Michael A Riviere

University of Minnesota Twin Cities, Minneapolis, MN, United States

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Publications (10)33.09 Total impact

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    ABSTRACT: : 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. : 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. : 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.
    Journal of pediatric gastroenterology and nutrition 07/2010; 51(1):46-54. · 2.18 Impact Factor
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    Phillip A Wilmarth, Michael A Riviere, Larry L David
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    ABSTRACT: 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 betaA4 was found in human lens. The minor crystallin gammaN was detected for the first time in bovine and chicken lenses. Chicken gammaS was identified and is the first member of the gamma-crystallin family observed in avian lenses. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1007/s12177-009-9042-6) contains supplementary material, which is available to authorized users.
    Journal of Ocular Biology Diseases and Informatics 12/2009; 2(4):223-234.
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    ABSTRACT: 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. 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. 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. 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.
    Investigative ophthalmology & visual science 08/2009; 50(12):5828-36. · 3.43 Impact Factor
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    ABSTRACT: To produce two-dimensional electrophoresis (2-DE) maps for ovine crystallins and examine changes in ovine crystallins during cataract formation. 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. 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. 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.
    Investigative Ophthalmology &amp Visual Science 04/2008; 49(3):1016-22. · 3.44 Impact Factor
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    ABSTRACT: Identifying deamidated peptides using low-resolution mass spectrometry is difficult because traditional database search programs cannot accurately detect modified peptides when the mass differences are only 0.984 Da. In this study, we utilized differential reversed-phase elution behavior of deamidated and corresponding unmodified peptide forms to significantly improve deamidation detection on a low-resolution LCQ ion trap instrument. We also improved the mass measurements of unmodified and deamidated peptide forms by averaging survey scans across each chromatogram peak. Tryptic digests of a series of normal (3-day old, 2-year old, 18-year old, 35-year old, and 70-year old) and cataractous (93-year old) human lens samples were used to produce large numbers of potentially deamidated peptides. The complex peptide mixtures were separated by strong cation exchange (SCX) chromatography followed by reversed-phase (RP) chromatography. Synthetic peptides were used to show that unmodified and deamidated peptides coeluted during the SCX separation and were completely resolved with the RP conditions used. Retention time shifts (RTS) and mass differences (DeltaM) of deamidated lens peptides and their corresponding unmodified forms were manually determined for the 70-year old lens sample. These values were used to assign correct or incorrect deamidation identifications from SEQUEST searches where deamidation was specified as a variable modification. Manual validation of SEQUEST identifications from synthetic peptides, 3-day old, and 70-year old samples had an overall 42% deamidation detection accuracy. Filtering SEQUEST identifications using RTS and DeltaM constraints resulted in >93% deamidation detection accuracy. An algorithm was developed to automate this method, and 72 Crystallin deamidation sites, 18 of which were not previously reported in human lens tissue, were detected.
    Journal of Proteome Research 09/2007; 6(9):3819-26. · 5.06 Impact Factor
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    ABSTRACT: The ocular vascular endothelium plays a key role in the development of several leading retinal causes of blindness in Western nations. Choroidal endothelial cells are integral to the subretinal neovascular lesions that characterize the exudative form of late age-related macular degeneration (AMD), and retinal endothelial cells participate in the initiation of diabetic retinopathy and posterior uveitis. Vascular endothelial cells at different sites exhibit considerable molecular diversity. This diversity has implications for understanding the pathogenesis of tissue-specific diseases and for the development of targeted therapies to treat these conditions. Previous work from our group has identified significant differences in the gene transcript profiles of human retinal and choroidal endothelial cells. Because the proteome ultimately determines the behavior of any given cell, however, it is critical to determine whether molecular differences exist at the level of protein expression. Retinal and choroidal endothelial cells were separately isolated from five sets of human eyes by enzymatic digestion with type II collagenase followed by anti-CD31 antibody-conjugated