High-Resolution Complexome Profiling by Cryoslicing BN-MS Analysis

The vast majority of conventional BN-MS studies as well as the recently established high-resolution csBN-MS approach have been applied to mitochondrial and plastid preparations that are (i) readily available, (ii) have limited complexity, and (iii) express target (membrane) protein complexes at high densities. This protocol extends the application of high-resolution complexome profiling to non-mitochondrial membranes expressing low-abundant proteins, about which little information on their integration into complexes is available. For demonstration purposes, we chose an endosome-enriched membrane preparation from mouse kidney obtained by density gradient centrifugation.

Optimization of this preparation has been guided by the marker protein TPC1 that forms intracellular ion channels predominantly localized to early and recycling endosomes¹². It is also highly expressed in renal proximal tubular cells, as shown by immunohistochemical analysis of kidney tissue sections (Figure 1A). These membranes were gently solubilized (ComplexioLyte 47 at a low protein:detergent ratio of 1:8) and concentrated on a sucrose cushion by ultracentrifugation. The latter turned out to be an important step for removing excess lower molecular weight components (i.e., detergents, lipids, salts, organic polymers, and metabolites) that tend to negatively impact on resolution of preparative BN-PAGE separations.

Complex separation on a native 1%-13% (w/v) polyacrylamide gradient gel (Figure 1B, middle panel) showed strongly stained protein bands with very little migration artifacts. SDS-PAGE separation of a narrow BN-PAGE gel strip (Figure 1B, frame boxed in red) as a second dimension followed by western blot analysis showed a well-resolved pattern of distinct TPC1-associated complex populations (Figure 1B, upper panel, marked by red arrows), most likely resulting from association with additional protein subunits and/or posttranslational modifications (such as glycosylation¹²). A 3 cm section of interest was excised, fixed, and processed for cryomicrotome slicing as described¹¹. The individual steps of this procedure (in particular, the precise alignment of the broad gel section), which is of critical importance for preserving resolution during sampling, are documented in the accompanying video. The embedded gel section was finally cut into 101 gel slices with a uniform thickness of 0.25 mm (Figure 1B, lower panel), which were separately digested and analyzed by high performance LC-coupled mass spectrometry.

In addition to size resolution, the quality of protein quantification is key for successful complexome profiling. With the MS set-up and settings used, analysis of the samples was quite comprehensive, resulting in an average identification of more than 1,000 proteins and 10,000 peptides (8,200 of which were protein-specific) per slice, and around 3,000 proteins and 43,000 peptides (38,500 of which were protein-specific) in total. Nevertheless, due to the stochastic nature of data-dependent MS/MS sequencing and its limitations in dynamic range, intensity information was still fragmentary for less abundant proteins. Therefore, an elaborate MS data processing procedure¹¹was performed that is based on precise assignment of peptide signals (peak volumes [PVs] = peptide-related signal intensities integrated over m/z and time) over the entire series of datasets.

As shown in Figure 2A,B, deviations of peptide signals in mass and retention time that remained after calibration were identical for MS-sequenced and for indirectly assigned PVs (with very narrow tolerances of <1 ppm and <0.5 min for 95% of the PVs), indicative of a very low rate for false-positive PV assignment. Remaining outliers were filtered based on their consistency with other related PVs. Since all MS measurements were performed consecutively on the same LC-MS set-up without changes in parameters or hardware components, run-to-run variations (determined as the median of all PV intensities in a sample relative to those in the neighboring slices) were small and easily eliminated by rescaling of the PV datasets (Figure 2C). The resulting peptide intensity information was then used to reconstruct 2,545 protein relative abundance profiles. As shown in Figure 2D, more than 75% of these protein profiles were based on at least three independent protein-specific peptides.

Next, the protocol assessed relevance of the step size of BN-PAGE gel sampling for the resolution of protein complexes. For this purpose, slice datasets were joined by summing the PV information from two, three, or four consecutive slices, thus simulating the outcome for step sizes of 0.5 mm, 0.75 mm, and 1 mm (compared to the original sampling of 0.25 mm). Figure 3 illustrates the resulting abundance profiles for protein TPC1 as an example (A-D). At 0.25 mm, relative intensities and size separation of TPC1-associated complex populations (Figure 3A) were nicely in agreement with the results from western blot analysis (Figure 1B, upper panel); although, the profile showed some noise, mostly resulting from missing values ("gaps") in the PV matrix used for quantification.

