ABRF-PRG05: de novo peptide sequence determination.

A common request of proteomics core facilities is protein identification. However, in some instances primary sequence information for the protein in question is not present in public databases. In other cases, the amino acid sequence of a protein may differ in some way from the sequence predicted from the gene sequence in a database as a result of gene mutation, gene splicing, and/or multiple posttranslational modifications. Thus, it may be necessary to determine the sequence of one or more peptides de novo in order to identify and/or adequately characterize the protein of interest. The primary goal of this study was to give participating laboratories an opportunity to evaluate their proficiency in sequencing unknown peptides that are not included in any published database. Samples containing 3-6 pmol each of five synthetic peptides with amino acid sequences that were not present in public databases were sent to 106 laboratories. One nonstandard amino acid was present in one of the peptides. From a comparison of the results obtained by different strategies, participating laboratories will be able to gauge their own capabilities and establish realistic expectations for the approaches that can be used for this determination.

ABRF-PRG04: differentiation of protein isoforms.

Accurate protein identification sometimes requires careful discrimination between closely related protein isoforms that may differ by as little as a single amino acid substitution or post-translational modification. The ABRF Proteomics Research Group sent a mixture of three picomoles each of three closely related proteins to laboratories who requested it in the form of intact proteins, and participating laboratories were asked to identify the proteins and report their results. The primary goal of the ABRF-PRG04 Study was to give participating laboratories a chance to evaluate their capabilities and practices with regards to sample fractionation (1D- or 2D-PAGE, HPLC, or none), protein digestion methods (in-solution, in-gel, enzyme choice), and approaches to protein identification (instrumentation, use of software, and/or manual techniques to facilitate interpretation), as well as determination of amino acid or post-translational modifications. Of the 42 laboratories that responded, 8 (19%) correctly identified all three isoforms and N-terminal acetylation of each, 16 (38%) labs correctly identified two isoforms, 9 (21%) correctly identified two isoforms but also made at least one incorrect identification, and 9 (21%) made no correct protein identifications. All but one lab used mass spectrometry, and data submitted enabled a comparison of strategies and methods used.

Proteomic characterization of isolated retinal pigment epithelium microvilli.

Polarized epithelial cells are characterized by displaying compartmentalized functions associated with differential distribution of transporters, structural proteins, and signaling molecules on their apical and basolateral surfaces. Their apical surfaces frequently elaborate microvilli, which vary in structure according to the specific type and function of each epithelium. The molecular basis of this heterogeneity is poorly understood. However, differences in function will undoubtedly be reflected in the specific molecular composition of the apical surface in each epithelial subtype. We have exploited a method for isolating microvilli from the mouse eye using wheat germ agglutinin (WGA)-agarose beads to begin to understand the specific molecular composition of apical microvilli of the retinal pigment epithelium (RPE) and expand our knowledge of the potential function of this interface. Initially, apical RPE plasma membranes bound to WGA beads were processed for morphological analysis using known apical and basolateral surface markers. The protein composition of the apical microvilli was then established using proteomic analysis. Over 200 proteins were identified, including a number of proteins previously known to be localized to RPE microvilli, as well as others not known to be present at this surface. Localization of novel proteins identified with proteomics was confirmed by immunohistochemistry in both mouse and rat eye tissue. The data generated provides new information on the protein composition of the RPE apical microvilli. The isolation technique used should be amenable for isolating microvilli in other epithelia as well, allowing new insights into additional functions of this important epithelial compartment.

Support for a proposed retinoid-processing protein complex in apical retinal pigment epithelium.

The interaction of cellular retinaldehyde-binding protein (CRALBP) with ERM (ezrin, radixin, moesin)-binding phosphoprotein 50 (EBP50) in retinal pigment epithelium (RPE) microsomes has led to the hypothesis that a retinoid-processing protein complex exists in apical RPE. Mouse RPE apical processes were isolated on wheat germ agglutinin-coated agarose beads. Proteomic analyses of the isolated apical RPE demonstrated the presence of CRALBP, EBP50, 11-cis-retinol dehydrogenase, cellular retinol-binding protein 1, and interphotoreceptor retinoid-binding protein. The results support the hypothesis that a visual cycle protein complex may serve in the localization and release of 11-cis-retinoid in the apical RPE.

Protein database, human retinal pigment epithelium.

The retinal pigment epithelium (RPE) is a single cell layer adjacent to the rod and cone photoreceptors that plays key roles in retinal physiology and the biochemistry of vision. RPE cells were isolated from normal adult human donor eyes, subcellular fractions were prepared, and proteins were fractionated by electrophoresis. Following in-gel proteolysis, proteins were identified by peptide sequencing using liquid chromatography tandem electrospray mass spectrometry and/or by peptide mass mapping using matrix-assisted laser desorption ionization time-of-flight mass spectrometry. Preliminary analyses have identified 278 proteins and provide a starting point for building a database of the human RPE proteome.

