Distinct energy metabolism of auditory and vestibular sensory epithelia revealed by quantitative mass spectrometry using MS2 intensity

Abstract

Measuring the abundance of many proteins over a broad dynamic range requires accurate quantitation. We show empirically that, in MS experiments, relative quantitation using summed dissociation-product ion-current intensities is accurate, albeit variable from protein to protein, and outperforms spectral counting. By applying intensities to quantify proteins in two complex but related tissues, chick auditory and vestibular sensory epithelia, we find that glycolytic enzymes are enriched threefold in auditory epithelia, whereas enzymes responsible for oxidative phosphorylation are increased at least fourfold in vestibular epithelia. This striking difference in relative use of the two ATP-production pathways likely reflects the isolation of the auditory epithelium from its blood supply, necessary to prevent heartbeat-induced mechanical disruptions. The global view of protein expression afforded by label-free quantitation with a wide dynamic range reveals molecular specialization at a tissue or cellular level.

Fig. 1.

Quantitation with MS2 intensities. (A) Illustration depicting spectral counts and intensities from two peptides (A1 and B1), each derived from a different protein, one of 10-fold greater abundance. If digest efficiency, chromatography behavior, ionization, and charge are equal, peptide A1 will have 10 times greater MS1 intensity than peptide B1. In MS2 spectra, A1 and B1 are both counted as one spectral count; by contrast, if sampling, collision efficiency, and ion purity are similar, peptide A1 will produce 10 times more total MS2 intensity than peptide B1. CID, collision-induced dissociation. (B and C) Detection of spiked proteins in complex protein mixture. Ten purified proteins (ALB, serum albumin; AMY, α-amylase; CA, carbonic anhydrase; CAT, catalase; LACTA, α-lactalbumin; LGB, β-lactoglobulin; OVAL, ovalbumin; PYG, phosphorylase; TF, transferrin) were added in various mass ratios to an E. coli extract (total protein, 30 μg) and the mixture was subjected to LC-MS/MS. Normalized spectral counts (B) and normalized intensity (C) were calculated for each protein at each dilution. Perfect correspondence between the amount spiked and the normalized detection value gives the unity line (dashed). (D) Relationship between normalized counts and normalized intensity for 1,018 E. coli proteins detected in at least three of 15 runs from the experiment of B and C. Fit is a second-order polynomial (i = 1.3 + 1.8 c + 0.07 c²).

Fig. 2.

Protein abundance in chick auditory and vestibular epithelia. (A–C) Morphology of utricle (A, UTR) and cochlea (B, COCH). Red dashed lines in A and B indicate approximately where tissue peels were taken. SEM image of peeled cochlea preparation (C) shows the tectorial membrane. (D) Reproducibility of MS quantitation. Eight UTR samples were randomized into two groups; normalized intensities were plotted for each detected protein in which SEM/mean is lower than 0.5. No proteins were significantly enriched (SAM analysis; all q-values >75%). (E) Sample-to-sample variation for a given protein varies more widely for the between-tissue (i.e., COCH/UTR) comparison than for the in-tissue (UTR/UTR) comparison. “COCH adj/UTR” indicates distribution after CALB1 and glycolytic enzyme i values were adjusted in cochlea to the utricle levels and all i values were recalculated. μ is midpoint of Gaussian fit to data. (F) Quantitation of individual proteins detected both in cochlea and utricle samples. Normalized intensities after tectorins were removed are plotted for each protein; tectorin α and β (TECTA and TECTB) intensities are also plotted to illustrate their enrichment in cochlea. Dashed line is unity line. Points are colored if SAM analysis indicated significant enrichment and enrichment greater than twofold. Only data with SEM/mean of less than 0.5 are displayed. (G) Relative mass abundances of cochlea proteins. (H) Relative mass -abundances of cochlea proteins with tectorins removed. (I) Relative mass -abundances of utricle proteins. Although not all labels are legible, the data are present in Dataset S4.

