Extended reading frame of a ubiquitin gene encodes a stable, conserved, basic protein

Antibodies specific for the 80-amino acid hypothetical protein encoded by the in-frame, 3'-extension of a human ubiquitin gene were produced in rabbits by immunization with a 14-residue synthetic peptide. When used to probe HeLa cell extracts for the non-ubiquitin product of this natural fusion gene, the antipeptide sera detected a protein with an apparent molecular weight of 16,000 Da by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. An immunoreactive protein of identical mobility was detected in organisms ranging from Acanthamoeba to man, indicating that the extension protein, like ubiquitin, is highly conserved. The immunoreactive protein was isolated from calf thymus, and direct sequencing revealed the first 16 amino acids to be identical to those predicted from the extension portion of the human cDNA. Thus, ubiquitin was no longer present at the amino terminus. The purified bovine extension protein failed to react with a ubiquitin-specific antibody indicating the absence of isopeptide-linked ubiquitin as well. Moreover, by denaturing gel permeation chromatography the extension has a molecular weight of 10,000 Da, a value that corresponds more closely to the size of the extension alone (9,000 Da) than to the intact fusion protein (17,500 Da). The extension protein, which was found in both cytoplasmic and nuclear fractions of HeLa cells, persisted at high levels when protein synthesis was blocked with cycloheximide or puromycin. These results show that the 80-residue extension protein is the stable, processed product of the ubiquitin fusion gene.     

The apparent molecular size of native alpha-crystallin B in non-lenticular tissues

The apparent molecular size of the native alpha-crystallin B in cytosol preparations from rat heart, brain and retina was determined by gel permeation chromatography, detecting the protein by immunochemical assay (ELISA), using an alpha-crystallin specific antiserum. Native alpha-crystallin from cytosol preparations of rat lens cortex was used as a reference. alpha-Crystallin B present in all three cytosol preparations from non-lenticular tissues eluted in a single symmetrical peak, with the same elution volume as alpha-crystallin from lens cortex cytosol preparations, corresponding to an apparent average molecular size of 0.8 x 10(6) Da. No other species could be detected. The results indicate that the alpha-crystallin aggregates characterized by an apparent average molecular mass of 0.8 x 10(6) Da, and considered to be the native, physiological form of the protein in the lens, are indeed not specific to lens tissue. Furthermore, the size of these alpha-crystallin aggregates is independent of their polypeptide composition. Aggregates found in the lens, composed of alpha A and alpha B polypeptides and their respective phosphorylated forms alpha Ap and alpha Bp, are similar in size to those found in heart, brain and retina, containing the alpha B but not the alpha A polypeptide.     

The eye lens has an active ubiquitin-protein conjugation system

Using exogenous 125I-ubiquitin, ubiquitin-lens protein conjugation was observed with supernatants of cultured rabbit lens epithelial cells and lens cortex tissue. Conjugation was ATP-dependent with the greatest variety and amount of conjugates larger than 150 kDa. In vivo production of ubiquitin-protein conjugates in cultured rabbit and beef lens epithelial cells and rabbit lens tissues of different developmental age was established using immunological detection. There were limited similarities between conjugates found in youngest as opposed to oldest tissue. Cultured rabbit cells contained 27 pmol/mg free ubiquitin and 18 pmol/mg conjugated ubiquitin. Levels of free ubiquitin in lens tissue epithelium, cortex, and core were 36, 5, and 5 pmol/mg, respectively. There were only 2 pmol/mg conjugated ubiquitin in each of these tissues. Hydrolysis of 125I-ubiquitin was catalyzed by supernatants of cultured lens cells, beef and human lens tissues, and reticulocytes. Degradation was greatest in epithelial tissues, and least in core. This corroborates studies which show that proteolytic capabilities are attenuated in older tissue. Decreased initiation of proteolysis by ubiquitination as well as diminished proteolysis in older lens tissue may be related to the accumulation of damaged proteins in aging lens tissue.     

