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-to-protein 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 light-sensitive 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.
Highlights
From the ‡Albert Eye Research Institute, 2310 Erwin Road, Durham NC 27710 §Institute for Genome Sciences and Policy, Duke University School of Medicine, Durham, NC 22708
We analyzed the patterns of subcellular protein distribution in photoreceptors by label-free quantitative mass spectrometry
Each peptide identified was allowed to be assigned to a single protein entry, and these assignments were made by ProteinProphet according to the rules of parsimony, and ProteinProphet scores are provided in supplemental Table 1. Both dependent analysis (DDA) and MSE data were searched against the NCBInr database with Rattus norvegicus taxonomy, with full 1ϫ reverse database appended for peptide false discovery rate determination, and duplicates removed using Protein Digest Simulator Basic
Summary
Unbiased quantitative label-free proteomics, utilizing liquid chromatography-tandem MS (LC-MS/MS), is a rapidly emerging methodology allowing comparison of protein contents across multiple samples, when utilizing the accurate-mass and time-tag approach for alignment of peptides across the samples (14 –20). We combined this technique with Western blot detection of individual proteins in sections, which provided an alternative to immunohistochemistry to study longitudinal protein distributions in these cells [26] This method resolved several long-standing controversies regarding the subcellular localization of photoreceptor-specific proteins, which arose from conflicting results of immunohistochemical studies (26 –28). We extended this methodology to analyze distribution profiles of hundreds of proteins by combining serial sectioning of the retina with quantitative mass spectrometry. We used this map to assess the subcellular distribution of all glycolytic enzymes and uncovered novel patterns for the two hexokinase isoforms and triosephosphate isomerase, each likely reflecting the unusually high metabolic activity of photoreceptor cells
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
