Proteomic Profiling of a Layered Tissue Reveals Unique Glycolytic Specializations of Photoreceptor Cells

Abstract

The retina is a highly ordered tissue whose outermost layers are formed by subcellular compartments of photoreceptors generating light-evoked electrical responses. We studied protein distributions among individual photoreceptor compartments by separating the entire photoreceptor layer of a flat-mounted frozen retina into a series of thin tangential cryosections and analyzing protein compositions of each section by label-free quantitative mass spectrometry. Based on 5038 confidently identified peptides assigned to 896 protein database entries, we generated a quantitative proteomic database (a "map") correlating the distribution profiles of identified proteins with the profiles of marker proteins representing individual compartments of photoreceptors and adjacent cells. We evaluated the applicability of several common peptide-toprotein quantification algorithms in the context of our database and found that the highest reliability was obtained by summing the intensities of all peptides representing a given protein, using at least the 5-6 most intense peptides when applicable. We used this proteome map to investigate the distribution of glycolytic enzymes, critical in fulfilling the extremely high metabolic demands of photoreceptor cells, and obtained two major findings. First, unlike the majority of neurons rich in hexokinase I, but similar to other highly metabolically active cells, photoreceptors express hexokinase II. Hexokinase II has a very high catalytic activity when associated with mitochondria, and indeed we found it colocalized with mitochondria in photoreceptors. Second, photoreceptors contain very little triosephosphate isomerase, an enzyme converting dihydroxyacetone phosphate into glyceraldehyde-3-phosphate. This may serve as a functional adaptation because dihydroxyacetone phosphate is a major precursor in phospholipid biosynthesis, a process particularly active in photoreceptors because of the constant renewal of their lightsensitive membrane disc stacks. Overall, our approach for proteomic profiling of very small tissue amounts at a resolution of a few microns, combining cryosectioning and liquid chromatography-tandem MS, can be applied for quantitative investigation of proteomes where spatial resolution is paramount.

Pyruvate kinase and aspartate-glutamate carrier distributions reveal key metabolic links between neurons and glia in retina

“…1B). Consistent with this finding, no PKLR was found in a proteomic analysis of rat retina (19). PKM1 is in brain, muscle, and retina, whereas PKM2 is most abundant in retina (Fig.…”

Mitochondria Contribute to NADPH Generation in Mouse Rod Photoreceptors

“…It had been suggested (27) that the pentose phosphate pathway in the rod outer segment could potentially supply all the NADPH reducing equivalents necessary for the reduction of all-trans-retinal to retinol. More recent studies have shown that glucose enters through transporter Glut-1 into the inner segment (28), where it is phosphorylated to glucose-6-phosphate by hexokinases I and II (29). Glucose-6-phosphate can then be utilized through the pentose phosphate pathway to generate NADPH from NADP ϩ .…”

Comprehensive Proteomic and Metabolomic Signatures of Nontypeable Haemophilus influenzae-Induced Acute Otitis Media Reveal Bacterial Aerobic Respiration in an Immunosuppressed Environment

“…This method provides a more precise measure of protein relative abundance, because all peptides available are used (40). Intensities of each protein identified were averaged within each treatment group, and fold-changes calculated between groups.…”