Frameshift events associated with the lysyl-tRNA and the rare arginine codon, AGA, in Escherichia coli: a case study involving the human Relaxin 2 protein

Human Relaxin 2 is an insulin-related peptide hormone with a mass of 19,084 Da. The mRNA contains a number of arginine codons that are rarely used by Escherichia coli to produce highly expressed proteins. As a result, expressing this recombinant protein in E. coli is problematic. When human Relaxin 2 was expressed in E. coli BL21 (DE3), several forms of the protein were made. One species had the expected molecular weight (19,084 Da). A second species observed had a molecular weight of 21,244 Da. A third minor species had a molecular weight of 17,118 Da. These aberrant molecular weights can be explained as follows. First, a sequence CGA-AAA-AAG-AGA, containing the rare arginine codons CGA and AGA was the site of the +1 frameshift that generated the 21,244 Da species. Since there was a limited supply of this arginyl-tRNA, the peptidyl-tRNA moved +1 nucleotide to occupy the codon and resumed protein synthesis. Second, a -1 frameshift associated with 'slippery A' sequence XXA-AAA-AAG accounted for 10% of the product with a mass of 17,118 Da. Presumably, the shift to -1 also occurred because there was a paucity of the arginyl-tRNAArgucu. Introduction of a plasmid coding for the cognate tRNA for AGA and site directed mutagenesis prevented the formation of both frameshift species.     

Fe2+ oxidation of alpha-crystallin produces a 43,000 Da aggregate composed of A and B chains cross-linked by non-reducible covalent bonds

The structure of a 43,000 Da aggregate generated from bovine lens alpha-crystallin polypeptides of 20,000 Da using Fe2+ catalyzed oxidation, was studied by sequence analysis of a 30,000 Da proteolytic fragment. Three polypeptide components were simultaneously sequenced in the electroblotted 30,000 Da fragment, corresponding to Phe114-Ser130... of the alpha A chain, and His 111-Ser135... and Ser 35-Leu49... of the alpha B chain. The relative proportions of the components suggests that the three polypeptides are present in equimolar amounts. It is concluded that the 30,000 Da fragment and, therefore, the 43,000 Da aggregate is constructed of both the A and B polypeptide chains covalently cross-linked with non-reducible bonds. At least one of these cross-links is present towards the carboxy-terminus of the A and B chains after Phe114 and His111, respectively.     

LC-MS display of the total modified amino acids in cataract lens proteins and in lens proteins glycated by ascorbic acid in vitro

We previously reported chromatographic evidence supporting the similarity of yellow chromophores isolated from aged human lens proteins, early brunescent cataract lens proteins and calf lens proteins ascorbylated in vitro [Cheng, R. et al. Biochimica et Biophysica Acta 1537, 14-26, 2001]. In this paper, new evidence supporting the chemical identity of the modified amino acids in these protein populations were collected by using a newly developed two-dimensional LC-MS mapping technique supported by tandem mass analysis of the major species. The pooled water-insoluble proteins from aged normal human lenses, early stage brunescent cataract lenses and calf lens proteins reacted with or without 20 mM ascorbic acid in air for 4 weeks were digested with a battery of proteolytic enzymes under argon to release the modified amino acids. Aliquots equivalent to 2.0 g of digested protein were subjected to size-exclusion chromatography on a Bio-Gel P-2 column and four major A330nm-absorbing peaks were collected. Peaks 1, 2 and 3, which contained most of the modified amino acids were concentrated and subjected to RP-HPLC/ESI-MS, and the mass elution maps were determined. The samples were again analyzed and those peaks with a 10(4) - 10(6) response factor were subjected to MS/MS analysis to identify the daughter ions of each modification. Mass spectrometric maps of peaks 1, 2 and 3 from cataract lenses showed 58, 40 and 55 mass values, respectively, ranging from 150 to 600 Da. Similar analyses of the peaks from digests of the ascorbylated calf lens proteins gave 81, 70 and 67 mass values, respectively, of which 100 were identical to the peaks in the cataract lens proteins. A total of 40 of the major species from each digest were analyzed by LC-MS/MS and 36 were shown to be identical. Calf lens proteins incubated without ascorbic acid showed several similar mass values, but the response factors were 100 to 1000-fold less for every modification. Based upon these data, we conclude that the majority of the major modified amino acids present in early stage brunescent Indian cataract lens proteins appear to arise as a result of ascorbic acid modification, and are presumably advanced glycation end-products.     

