) Google Scholar) and 437 (14.Wessels H.J. Vogel R.O. Lightowlers R.N. Spelbrink J.N. Rodenburg R.J. van den Heuvel L.P. van Gool A.J. Gloerich J. Smeitink J.A. Nijtmans L.G. Analysis of 953 human proteins from a mitochondrial HEK293 fraction by complexome profiling.PLoS One. 2013; 8: e68340Crossref PubMed Scopus (41) Google Scholar) mitochondrial proteins. In these studies, 82/73 (including 8 single-peptide hits) and 55/54 (including 7 single-peptide hits) of the 90 known OXPHOS complex subunits were identified/clustered, respectively. Furthermore, TMEM126B was identified as a novel and essential subunit of an OXPHOS complex I assembly complex (13.Heide H. Bleier L. Steger M. Ackermann J. Dröse S. Schwamb B. Zörnig M. Reichert A.S. Koch I. Wittig I. Brandt U. Complexome profiling identifies TMEM126B as a component of the mitochondrial complex I assembly complex.Cell Metab. 2012; 16: 538-549Abstract Full Text Full Text PDF PubMed Scopus (198) Google Scholar). Notably, all of these studies achieved clustering of protein profiles for the dominating populations of complexes, although they largely failed to obtain information on sub-complexes and super-complexes, most likely as a consequence of the strong undersampling in the first dimension (well below the resolution of BN-PAGE) and a limited dynamic range of MS-based identification and quantification. To improve the resolution of BN-MS for analysis of protein super-complexes and their subunit composition, we have recently started to develop sub-millimeter sampling of BN gel lane sections by using cryo-microtome slicing (15.Schwenk J. Harmel N. Brechet A. Zolles G. Berkefeld H. Müller C.S. Bildl W. Baehrens D. Hüber B. Kulik A. Klöcker N. Schulte U. Fakler B. High resolution proteomics unravel architecture and molecular diversity of native AMPA receptor complexes.Neuron. 2012; 74: 621-633Abstract Full Text Full Text PDF PubMed Scopus (300) Google Scholar, 16.Turecek R. Schwenk J. Fritzius T. Ivankova K. Zolles G. Adelfinger L. Jacquier V. Besseyrias V. Gassmann M. Schulte U. Fakler B. Bettler B. Auxiliary GABAB receptor subunits uncouple G protein βγ subunits from effector channels to induce desensitization.Neuron. 2014; 82: 1032-1044Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar). Here, we describe a new methodology for comprehensive and high resolution complexome profiling that combines this high resolution gel sampling method with a sensitive and precise label-free MS quantification workflow. Protein profiles determined in a mammalian mitochondrial membrane preparation showed a highly effective mass resolution ( pyro-Glu (N-terminal Glu), Glu > pyro-Glu (N-terminal Glu), oxidation (Met), and propionamide (Cys) were chosen as variable modifications; peptide, and fragment mass tolerance were set to ± 5 ppm and ± 0.8 Da, respectively, and one missed tryptic cleavage was allowed. The expectation value cutoff for peptide identification was set to 0.5. Related identified proteins (subset or species homologs) were grouped together using the name of the predominant member. The final list of identified mitochondrial proteins (supplemental table 1) was obtained after filtering the search results obtained for the 230 samples for proteins that were (i) localized or linked to mitochondria according to UniProtKB/Swiss-Prot database annotation or to the MitoCarta database (entries manually updated to the current UniProtKB/Swiss-Prot protein information), and (ii) whose identification was based on at least two independent protein-specific peptides (at least one with maximum expectation value <0.05) found in at least two slice samples. As an exception, proteins were accepted with only one identified specific peptide (with maximum expectation value 1) and weighting (with the sum of the three other matrix elements (PVs)); very low consistent PV values with a sum score <0.2 were finally eliminated. PVs of each peptide were then normalized to their maximum over the 230 datasets to obtain relative peptide profiles. These were subsequently ranked for each protein by pairwise Pearson correlation (19.Bildl W. Haupt A. Muller C.S. Biniossek M.L. Thumfart J.O. Huber B. Fakler B. Schulte U. Extending the dynamic range of label-free mass spectrometric quantification of affinity purifications.Mol. Cell. Proteomics. 