Disease-causing mutations in the cellular retinaldehyde binding protein tighten and abolish ligand interactions.

Mutations in the human cellular retinaldehyde binding protein (CRALBP) gene cause retinal pathology. To understand the molecular basis of impaired CRALBP function, we have characterized human recombinant CRALBP containing the disease causing mutations R233W or M225K. Protein structures were verified by amino acid analysis and mass spectrometry, retinoid binding properties were evaluated by UV-visible and fluorescence spectroscopy and substrate carrier functions were assayed for recombinant 11-cis-retinol dehydrogenase (rRDH5). The M225K mutant was less soluble than the R233W mutant and lacked retinoid binding capability and substrate carrier function. In contrast, the R233W mutant exhibited solubility comparable to wild type rCRALBP and bound stoichiometric amounts of 11-cis- and 9-cis-retinal with at least 2-fold higher affinity than wild type rCRALBP. Holo-R233W significantly decreased the apparent affinity of rRDH5 for 11-cis-retinoid relative to wild type rCRALBP. Analyses by heteronuclear single quantum correlation NMR demonstrated that the R233W protein exhibits a different conformation than wild type rCRALBP, including a different retinoid-binding pocket conformation. The R233W mutant also undergoes less extensive structural changes upon photoisomerization of bound ligand, suggesting a more constrained structure than that of the wild type protein. Overall, the results show that the M225K mutation abolishes and the R233W mutation tightens retinoid binding and both impair CRALBP function in the visual cycle as an 11-cis-retinol acceptor and as a substrate carrier.

Drusen proteome analysis: an approach to the etiology of age-related macular degeneration.

Drusen are extracellular deposits that accumulate below the retinal pigment epithelium on Bruch's membrane and are risk factors for developing age-related macular degeneration (AMD). The progression of AMD might be slowed or halted if the formation of drusen could be modulated. To work toward a molecular understanding of drusen formation, we have developed a method for isolating microgram quantities of drusen and Bruch's membrane for proteome analysis. Liquid chromatography tandem MS analyses of drusen preparations from 18 normal donors and five AMD donors identified 129 proteins. Immunocytochemical studies have thus far localized approximately 16% of these proteins in drusen. Tissue metalloproteinase inhibitor 3, clusterin, vitronectin, and serum albumin were the most common proteins observed in normal donor drusen whereas crystallin was detected more frequently in AMD donor drusen. Up to 65% of the proteins identified were found in drusen from both AMD and normal donors. However, oxidative protein modifications were also observed, including apparent crosslinked species of tissue metalloproteinase inhibitor 3 and vitronectin, and carboxyethyl pyrrole protein adducts. Carboxyethyl pyrrole adducts are uniquely generated from the oxidation of docosahexaenoate-containing lipids. By Western analysis they were found to be more abundant in AMD than in normal Bruch's membrane and were found associated with drusen proteins. Carboxymethyl lysine, another oxidative modification, was also detected in drusen. These data strongly support the hypothesis that oxidative injury contributes to the pathogenesis of AMD and suggest that oxidative protein modifications may have a critical role in drusen formation.

Evidence that light modulates protein nitration in rat retina.

As part of ongoing efforts to better understand the role of protein oxidative modifications in retinal pathology, protein nitration in retina has been compared between rats exposed to damaging light or maintained in the dark. In the course of the research, Western methodology for detecting nitrotyrosine-containing proteins has been improved by incorporating chemical reduction of nitrotyrosine to aminotyrosine, allowing specific and nonspecific nitrotyrosine immunoreactivity to be distinguished. A liquid chromatography MS/MS detection strategy was used that selects all possible nitrotyrosine peptides for MS/MS based on knowing the protein identity. Quantitative liquid chromatography MS/MS analyses with tetranitromethane-modified albumin demonstrated the approach capable of identifying sites of tyrosine nitration with detection limits of 4-33 fmol. Using two-dimensional gel electrophoresis, Western detection, and mass spectrometric analyses, several different nitrotyrosine-immunoreactive proteins were identified in light-exposed rat retina compared with those maintained in the dark. Immunocytochemical analyses of retina revealed that rats reared in darkness exhibited more nitrotyrosine immunoreactivity in the photoreceptor outer segments. After intense light exposure, immunoreactivity decreased in the outer segments and increased in the photoreceptor inner segments and retinal pigment epithelium. These results suggest that light modulates retinal protein nitration in vivo and that nitration may participate in the biochemical sequela leading to light-induced photoreceptor cell death. Furthermore, the identification of nitrotyrosine-containing proteins from rats maintained in the dark, under non-pathological conditions, provides the first evidence of a possible role for protein nitration in normal retinal physiology.