Essential factors determining codon usage in ubiquitin genes

Summary Ubiquitin is ubiquitous in all eukaryotes and its amino acid sequence shows extreme conservation. Ubiquitin genes comprise direct repeats of the ubiquitin coding unit with no spacers. The nucleotide sequences coding for 13 ubiquitin genes from 11 species reported so far have been compiled and analyzed. The G+C content of codon third base reveals a positive linear correlation with the genome G+C content of the corresponding species. The slope strongly suggests that the overall G+C content of codons of polyubiquitin genes clearly reflects the genome G+C content by AT/GC substitutions at the codon third position. The G+C content of ubiquitin codon third base also shows a positive linear correlation with the overall G+C content of coding regions of compiled genes, indicating the codon choices among synonymous codons reflect the average codon usage pattern of corresponding species. On the other hand, the monoubiquitin gene, which is different from the polyubiquitin gene in gene organization, gene expression, and function of the encoding protein, shows a different codon usage pattern compared with that of the polyubiquitin gene. From comparisons of the levels of synonymous substitutions among ubiquitin repeats and the homology of the amino acid sequence of the tail of monomeric ubiquitin genes, we propose that the molecular evolution of ubiquitin genes occurred as follows: Plural primitive ubiquitin sequences were dispersed on genome in ancestral eukaryotes. Some of them situated in a particular environment fused with the tail sequence to produce monomeric ubiquitin genes that were maintained across species. After divergence of species, polyubiquitin genes were formed by duplication of the other primitive ubiquitin sequences on different chromosomes. Differences in the environments in which ubiquitin genes are embedded reflect the differences in codon choice and in gene expression pattern between poly- and monomeric ubiquitin genes.     

High-level expression of the recombinant hybrid peptide cecropinA(1-8)-magainin2(1-12) with an ubiquitin fusion partner in Escherichia coli

The hybrid antibacterial peptide CA-MA [cecropinA(1-8)-magainin2(1-12)] with potent antimicrobial properties but no hemolytic activity is a potential alternative antibiotic. To explore a new approach for high-level expression of the hybrid peptide CA-MA in Escherichia coli, the sequence of ubiquitin (UBI) from housefly was inserted into the plasmid pQE30 to construct the vector pQEUBI. The cDNA fragment encoding CA-MA with preferred codons of E. coli was obtained by recursive PCR (rPCR) and cloned into the vector pQEUBI to express the fusion protein (His)(6)-UBI-CA-MA. The fusion protein was expressed in soluble form under the optimized conditions at high level (more than 36% of the total proteins). With (His)(6)-tag, the fusion protein was easily purified by Ni-NTA chromatography and 36 mg of fusion protein was purified from 1L of culture medium. The fusion protein was efficiently cleaved by ubiquitin C-terminal hydrolase (UCH), yielding recombinant CA-MA with high antimicrobial activity. After removing the contaminants by Ni-NTA chromatography, recombinant CA-MA was purified to homogeneity by reversed-phase HPLC and 6.8mg of pure active CA-MA was obtained from 1L culture medium. Analysis of recombinant CA-MA by MALDI-TOF-MS showed that the molecular weight of the purified recombinant CA-MA was 2559Da, which perfectly matches the mass (2559Da) calculated from the amino acid sequence. Analysis of CA-MA by circular dichroism (CD) revealed that the secondary structures of CA-MA in water solution were 17.4% alpha-helix and 82.6% random coil but no beta-sheet. Our results demonstrated that functional CA-MA can be produced in sufficient quantities using the ubiquitin fusion technique. This is the first report on the heterologous expression of a hybrid antibacterial peptide fused to ubiquitin in E. coli.     

