Title: Cryo-slicing Blue Native-Mass Spectrometry (csBN-MS), a Novel Technology for High Resolution Complexome Profiling
Abstract: 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. 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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 (<5% molecular weight difference) over the whole BN-PAGE separation range and together covered a major portion of the mitochondrial membrane proteome. Mitochondria were isolated from freshly dissected adult rat brains following the procedure by Rendon and Masmoudi (17.Rendon A. Masmoudi A. Purification of non-synaptic and synaptic mitochondria and plasma membranes from rat brain by a rapid Percoll gradient procedure.J. Neurosci. Methods. 1985; 14: 41-51Crossref PubMed Scopus (39) Google Scholar). 15 g of brain tissue were washed in isolation buffer (IB, 320 mm sucrose, 6 mm Tris-HCl, pH 7.5, 6 mm EDTA + protease inhibitors) and gently homogenized with 70 ml of IB using a glass potter. An equal volume of IB was added, and the homogenate was centrifuged for 5 min at 1,100 × g. Pellets were re-homogenized by repeating this procedure, and the combined supernatants were pelleted for 10 min at 17,000 × g, resuspended in IB, and pelleted again for 20 min. The washed pellet was homogenized in 30 ml of IB, loaded onto Ficoll step gradients (9 ml on 7 ml of 7.5% FicollTM (PM400, GE Healthcare, Germany) in IB/7 ml of 13% Ficoll in IB), and centrifuged at 100,000 × g for 30 min. The fractions at the 7.5/13% Ficoll interphase containing vesicle-enclosed mitochondria were collected, diluted with IB, and pelleted (10 min at 18,000 × g). These vesicles were lysed in 6 mm Tris-HCl, pH 8.1, and protease inhibitors (40 ml, 30 min), and the membranes were collected by ultracentrifugation (15 min at 75,000 × g). After resuspension in 3 ml of IB, the membranes were separated on an isotonic step gradient (1 ml/gradient) built by 10%, 20% and 40% Percoll (GE Healthcare) in IB by centrifugation for 20 min at 37,000 × g. Mitochondria were retrieved from the 20–40% Percoll interphase, washed twice in IB by dilution/centrifugation/resuspension cycles, and stored as a concentrated suspension (56 mg/ml) at −80 °C. Non-denaturing 1–13% (v/v acrylamide) gradient gels (14 × 11 cm, 1.5-mm spacer) were cast using a peristaltic pump fed by a linear gradient mixer filled with two solutions with freshly added ammonium persulfate and TEMED as follows: solution A (13 ml), 11.5% acrylamide, 0.45% bisacrylamide, 0.75 m aminocaproic acid, 50 mm BisTris, pH 7.0, 10% glycerol; and solution B (13 ml), 0.95% acrylamide, 0.025% bisacrylamide, 0.75 m aminocaproic acid, 50 mm BisTris, pH 7.0, 0.2% ComplexioLyte 47 detergent (Logopharm, Germany), and polymerized overnight. Gels were run in water-cooled (10 °C) Penguin M electrophoresis systems (PEQLAB, Germany) using the cathode and anode buffer system as described previously (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). 1 mg of mitochondrial membranes was solubilized in 0.8 ml of detergent buffer (1% ComplexioLyte 47 (protein-detergent ratio of 1:8) with salts replaced by 0.5 m aminocaproic acid, 50 mm imidazole, pH 7.0, 1 mm EDTA, 1 mm EGTA, and protease inhibitors) for 30 min on ice and cleared by ultracentrifugation (11 min at 79,000 × g, S80-AT3 rotor with tube adaptors). The solubilizate was supplemented with 10% glycerol, 0.1% Coomassie G-250 and directly loaded onto the gel (20 μg/mm2 gel cross-section). Voltage was initially set to 100 V for 30 min, ramped to 500 V during 1 h, and kept at 500 V for 8 h. After BN-PAGE separation, gel lanes were excised (5-mm strip was dissevered for 2D analysis, Fig. 1A) and frozen at −20 °C. For 2D-BN-SDS-PAGE, the narrow strip was incubated twice for 5 min in 5 ml of 2× Laemmli buffer and placed on a 13% SDS-polyacrylamide gel silver-stained after the run. The broad gel strip was fixed twice (for 5 min in 30% ethanol, 15% acetic acid), washed