Classification of subcellular location by comparative proteomic analysis of native and density-shifted lysosomes

Abstract

One approach to the functional characterization of the lysosome lies in the use of proteomic methods to identify proteins in subcellular fractions enriched for this organelle. However, distinguishing between true lysosomal residents and proteins from other cofractionating organelles is challenging. To this end, we implemented a quantitative mass spectrometry approach based on the selective decrease in the buoyant density of liver lysosomes that occurs when animals are treated with Triton-WR1339. Liver lysosome-enriched preparations from control and treated rats were fractionated by isopycnic sucrose density gradient centrifugation. Tryptic peptides derived from gradient fractions were reacted with isobaric tag for relative and absolute quantitation eight-plex labeling reagents and analyzed by two-dimensional liquid chromatography matrix-assisted laser desorption ionization time-of-flight MS. Reporter ion intensities were used to generate relative protein distribution profiles across both types of gradients. A distribution index was calculated for each identified protein and used to determine a probability of lysosomal residence by quadratic discriminant analysis. This analysis suggests that several proteins assigned to the lysosome in other proteomics studies are not true lysosomal residents. Conversely, results support lysosomal residency for other proteins that are either not or only tentatively assigned to this location. The density shift for two proteins, Cu/Zn superoxide dismutase and ATP-binding cassette subfamily B (MDR/TAP) member 6, was corroborated by quantitative Western blotting. Additional balance sheet analyses on differential centrifugation fractions revealed that Cu/Zn superoxide dismutase is predominantly cytosolic with a secondary lysosomal localization whereas ATP-binding cassette subfamily B (MDR/TAP) member 6 is predominantly lysosomal. These results establish a quantitative mass spectrometric/subcellular fractionation approach for identification of lysosomal proteins and underscore the necessity of balance sheet analysis for localization studies.

Fig. 1.

Distribution of organelle markers and protein in sucrose density gradient fractions. Differential centrifugation L fractions prepared from livers of control or Triton WR-1339 treated rats were fractionated by isopycnic sucrose density gradient centrifugation. Each gradient represents an individual animal. Indicated fractions were combined following marker analysis as shown to create four pools per gradient.

Fig. 2.

Workflow for sample processing and iTRAQ labeling. Liver homogenates were processed and used for quantitative mass spectrometry as described in text.

Fig. 3.

Distribution of different classes of proteins in sucrose density gradient pooled fractions. Relative intensities were calculated as described in Methods. Error bars show the 95% confidence intervals calculated using Prism5.03 (GraphPad Software, Inc). The profile for β-galactosidase is based on enzyme activity and protein measurements conducted following fractions were pooled. Note that the specific reporter ion intensities or activity are normalized to protein levels. Thus, even though most of the lysosomal proteins sediment in the denser fractions in the control gradients as shown in Fig. 1, these fractions also contain the bulk of the protein, resulting in similar relative specific activities among the pooled fractions.

Fig. 4.

Distribution profiles for curated lysosomal proteins. Top Panels shows mean distribution profiles for all curated lysosomal proteins. ACP5 is depicted as a dotted black line, all others as solid black lines. Remaining panels show of individual spectra (black lines) that are used to calculate the mean distribution profiles (thick red line) for classical lysosomal acid phosphatase (ACP2), tartrate-resistant acid phosphatase (ACP5), and cathepsin D (CTSD).

Fig. 5.

Classification of proteins using quadratic discriminant analysis (QDA). Each protein identified in a given experiment is represented by a separate symbol: black circles, curated lysosomal proteins; red squares, others. The gray and red dots depict the bootstrap points of the curated lysosomal and other data set, respectively. Curves represent the boundary lines where the posterior probability for assignment as lysosomal or not lysosomal is equal for different classification routines (see text for details). Statistical analysis was conducted as follows: For each protein we generated 10,000 points using a bivariate normal parametric bootstrap procedure using the two sample means of pC_1,2 and pT_1,2 (calculated from individual spectra associated with each protein) and their variances and the covariance. Formally, denote by p̂_C and p̂_T the sample means of the n reporter ion intensity measurements for a particular protein and let s_C², s_T², and s_CT denote, respectively, the variances of the n spectra and their covariance. Then the precision of the estimates of p̂_C and p̂_T are given by s_C²/n, s_T²/n, and s_CT/n. These three parameters, which reflect the precision of the estimates, are used to generate the aforementioned bivariate normal random variables. (For those proteins with fewer than three spectra, we used estimates of s_C², s_T², and s_CT derived from the average values of these parameters across all proteins.) This process was carried out for each of the proteins (resulting in 5,440,000 and 4,500,000 points for Experiments 1 and 2, respectively), and these points were used to carry out the discriminant analysis. This procedure effectively places greater weight on proteins which are more accurately characterized, i.e. those with smaller variances and covariances.

Fig. 6.

Classifications of different data sets. Selected proteins identified in Experiment I are represented according to their Stage 2 assignments: Black circles, lysosomal; red squares, nonlysosomal; gray diamonds, ambiguous. Dashed line represents points with a predicted posterior probability of 0.5. Top Left Panel, contained in the initial curated lysosomal set. Top Right Panel, not in the initial curated lysosomal set. Middle Left Panel, listed in Table I of Bagshaw et al. (27) Middle Right Panel, listed in Table I of Schröeder et al. (28), Bottom Panels. Bottom Left Panel, proteins assigned to mouse mitochondria with confidence levels of 1–5 (http://www.broadinstitute.org/pubs/MitoCarta/index.html) (29). Bottom Right Panel: listed in the rat and/or mouse pages of the PeroxisomeDB 2.0 (http://www.peroxisomedb.org/) (30).

Fig. 7.

Posterior probabilities of select proteins for assignment as lysosomal. Symbols are as in Fig. 6. Error bars indicate 95% confidence intervals. For clarity of presentation, the mouse gene name is used when the rat and mouse ortholog differ. Mouse/rat substitutions are as follows: NPC1/B2RYH4_RAT, LAMP1/LAMP1_RAT, GNS/Q32KJ5_RAT, HEXB/HEXB_RAT, GUSB/BGLR_RAT.

Fig. 8.

Distribution of lysosomal candidates in control and triton-treated rat liver differential centrifugation fractions. For each plot, area is proportional to total signal. Left Panels, Controls, Right Panels, Triton WR-1339 treated. Ordinate, relative specific activity (percentage of total recovered activity or signal normalized to percentage of total recovered protein). Abscissa, relative protein content of fraction (cumulative from left to right). Fractions are: N, nuclear; M, heavy mitochondrial; L, light mitochondrial; P, microsomal and; S, high speed supernatant. Markers were measured by activity assays whereas ABC6 and SOD1 were measured by quantitative Western blotting analyzing equal amounts of protein (2 and 4 μg) for each fraction.

Fig. 9.

Distribution of ABCB6 and SOD1 in sucrose density gradients of control and triton-treated rat liver ML fractions. Red and black symbols represent analyses of samples from Triton-WR1339-treated and control animals, respectively. The distribution of ABCB6 and SOD1 was determined by quantitative Western blotting analyzing equivalent volume proportions (corresponding to 0.5 and/or 1 mg wet weight liver) for each fraction. Western blotting for SOD1 revealed a single band that migrated with electrophoretic mobility between the 11 and 31 kDa size markers (data not shown), which is consistent with the known mass of the rat protein (∼20 kDa). Western blotting for ABCB6 revealed several bands but the one shown which is shifted in response to Triton-treatment was the only protein migrating between the 59 and 110 kDa size standards (data not shown), consistent with the observed size of rat ABCB6 (∼80 kDa).