Protein standard absolute quantification (PSAQ) method for the measurement of cellular ubiquitin pools

The protein ubiquitin is an important post-translational modifier that regulates a wide variety of biological processes. In cells, ubiquitin is apportioned among distinct pools, which include a variety of free and conjugated species. Although maintenance of a dynamic and complex equilibrium among ubiquitin pools is crucial for cell survival, the tools necessary to quantify each cellular ubiquitin pool have been limited. We have developed a quantitative mass spectrometry approach to measure cellular concentrations of ubiquitin species using isotope-labeled protein standards and applied it to characterize ubiquitin pools in cells and tissues. Our method is convenient, adaptable and should be a valuable tool to facilitate our understanding of this important signaling molecule.     

Age-related changes in human crystallins determined from comparative analysis of post-translational modifications in young and aged lens: does deamidation contribute to crystallin insolubility?

We have employed recently developed blind modification search techniques to generate the most comprehensive map of post-translational modifications (PTMs) in human lens constructed to date. Three aged lenses, two of which had moderate cataract, and one young control lens were analyzed using multidimensional liquid chromatography mass spectrometry. In total, 491 modification sites in lens proteins were identified. There were 155 in vivo PTM sites in crystallins: 77 previously reported sites and 78 newly detected PTM sites. Several of these sites had modifications previously undetected by mass spectrometry in lens including carboxymethyl lysine (+58 Da), carboxyethyl lysine (+72 Da), and an arginine modification of +55 Da with yet unknown chemical structure. These new modifications were observed in all three aged lenses but were not found in the young lens. Several new sites of cysteine methylation were identified indicating this modification is more extensive in lens than previously thought. The results were used to estimate the extent of modification at specific sites by spectral counting. We tested the long-standing hypothesis that PTMs contribute to age-related loss of crystallin solubility by comparing spectral counts between the water-soluble and water-insoluble fractions of the aged lenses and found that the extent of deamidation was significantly increased in the water-insoluble fractions. On the basis of spectral counting, the most abundant PTMs in aged lenses were deamidations and methylated cysteines with other PTMs present at lower levels.     

Aging and cellular maturation cause changes in ubiquitin-eye lens protein conjugates

The eye lens is a useful tissue for studying phenomena related to aging since it can be separated into differentially aged or matured zones. This work establishes correlations between ubiquitin-lens protein conjugating capabilities and age, as well as the stage of maturation of bovine lens tissue. When exogenous 125I-ubiquitin was combined with supernatants of epithelial (least mature), cortex, and core (most mature) tissue, ATP-dependent conjugation of 125I-ubiquitin to lens proteins was most effective with the epithelial tissue preparation. Conjugate formation was greatest when lenses were obtained from young animals. Supernatants from cultured bovine lens epithelial (BLE) cells conjugated more 125I-ubiquitin to lens proteins than any tissue preparation. In all cases the predominant conjugates formed in these cell-free assays were of high molecular mass, although conjugates with masses in the 25-70 kDa range were also observed. Lens tissue and cultured BLE cell preparations were also probed with antibodies to ubiquitin to detect in vivo ubiquitin-lens protein conjugates. There was more free ubiquitin and ubiquitin conjugates in tissue from young as compared with older lenses. The greatest levels of conjugates were observed in cultured BLE cells. Specificity in the ubiquitination system is indicated since some of the conjugates formed in vivo appear identical to those formed in the cell-free assays and in reticulocytes using exogenous 125I-ubiquitin. Upon development and maturation of lens tissue (i.e., core as opposed to epithelium), there is accumulation of lower molecular mass conjugates.     