briefly in ddH2O, and equilibrated in tissue embedding solution (Leica Tissue Freezing Medium, VWR, Germany) for 20 min under gentle movement. The equilibrated gel strip was cut into three sections (Fig. 1A, colored boxes), and each of them was embedded for cryo-microtome slicing as illustrated in Fig. 2. Gel pieces were carefully placed on a frozen block of embedding solution in casting molds (3 × 3 cm, half-filled) with the protein migration front perfectly aligned with one of the mold walls to avoid any distortion of the gel. The gel position was fixed with needles before overlaying it with embedding solution and freezing. The frozen blocks were mounted on precooled probe supports and equilibrated to a temperature of −19 °C in the cryo-microtome (Leica CM1950, Germany). Slicing was done by slow manual driving of the specimen feed set to a step size of 0.3 mm (angle of 0°). Around 80 successive slices from each block were separately collected and stored frozen. Prior to digestion, the embedding medium was largely removed by washing the gel strips twice in 30% ethanol, 15% acetic acid and ddH2O, once in 100% ethanol and again twice in ddH2O. In-gel digestion was carried out according to the procedure described previously (18.Pandey A. Andersen J.S. Mann M. Use of mass spectrometry to study signaling pathways.Sci. STKE. 2000; 2000: pl1PubMed Google Scholar) using sequencing grade modified trypsin (Promega, Germany; 1:200 in 25 mm NH4HCO3).Fig. 2.Embedding and cryo-slicing of BN gels. Documentation of the key steps of sample preparation. A, trimming of gel sections after equilibration. B, their positioning and embedding for freezing. C, mounting and slicing of frozen gel blocks with a cryo-microtome. During all steps, the precise perpendicular orientation of the gel section with respect to the migration front must be maintained. D, left panel, removal of embedding medium from gel slices. Right panel, gel slices after thawing (view on the gel slice cross-section). Slices were numbered 1–230 starting from the low molecular weight end.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Extracted vacuum-dried peptides were re-dissolved in 20 μl of 0.5% trifluoroacetic acid for each of the 230 samples and transferred into individual well plate cavities. Using an UltiMate 3000 RSLCnano (Dionex/Thermo Scientific, Germany), 5 μl of each sample were trapped on a C18 PepMap100 precolumn (particle size 5 μm; Dionex/Thermo Scientific) with eluent A (5 min; 20 μl/min) and separated in a PicoTipTM Emitter (inner diameter 75 μm; tip 8 μm; New Objective) manually packed with ReproSil-Pur 120 ODS-3 (C18; particle size 3 μm; Dr. Maisch HPLC, Germany) with an aqueous-organic gradient (eluent A: 0.5% (v/v) acetic acid; eluent B: 0.5% (v/v) acetic acid in 80% (v/v) acetonitrile; nano/cap pump gradient: 5 min 3% B, 60 min from 3% B to 30% B, 15 min from 30% B to 99% B, 5 min 99% B, 5 min from 99% B to 3% B, 15 min 3% B; flow rate 300 nl/min). Eluting peptides were electrosprayed (2.3 kV; transfer capillary temperature 250 °C) in positive ion mode into an Orbitrap Elite tandem mass spectrometer equipped with a Nanospray Flex Ion Source (Thermo Scientific; total acquisition time 105 min according to the gradient). FT full MS (m/z 370 to 1,700; resolution 240,000 FWHM; profile data) and data-dependent ion trap MS/MS spectra (maximum of 10 per scan cycle; normal scan rate; centroid data) were acquired with target values of 1,000,000 and 10,000, and maximum injection times of 500 and 200 ms, respectively. Injection waveforms were enabled for all spectra. The following data-dependent settings were used: dynamic exclusion enabled (repeat count, 1; exclusion list size, 500; repeat/exclusion duration, 30 s; exclusion mass width, ± 20 ppm); ion trap MS/MS ion injection time predicted; preview mode for FT-MS master scans, charge state screening, mono-isotopic precursor selection, and charge state rejection (+1) enabled; minimum signal threshold, 500 counts; activation type, collision-induced dissociation (default charge state, 2; isolation width:, 2.0 m/z; normalized collision energy, 35; activation Q, 0.25; activation time, 10 ms). Peak lists were extracted from fragment ion spectra using the msconvert.exe tool (part of ProteoWizard, version 2.2.3214; default parameters). For each dataset, all precursor m/z values were shifted by the median m/z offset of all peptides assigned to proteins in a preliminary database search with 15 ppm peptide mass tolerance. Corrected peak lists were searched with Mascot 2.5.1 (Matrix Science, UK) against the UniProtKB/Swiss-Prot database (release 2015_02; only P00761, P00766, and P02769 and all rat, mouse, and human entries) supplemented with 349 rat UniProtKB/TrEMBL or NCB RefSeq entries (identified by BLAST searches of mitochondrial proteins for which no rat homologs existed in the UniProtKB/Swiss-Prot database). Acetyl (protein N terminus), carbamidomethyl (Cys), Gln > 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 <0.05 and in at least two slice samples) when at the same time more than 10% of the detectable tryptic peptides of that protein were identified (see supplemental table 1). The raw data and database search results (mzIdentML format) have been submitted to the PRIDE repository (project accession code PXD002681). Label-free quantification of proteins was carried out following the principles described previously (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) with further optimizations to address the specific challenges arising from combining 230 complex datasets. Peptide signal intensities were integrated over time and m/z as peak volumes (PVs) from FT full scans using MaxQuant version 1.4.1.2 with integrated off-line mass calibration (20.Cox J. Michalski A. Mann M. Software lock mass by two-dimensional minimization of peptide mass errors.J. Am. Soc. Mass Spectrom. 2011; 22: 1373-1380Crossref PubMed Scopus (110) Google Scholar). For correcting peptide PV elution time shifts, individual datasets were aligned one-by-one to reference peptide elution times using LOESS regression. These reference times were dynamically calculated from the median peptide elution times over all previously aligned datasets and updated accordingly after each alignment. The process started with the largest dataset as seed and successively aligned the dataset having the highest overlap with the reference peptide list. PVs were then assigned to peptides based on their m/z and elution time, values that were obtained either directly from MS/MS-based identification or indirectly, i.e. from identifications in parallel datasets (termed "inserted"). Alignment and assignment were carried out by software developed in-house using parameters that resulted in effective m/z and elution time matching tolerances of ± 2.5 ppm and ± 1 min, respectively. The assigned PV data has been formatted as mzQuantML file and submitted to the PRIDE repository (project accession code PXD002681). To correct for run-to-run variations in peptide recovery and ionization efficiency of the LC-MS setup, PVs of each dataset n were rescaled by the median of all peptide scale factors s with s = (∑n − 2n + 2 PVn)/PVn (see supplemental figure 1). The PV data for each protein were filtered for outliers and false-positive assignments using a new correlation-based method. For each protein and over all 230 datasets, each PV was checked for its consistency with other PVs of the same protein (lines and columns in supplemental table 2) by finding all 2-by-2 sub-matrices [BADC] containing the respective PV and calculating their scores s = (A/B)/(C/D) (i.e. the ratios of the pairs of connected PV ratios). The consistency of each PV was then determined from the sum of all of its associated sub-matrix scores s after reciprocal normalization (s = 1/s if s >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