Michael A. Riviere

Techniques for accurate protein identification in shotgun proteomic studies of human, mouse, bovine, and chicken lenses

Analysis of shotgun proteomics datasets requires techniques to distinguish correct peptide identifications from incorrect identifications, such as linear discriminant functions and target/decoy protein databases. We report an efficient, flexible proteomic analysis workflow pipeline that implements these techniques to control both peptide and protein false discovery rates. We demonstrate its performance by analyzing two-dimensional liquid chromatography separations of lens proteins from human, mouse, bovine, and chicken lenses. We compared the use of International Protein Index databases to UniProt databases and no-enzyme SEQUEST searches to tryptic searches. Sequences present in the International Protein Index databases allowed detection of several novel crystallins. An alternate start codon isoform of βA4 was found in human lens. The minor crystallin γN was detected for the first time in bovine and chicken lenses. Chicken γS was identified and is the first member of the γ-crystallin family observed in avian lenses.

Susceptibility of Ovine Lens Crystallins to Proteolytic Cleavage during Formation of Hereditary Cataract

PURPOSE To produce two-dimensional electrophoresis (2-DE) maps for ovine crystallins and examine changes in ovine crystallins during cataract formation. METHODS Soluble and insoluble fractions were isolated from normal, whole lenses of 26-week-old sheep, the proteins separated by 2-DE, and the spots digested with trypsin and subjected to tandem mass spectral analysis. Spot identifications were made by using mass spectrometry data from each spot digestion and data from 2-DE maps of proteins from soluble and insoluble cortices of 10-month-old ovine lens. Ovine alphaA-, alphaB-, and betaB3-crystallin cDNAs were sequenced, whereas other ovine crystallins were identified by using bovine sequences. Proteins were then isolated from whole lenses of 26-week-old lambs with mature hereditary cataracts, and the changes in the crystallins were determined by 2-DE. The masses of truncated crystallins were determined after elution from 2-DE gels. RESULTS The ovine lens contained the normal complement of crystallins and, similar to other mammalian lenses, underwent partial proteolysis of betaB1-, betaA3-, and betaB3-crystallin during maturation. Cataract development was associated with enhanced truncation of alpha- and beta-crystallins. C-terminal truncations of alphaA- and alphaB-crystallin and N-terminal truncation of betaB2-crystallin were observed as well as a loss of gamma-crystallin. CONCLUSIONS These data provide the first 2-DE gel maps for ovine lens crystallins and indicated that ovine lens crystallins are truncated during lens maturation. The differences in proteolysis appearing in normal and cataractous lenses suggested that calpain isoforms may be differentially activated during lens maturation and cataract. The ovine hereditary cataract is a useful nonrodent model to study the role of calpain proteolysis in cataract formation.

Human and monkey lenses cultured with calcium ionophore form αB-crystallin lacking the C-terminal lysine, a prominent feature of some human cataracts.

PURPOSE Elevation of lens calcium occurs in both human and experimental animal cataracts, and opacification may result from calcium-activated proteolysis. The purpose of the present study was to determine whether calcium accumulation in cultured human and Macaca mulatta lenses results in proteolysis of crystallins, the major lens proteins. METHODS Two-dimensional electrophoresis and mass spectrometry were used to construct detailed maps of human and monkey lens crystallins so that proteolysis after calcium accumulation could be monitored and the altered crystallins identified. Human and macaque lenses cultured in A23187 showed elevated lenticular calcium and superficial cortical opacities. The carboxypeptidase E (CPE) gene is expressed in human lens, and its presence in lens fibers was demonstrated by Western blot. To investigate whether CPE could cause similar truncation, purified alphaB-crystallin and CPE were incubated in vitro. RESULTS The major change observed in the crystallins of these cultured lenses was the accumulation of alphaB(1-174)-crystallin resulting from the loss of a C-terminal lysine. This result was significant, because similar appearance of alphaB(1-174) is a prominent change in some human cataracts. alphaB-crystallin and CPE incubation result in the formation of alphaB(1-174)-crystallin. This truncation was specific to alphaB(1-174)-crystallin, since other crystallins were not proteolyzed. Although a weaker activator than zinc, calcium activated CPE in vitro. CONCLUSIONS Since zinc concentrations did not increase during culture in A23187, calcium uptake in the lens may be responsible for CPE activation and alphaB(1-174) formation during cataract.

Proteomic analysis of biopsied human colonic mucosa.

Background and Objectives: Pediatric gastroenterologists have a unique opportunity to study the proteins in the gastrointestinal tract. To assess the power of proteomic studies we compared 2 methods for analysis of proteins in normal human colonic mucosa: 2-dimensional gel electrophoresis (2DE) and 2-dimensional liquid chromatography (2DLC) in conjunction with mass spectrometry. We used Ingenuity Pathway Analysis to examine these proteins regarding function, location, and relation to disease. Results: 2DLC identified 550 proteins, whereas 2DE identified 107 proteins, 18 of which were not observed with 2DLC. The function associated with the largest number of proteins for both methods was cancer (236 proteins with 2DLC, 61 proteins with 2DE). The largest group of proteins was from the cytoplasm (49.3% from 2DE and 49.1% from 2DLC). Two hundred seventy of the total 568 proteins were related to 26 different categories of human disease and 200 of these 270 were described in large intestine, 227 were described in blood, and 149 were described in serum or plasma. Conclusions: These methods are complementary, although many more proteins were identified with 2DLC. This suggests that 2DLC should have greater utility in examining changes in the proteome of the colonic mucosa during disease than 2DE. However, some proteins found were unique to 2DE, and thus the methods chosen for a given analysis must be matched with the proteins to be studied. When pediatric gastroenterologists use proteomic methods, there is a new opportunity to increase our understanding of the gastrointestinal tract in health and disease.