Joining of two slices corresponding to 0.5 mm maintained the correct intensities and separation of the TPC1-associated complexes and removed quantification noise (Figure 3B). In contrast, larger step sizes of 0.75 mm and 1 mm (Figure 3C,D) led to a loss of size resolution and abolished discrimination of TPC1 complex subpopulations. It should be noted that the vast majority of published conventional BN-MS analyses use manually cut 2 mm slices (around 60 to cover the entire gel lane)^7,8,9,10.

Conversion of migration distance or slice index to molecular size is generally based on markers, either commercially available native standard proteins or well-characterized endogenous protein complexes with known subunit composition (mostly [super]complexes of the mitochondrial oxidative respiratory chain [OXPHOS])¹³. However, since BN-PAGE separation is based on the effective molecular cross-section that is determined not only by the molecular mass but also by the 3D structure and number of associated lipids, detergent, and Coomassie molecules, individual proteins may show larger deviations. Therefore, it was chosen to use larger sets of protein complexes as markers¹¹. The plot in Figure 4 shows 23 selected markers with a representative subunit shown as a black circle, indicating the log10 values of its predicted molecular mass (according to the UniProtKB/Swiss-Prot database) vs. the slice index of the corresponding profile peak maximum. The latter were obtained from automatized Gaussian fits to the relative abundance data, as shown in the inset of Figure 4 showing the example with chaperone BCS1. Linear regression (red line) provided a function to convert slice index values to apparent molecular sizes, which ranged from 160-630 kDa, along the investigated gel section.

Finally, the analysis provided information on well-characterized complexes and demonstrated the existence of novel subunits and complex assemblies. Examples highlighting different aspects of the complexome are shown in Figure 5 (A-C: proteins expressed or preferably located in endosomal compartments; D-F: complexes from other subcellular localizations). The iron-transporting protein ferritin is known to form complexes from 24 light (FRIL1) and/or heavy (FRIH) subunits, with a total molecular weight of 440 kDa¹⁴ (Figure 5A, filled arrow). The subunit profiles (Figure 5A) suggest the existence of at least two smaller forms of the complex (with an apparent mass of 360 kDa and 340 kDa; open arrows) with distinct heavy/light chain stoichiometries (better visible after rescaling of abundances, inset of Figure 5A) that are abundantly present in endosomes.

In contrast, nicalin-nomo1 complexes¹⁵ (Figure 5D), the gamma-secretase core complex¹⁶ (Figure 5B), and the GPI-transamidase machinery¹⁷ (Figure 5E) show fixed abundance ratios of their core subunits over the entire size range and independent from association with additional proteins. This indicates that their subunits are exclusive to each other. Vacuolar H⁺-ATPases are multiprotein complexes assembled from a pool of more than 20 subunits in a modular manner with a total molecular weight of around 900 kDa. Figure 5C reveals sub-complexes with distinct compositions from at least 17 subunits, either representing biological (dis)assembly intermediates or subcomplexes generated by the experimental conditions, some of which have also been observed in a recent BN-MS study¹⁸. Another multi-protein complex example is the proteasome¹⁹ (Figure 5F). Close inspection of the abundance profiles of the alpha and beta subunits forming the 20S proteasome core suggests two major complex populations with subtle differences in size (590 kDa and 575 kDa, indicated by grey arrows) and integration of three beta subunits.

In summary, csBN-MS complexome profiling of endosome-enriched kidney membranes provide comprehensive and detailed results regarding the (i) integration of uniform, low-abundant target proteins into complexes, (ii) general complex subunit composition and stoichiometry, and (iii) complex heterogeneities, substructures, and (dis)assembly intermediates.