Mass spectrometric determination of a novel modification of the N-terminus of histidine-tagged proteins expressed in bacteria.

Two proteins, FKBP, and Spo0F, were expressed in bacteria as histidine-tagged fusion proteins and isolated under native conditions. MALDI-TOF-MS analysis revealed that each protein preparation contained two components, neither of which corresponded to the molecular weights predicted from DNA sequences. The difference in molecular weight between the two FKBP components and two Spo0F components was approximately 178 +/- 14 Da. Site-specific proteolytic cleavage resulted in the release of histidine-tagged peptide from the recombinant proteins. MALDI mass spectra of the cleaved proteins showed a single molecular ion peak for each species with the predicted molecular weights. The histidine-tagged peptide released from both fusion proteins displayed two distinct peaks by MALDI-FT-MS corresponding to monoisotopic molecular weights of 2269. 027 Da and 2447.087 Da, respectively, which were both inconsistent with the predicted peptide sequence M-G-H-H-H-H-H-H-H-H-H-H-S-S-G-H-I-E-G-R of 2400.055 Da. The peptide at 2269.027 Da was sequenced by ESI-MS-MS and found to be a truncated histidine tag resulting from an initiator methionine deletion. ESI-MS-MS analysis of the peptide at 2447.087 Da indicated a moiety of 178.0 Da attached to the second residue glycine of the histidine tag. This alteration of the N-terminus does not fit any known modifications. A synthetic peptide with the identical sequence of the isolated his-tag M-G-H-H-H-H-H-H-H-H-H-H remained unmodified during the protein purification process, suggesting that modification of the initiator methionine was carried out in vivo, rather than the result of a chemical reaction from the isolation procedure.     

Physicochemical characterization and phylogenetic comparison of fish lens proteins.

The composition of soluble eye lens proteins from four chondropterygiian and fourteen teleostean fishes were analyzed for heterogeneity in MW and pI. Lens proteins from all the fish species studies are distributed in the pI range 4.3-9.0 with polypeptides in the range 17,500-31,000 Da. Phylogenetic trees are constructed based on the observations.     

Characterisation of sphingolipids in the human lens by thin layer chromatography–desorption electrospray ionisation mass spectrometry

The lipidome of the human lens is unique in that cholesterol and dihydrosphingomyelin are the dominant classes. Moreover, the lens lipidome is not static with dramatic changes in several sphingolipid classes associated with both aging and cataract. Accordingly, there is a clear need to expand knowledge of the molecular species that constitute the human lens sphingolipidome. In this study, human lens lipids have been extracted and separated by thin-layer chromatography (TLC). Direct analysis of the TLC plates by desorption electrospray ionisation–mass spectrometry (DESI–MS) allowed the detection over 30 species from 11 classes of sphingolipids. Significantly, novel classes of lens lipids including sulfatides, dihydrosulfatides, lactosylceramide sulfates and dihydrolactosylceramide sulfates were identified.     

Evidence for independent recruitment of zeta-crystallin/quinone reductase (CRYZ) as a crystallin in camelids and hystricomorph rodents

Zeta-crystallin/quinone reductase (CRYZ) is an NADPH oxidoreductase expressed at very high levels in the lenses of two groups of mammals: camelids and some hystricomorph rodents. It is also expressed at very low levels in all other species tested. Comparative analysis of the mechanisms mediating the high expression of this enzyme/crystallin in the lens of the Ilama (Lama guanacoe) and the guinea pig (Cavia porcellus) provided evidence for independent recruitment of this enzyme as a lens crystallin in both species and allowed us to elucidate for the first time the mechanism of lens recruitment of an enzyme- crystallin. The data presented here show that in both species such recruitment most likely occurred through the generation of new lens promoters from nonfunctional intron sequences by the accumulation of point mutations and/or small deletions and insertions. These results further support the idea that recruitment of CRYZ resulted from an adaptive process in which the high expression of CRYZ in the lens provides some selective advantage rather than from a purely neutral evolutionary process.      