Fig. 3.

Comparison of MS and microarray quantitation. (A) Quantitation of individual transcripts detected in cochlea and utricle samples. Points are colored if SAM analysis indicated significant enrichment and enrichment is greater than twofold. Mean ± SEM are plotted. (B) Venn diagram showing overlap of detection by Affymetrix arrays and MS. Affymetrix: 13,078 probesets mapping to 9,422 genes. MS: 1,029 proteins from 1,028 genes, mapping to 1,715 probesets. (C) Distribution of utricle/cochlea ratios for entries detected by Affymetrix microarrays and MS. (D) Correlation between cochlea/utricle rratio measured by Affymetrix array and by MS.

Fig. 4.

Up-regulation of glycolysis and down-regulation of oxidative phosphorylation in auditory sensory epithelium. (A and B) Distribution of peptides detected in cochlea (red) or utricle (blue) for proteins of glycolytic (A) and mitochondrial energy metabolism (B) pathways. Bar height indicates summed intensity for all peptides detected that span the indicated amino acid residues. Dashed lines indicate length of protein in amino acids. (C) Selected microarray cochlea:utricle expression levels. If up-regulation in cochlea or utricle is significant by SAM analysis, the bar is colored red or blue, respectively. Dashed line indicates twofold up-regulation in cochlea. (D) Flowchart of glycolysis, glycogen metabolism, mitochondrial energy production, and mitophagy. Legend indicates up-regulation detected by MS and/or microarray. Genes regulated by HIF1A are indicated.

Fig. 5.

Glycolytic protein expression and rate of glycolysis are increased in auditory sensory epithelium. (A) Protein immunoblot for glycolytic proteins GAPDH and ENO1, mitochondrial proteins MDH2, CYCS, and ATP5A1, and calcium buffer CALB1 as a positive control. (B) Glycolytic rates in utricle, cochlea, and brain measured by [³H]glucose use, normalized to total protein; cochlea-specific activity is significantly different from utricle and brain (two-tailed t test, **P < 0.005 and ****P < 0.0001). (C–J) Immunocytochemical detection of glycolytic and mitochondrial energy metabolism proteins in utricle (C, D, G, and H) and cochlea (E, F, I, and J). For each antibody, merge is on the left, actin counterstain in the middle, and antibody labeling on the right.

Fig. 6.

Relationship of blood vessels to vestibular and auditory sensory epithelia. Nomarski images of chick utricle (A) and cochlea (B). Tectorial membrane has been removed. Arrows indicate direction of mechanical stimulus. Panels are 1,272 μm wide. Chick utricle (C) and cochlea (D) stained with phalloidin (to detect actin) and DAPI (to detect nuclei). Hair cell (HC) and supporting cell (SC) layers are indicated, as is stroma in utricle, and basilar membrane and auditory nerve ganglion (ANG) in cochlea. White asterisks indicate blood vessels. Panels are 317 μm wide.

Fig. P1.

Proposed mechanism for enhancement of glycolysis and suppression of oxidative phosphorylation in cochlea. In the utricle (Left), a ready supply of O₂ presumably degrades hypoxia-inducible factor α (HIF1A), a protein associated with oxygen-independent energy production (i.e., glycolysis), maintaining normal oxygen-involving energy-producing reactions (i.e., oxidative phosphorylation). By contrast, in the cochlea (Right), a bevy of antiangiogenesis factors preventing blood-vessel infiltration are expressed. In response, O₂ levels diminish, and HIF1A is presumably stabilized. In turn, HIF1A increases levels of glycolytic enzymes, thereby increasing glycolysis rates. HIF1A also degrades mitochondria (responsible for oxygen-dependent energy production). Because sound stimuli activate hair cells through vibration of the underlying ECM, the absence of blood vessels in the cochlea prevents disruption of cochlear sensitivity by the heartbeat.