2012; 11 (M111.007955)Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar). The average of at least two (one for the exception of proteins identified by one specific peptide (see under "Protein Identification")) and up to 6 or 50% (whichever value is greater) best correlating protein-specific peptide profiles over a window of three consecutive slices was used to calculate the relative abundance values of each protein profile. Finally, these protein profiles were least squares-fitted to the normalized (molecular) abundance (abundancenorm) values (19.Bildl W. Haupt A. Muller C.S. Biniossek M.L. Thumfart J.O. Huber B. Fakler B. Schulte U. Extending the dynamic range of label-free mass spectrometric quantification of affinity purifications.Mol. Cell. Proteomics. 2012; 11 (M111.007955)Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar) determined for the respective protein in the 230 datasets as a rough estimate of molecular abundance (supplemental table 3). Individual protein profiles were analyzed by finding peaks in abundance mass profiles (up to three per protein) using continuous wavelet transformation (Mexican Hat) and fitting a Gaussian function to these peaks (Mathematica, Wolfram Research, version 9.0.1) and Python/SciPy (versions 2.7.6 and 0.15.1). Thus, each protein or protein complex was characterized by the position of its abundance peak maximum and by the FWHM value (both parameters were given in slice number units). The smallest FWHM values represented a measure for the effective size resolution of the approach (supplemental table 4). The slice numbers were converted to apparent molecular sizes (apparent mass in kDa) using the result of the linear regression of the slice number positions of 31 reference protein complex peaks versus their log10(Mr) values (as reported in UniProtKB/Swiss-Prot, see supplemental table 5). The relative abundance-mass profiles of OXPHOS complexes I–V were determined by the median of the relative abundance profiles of the respective core complex protein subunits (supplemental table 6). For identification of novel protein complexes, defined complex assemblies (supplemental table 4) were screened against the profiles of all proteins determined by the csBN-MS approach using Pearson correlation; the respective results were verified by manual inspection. In addition, single linkage hierarchical clustering of the individual protein abundance profiles was performed with Pearson correlation as distance metric using Python/SciPy (supplemental figure 2). The Saccharomyces cerevisiae strain Tom22His and the corresponding wild-type strain YPH499 were described before (21.Meisinger C. Ryan M.T. Hill K. Model K. Lim J.H. Sickmann A. Müller H. Meyer H.E. Wagner R. Pfanner N. Protein import channel of the outer mitochondrial membrane: a highly stable Tom40-Tom22 core structure differentially interacts with preproteins, small tom proteins, and import receptors.Mol. Cell. Biol. 2001; 21: 2337-2348Crossref PubMed Scopus (143) Google Scholar). The yeast strain expressing Por1HA in the YPH499 background was generated by chromosomal integration of a coding region for a triple HA tag and a HIS3 cassette in front of the stop codon of POR1 by homologous recombination (22.Wenz L.S. Opaliński L. Schuler M.H. Ellenrieder L. Ieva R. Böttinger L. Qiu J. van der Laan M. Wiedemann N. Guiard B. Pfanner N. Becker T. The presequence pathway is involved in protein sorting to the mitochondrial outer membrane.EMBO Rep. 2014; 15: 678-685PubMed Google Scholar). Yeast cells were grown on yeast extract/peptone/glycerol medium (1% (w/v) yeast extract, 2% (w/v) bactopeptone, 3% (v/v) glycerol) at 24–30 °C. Mitochondria were isolated by differential centrifugation as described (23.Meisinger C. Pfanner N. Truscott K.N. Isolation of yeast mitochondria.Methods Mol. Biol. 2006; 313: 33-39PubMed Google Scholar). For purification of Por1-containing complexes, Por1HA mitochondria were lysed under native conditions with digitonin buffer (0.3% (w/v) digitonin, 10% (v/v) glycerol, 50 mm NaCl, 0.1 mm EDTA, 20 mm Tris/HCl pH 7.4) at a protein/detergent ratio of 1:3 (1 mg of protein/ml) for 15 min on ice. After removal of insoluble material by centrifugation (10 min, 20,000 × g, 4 °C), the samples were subjected to affinity purification via anti-HA chromatography (Roche Diagnostics, Switzerland). After excessive washing with digitonin buffer containing 0.1% (w/v) digitonin, bound proteins were eluted under native conditions by incubation with 1 mg/ml HA peptides (Roche Diagnostics). Tom22His-containing protein complexes were purified by nickel in complex with nitrilotriacetic acid-agarose (Qiagen, Germany) (22.Wenz L.S. Opaliński L. Schuler M.H. Ellenrieder L. Ieva R. Böttinger L. Qiu J. van der Laan M. Wiedemann N. Guiard B. Pfanner N. Becker T. The presequence pathway is involved in protein sorting to the mitochondrial outer membrane.EMBO Rep. 2014; 15: 678-685PubMed Google Scholar). Denatured proteins were analyzed by SDS-PAGE, and native s