Ubiquitin in homeostasis,development and disease

Ubiquitin is the most phylogenetically conserved protein known. This 8,500 Da polypeptide can be covalently attached to cellular proteins as a posttranslational modification. In most cases, the addition of multiple ubiquitin adducts to a protein targets it for rapid degradation by a multisubunit protease known as the 26S proteasome. While the ubiquitin/26S proteasome pathway is responsible for the degradation of the bulk of cellular proteins during homeostasis, it may also be responsible for the rapid loss of protein during the programmed death of certain cells, such as skeletal muscle during insect metamorphosis. In addition, alterations in the expression and regulation of ubiquitin may play significant roles in pathological disorders. For example, dramatic increases in ubiquitin and ubiquitin-protein conjugates are observed in a wide variety of neurodegenerative disorders, including Alzheimer's disease. Patients suffering from the autoimmune disease systemic lupus erythematosus generate antibodies reacting with ubiquitin and ubiquitinated histones. At present, it is not known whether these changes in ubiquitin expression and regulation initiate pathological changes in these diseases or if they are altered as a consequence of these disorders.     

Structural characterization of adrenal chromogranin A and parathyroid secretory protein-I as homologs

We have isolated and purified adrenal chromogranin A (Ch A) for the purpose of making structural comparisons to parathyroid secretory protein-I (SP-I), because our earlier data indicated these two molecules may be the same protein. An improved purification step, using high-performance liquid chromatography (HPLC), has enabled us to demonstrate that both SP-I and Ch A consists of two species, one of approximately 72,000 Da and one of approximately 66,000 Da. The amino acid composition is the same for all four species. The difference in molecular mass is assumed to be due to carbohydrate content. Cyanogen bromide digestion of each of the four samples, followed by HPLC separation of the generated peptides, resulted in a chromatographic profile that was the same for each digest. Amino acid analysis of the eight peptide fragments obtained from each digest indicates that both species of Ch A and both species of SP-I yielded the same peptide mixtures following this cleavage reaction. One large (approximately 50,000 Da) CNBr peptide was obtained and seven smaller ones, one of which contains cysteine. The large fragment behaved similarly to the intact molecule in a radioimmunoassay. HPLC separation of tryptic digests of Ch A (72,000 Da) and SP-I (72,000 Da) also resulted in elution profiles that were very similar to each other. Amino acid analysis revealed 23 peptides common to each digest. Ch A contained four peptides ranging in size from 4 to 30 residues that were not observed in the SP-I digest. SP-I contained two peptides, each with about 30 residues, that were not found in the Ch A digest. Nothing unusual was noted in any of the uncommon peptides. Thus, both a chemical and an enzymatic digestion of these molecules followed by analysis of the peptides generated, indicates that SP-I and Ch A are nearly identical homologs.     

Molecular aging of lens crystallins and the life expectancy of the animal. Age-related protein structural changes studied in situ by Raman spectroscopy.

In order to investigate the relationship of molecular aging of lens crystallins to an animal's life expectancy or to the type of the lens, Raman spectra have been measured in situ for rabbit and guinea-pig lens nuclei at various stages of aging; these spectra have been compared with those of rat and mouse lens nuclei previously reported. Lens aging results in pronounced differences among the Raman spectra of the lens nuclei of the four species. It is shown that the rates of dehydration, inter- and intramolecular disulfide bond formation, and microenvironmental changes in the tryptophan residues of lens crystallins are different among the four species. Much faster changes occur for rat and mouse, which have a shorter life expectancy (2 years) and give rise to hard lens nuclei while slower changes occur for rabbit and guinea-pig, which have a longer life expectancy (5-7 years), and give soft lens nuclei. In addition, the Raman data reveal, for all the species investigated, that there are correlations among the rates of the dehydration, the inter- and intramolecular disulfide bond formation, and the microenvironmental changes in the tryptophan residues. Therefore, there seems to be a common mechanism for molecular aging of lens crystallins among the four species, although the rate of the molecular aging strongly depends upon the life expectancy of the animal and the type of the lens. The most important factor determining the rate of the molecular aging is probably the dehydration which decreases free water in the lens nucleus.     