Figure 1: Preparative BN-PAGE separation of solubilized endosome-enriched membranes from mouse kidney using intracellular channel TPC1 as a marker. (A) Immunohistochemical localization of TPC1 protein in renal proximal tubules by confocal microscopy. Green: anti-TPC1 antibody¹² staining visualized with secondary Cy3-biotinylated goat anti-rabbit IgG; red: biotinylated Lotus tetragonolobus lectin (LTL, 10 µg/mL, FITC-conjugated) marking the luminal surface of proximal tubulus cells. The inset shows staining of a corresponding section from a TPC1-KO kidney as a negative control. White scale bars are 20 µm. Also of note is strong TPC1 expression in intracellular vesicles, known from independent experiments to represent early and recycling endosomes¹². (B) Preparative BN-PAGE separation of 2.5 mg of solubilized endosome-enriched membranes on a 1%-13% (w/v) polyacrylamide gradient gel. A narrow lane (framed in red) was cut for subsequent SDS-PAGE/western blot analysis (upper panel), resolving different TPC1-associated complexes and glycosylation patterns (red arrows: anti-TPC1/anti-rabbit HRP/ECL prime; green: positions and predicted masses [MDa] of marker protein complexes identified by total protein staining [SYPRO Ruby blot stain]) of the blot. From right to left: Na⁺/K⁺-transporting ATPase, Cytochrome b-c1 complex dimer, ATP synthase, NADH:ubiquinone oxidoreductase. A 3 cm section of interest from the gel lane was excised, embedded in tissue embedding medium, mounted, and sliced into 101 sections (0.25 mm) along the protein migration front using a cryomicrotome (lower panel; see video link). Please click here to view a larger version of this figure.

Figure 2: Key parameters determining accuracy of MS signal assignment and quantification as well as depth of analysis. (A) Distribution of relative mass errors (in ppm) after m/z calibration of MS/MS sequenced (red bars) and indirectly assigned (i.e., based on closely matching mass and retention times, see protocol; blue bars) peptide signals. This suggests a final mass error of <1 ppm (for 95% of the signals/assigned PVs) and very low rate of false positive assignments. (B) Distribution of nano-HPLC retention time deviations from total average after elution time alignment of peptide signals, using Loess regression (see protocol) and color coding as used in (A). Time error is less than 30 s for >95% of peptide signals/assigned PVs. (C) Run-to-run variation of total MS intensities plotted relative to the average of the two neighboring samples. These scale factors were applied to the raw PV tables to minimize systematic technical errors. (D) Peptide information used for calculating relative abundance profiles of proteins. After filtering protein-specific peptides for outliers, poorly scored, or single identifications (see protocol), 2,545 protein abundance profiles were determined, >75% of which were based on at least three peptides with reasonable confidence. Please click here to view a larger version of this figure.

Figure 3: Critical impact of step size in gel sampling on complexome resolution of TPC1. Datasets were joined by summing the signal intensities in groups of 1, 2, 3, and 4 consecutive slices (A-D, respectively) and processed identically to simulate different step sizes in gel slicing as indicated. The TPC1 profile shows some (oversampling) noise at 0.25 mm but good size resolution of three complex populations (see also Figure 1B), which is largely preserved at a 0.5 mm step width. Discrimination of these populations becomes lost as 1 mm is approached. Please click here to view a larger version of this figure.

Figure 4: Determination of apparent molecular weight. 23 marker complexes with defined molecular composition (as indicated, according to UniProtKB/Swiss-Prot) were used as size markers. Logarithmic values of their expected molecular weights (in kDa) were plotted vs. the profile peak maximum slice index of the indicated representative protein subunit (filled circles in black). Linear regression fitting to this data (red line) provided a function converting slice index values to apparent molecular weights. Peak maxima were determined by automatized Gaussian fits to protein profile peaks as shown in the inset (right) for the chaperone protein BCS1 (primary data in blue, fit boundaries indicated by orange lines, fit function in red). In addition, these fits determined peak half-maximal widths (green line, 6.5 slices, or 1.6 mm for the example shown) with the sharpest focusing complexes spanning around a 1.5 mm gel. Please click here to view a larger version of this figure.

Figure 5: Examples of protein complex subunit profiles. Relative protein abundance vs. apparent molecular weight plotted for heavy and light chain of ferritin (A) revealing molecular heterogeneity of ferritin subunit stoichiometry, more clearly visible after rescaling of abundances (inset). Filled arrow and open arrows denote the full complex (440 kDa) and two subcomplexes, respectively. The gamma-secretase (B) subunits quantitatively integrated into a single-core complex population. The subcomplexes of vacuolar H⁺-ATPases (C) exhibited multiple assemblies with distinct subunit composition, all expressed in endosomes. The nomo1 and nicalin proteins (D) formed an exclusive complex (the GPI-transamidase), which is a multi-subunit enzymatic machinery forming several complexes. (E) The 20S proteasome core complex showing (F) a subtle subcomplex pattern with two populations indicated by arrows in grey, all originating from other subcellular localizations. Please click here to view a larger version of this figure.