Glycation by ascorbic acid oxidation products leads to the aggregation of lens proteins

Previous studies from this laboratory have shown that there are striking similarities between the yellow chromophores, fluorophores and modified amino acids released by proteolytic digestion from calf lens proteins ascorbylated in vitro and their counterparts isolated from aged and cataractous lens proteins. The studies reported in this communication were conducted to further investigate whether ascorbic acid-mediated modification of lens proteins could lead to the formation of lens protein aggregates capable of scattering visible light, similar to the high molecular aggregates found in aged human lenses. Ascorbic acid, but not glucose, fructose, ribose or erythrulose, caused the aggregation of calf lens proteins to proteins ranging from 2.2 x 10(6) up to 3.0 x 10(8 )Da. This compared to proteins ranging from 1.8 x 10(6) up to 3.6 x 10(8 )Da for the water-soluble (WS) proteins isolated from aged human lenses. This aggregation was likely due to the glycation of lens crystallins because [U-(14)C] ascorbate was incorporated into the aggregate fraction and because NaCNBH(3), which reduces the initial Schiff base, prevented any protein aggregation. Reactions of ascorbate with purified crystallin fractions showed little or no aggregation of alpha-crystallin, significant aggregation of beta(H)-crystallin, but rapid precipitation of purified beta(L)- and gamma-crystallin. The aggregation of lens proteins can be prevented by the binding of damaged crystallins to alpha-crystallin due to its chaperone activity. Depending upon the ratios between the components of the incubation mixtures, alpha-crystallin prevented the precipitation of the purified beta(L)- and gamma-crystallin fractions during ascorbylation. The addition of at least 20% of alpha-crystallin by weight into glycation mixtures with beta(L)-, or gamma-crystallins completely inhibited protein precipitation, and increased the amount of the high molecular weight aggregates in solution. Static and dynamic light scattering measurements of the supernatants from the ascorbic acid-modified mixtures of alpha- and beta(L)-, or gamma-crystallins showed similar molar masses (up to 10(8 )Da) and hydrodynamic diameter (up to 80( )nm). These data support the hypothesis, that if the lens reducing environment is compromised, the ascorbylation of lens crystallins can significantly change the short range interactions between different classes of crystallins leading to protein aggregation, light scattering and eventually to senile cataract formation.     

Tryptic digestion of ubiquitin standards reveals an improved strategy for identifying ubiquitinated proteins by mass spectrometry

Ubiquitination plays an essential role in maintaining cellular homeostasis by regulating a multitude of essential processes. The ability to identify ubiquitinated proteins by MS currently relies on a strategy in which ubiquitinated peptides are identified by a 114.1 Da diglycine (GG) tag on lysine residues, which is derived from the C-terminus of ubiquitin, following trypsin digestion. In the following study, we report a more comprehensive approach for mapping ubiquitination sites by trypsin digestion and MS/MS analysis. We demonstrate that ubiquitination sites can be identified by signature peptides containing a GG-tag (114.1 Da) and an LRGG-tag (383.2 Da) on internal lysine residues as well as a GG-tag found on the C-terminus of ubiquitinated peptides. Application of this MS-based approach enabled the identification of 96 ubiquitination sites from proteins purified from human MCF-7 breast cancer cells, representing a 2.4-fold increase in the number of ubiquitination sites that could be identified over standard methods. Our improved MS-based strategy will aid future studies which aim to identify and/or characterize ubiquitinated proteins in human cells.