magnetic bead separation. Cells were washed to remove serum peptides in the culture medium, and lysed by sonication in buffer containing 2% sodium dodecyl sulfate. Protein was then precipitated with acetone. Retinal and choroidal endothelial samples from each donor were labeled with Cy3 and Cy5, respectively, mixed with a Cy2-labeled pooled protein sample to facilitate spot matching across gels, and separated by two-dimensional difference gel electrophoresis (2D-DIGE). Following a global normalization, differentially abundant protein spots that were visible in at least four of five donor gels were detected by the significance analysis of microarrays method, with false discovery rate set at 5%. Corresponding spots were excised from additional DIGE-labeled or Coomassie-stained 2D electrophoretic gels. Protein identification was performed by liquid chromatography and tandem mass spectrometry. Of 123 protein spots detected by 2D-DIGE that qualified for statistical analysis, we found 31 spots that demonstrated a significant difference in abundance between retinal endothelial samples versus choroidal endothelial samples. For 17 proteins, over 50% of the spectral counts could be matched to a single protein in the digested spot. Eleven proteins were more abundant in retinal endothelial cells (i.e., inorganic pyrophosphatase, protein disulfide isomerase A3, calreticulin, peroxiredoxin-4, protein disulfide isomerase, serpin B9, F-actin capping protein subunit beta, coactosin-like protein, vimentin, cathepsin B, and a high molecular weight form of annexin A3). Six proteins were more abundant in choroidal endothelial cells (i.e., glutathione peroxidase 1, ubiquitin carboxyl-terminal hydrolase isozyme L1 (UCH-L1), heat-shock protein beta-1, superoxide dismutase (Cu-Zn), nucleoside diphosphate kinase A, and a low molecular weight form of annexin 3). Our data indicate that the proteomes of retinal and choroidal vascular endothelial cells are different. Several differentially expressed proteins are implicated in the regulation of angiogenesis; these include cathepsin B and UCH-L1, proteins with transcripts that were also differently expressed according to microarray. Our observations further suggest that angiogenesis within the retina, a component of severe diabetic retinopathy and posterior uveitis, may be controlled by different mechanisms to those regulating choroidal neovascularization, as occur in exudative AMD. Future studies to establish the role of these angiogenic proteins in disease may suggest potential new targets for tissue-specific therapies.
    Molecular vision 02/2007; 13:2058-65. · 1.99 Impact Factor
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    ABSTRACT: 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.
    Journal of Proteome Research 11/2006; 5(10):2554-66. · 5.06 Impact Factor
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    ABSTRACT: The human whole saliva proteome was investigated using two-dimensional liquid chromatography (2-DLC). The 2-DLC study was able to identify, with high confidence, 102 proteins including most known salivary proteins (35), and a large number of common serum proteins (67). Peptides from proline-rich proteins, abundant in saliva, had unusual cleavage sites and were frequently only partially tryptic. Three proteins not previously observed in human saliva were also detected. Significantly greater numbers of identified proteins, including high molecular weight, low molecular weight, and proline-rich proteins, were found with 2-DLC compared to previously reported two-dimensional gel electrophoresis studies.
    Journal of Proteome Research 10/2004; 3(5):1017-23. · 5.06 Impact Factor
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    ABSTRACT: To characterize the adult chicken lens proteome using mass spectrometry and two-dimensional gel electrophoresis (2-DE). Lens proteins from 10-week old chickens were separated by gel filtration and reversed-phase chromatography, and whole protein masses were measured with electrospray mass spectrometry. Water-soluble lens proteins were separated by 2-DE and identified by tandem mass spectrometry of in-gel digests. Whole protein masses were consistent with all major chicken lens crystallin sequences, except for beta B2 and beta B3. Subsequent cDNA sequencing revealed errors in published sequences translating into 2- and 7-amino-acid differences, respectively, for beta B2 and beta B3, which were in better agreement with the measured masses. Previously uncharacterized forms of beta A2 and beta B2 were observed. The novel form of beta A2 had four fewer amino acids, was more abundant, and resulted from translation at a second start codon. The novel form of beta B2 contained 14 additional amino acids in the interdomain linker and resulted from alternate splicing within intron 4 of the transcript. All examined crystallins, except beta A3, for which data could not be obtained, were N-terminally acetylated, and all beta-crystallins lacked an initial methionine, except for the smaller beta A2 form. In-gel digests identified 29 proteins on the 2-DE map and indicated that truncation occurs within N-terminal extensions of beta-crystallins during lens maturation. The complementary techniques 2-DE, mass spectrometry, and DNA sequencing were used to provide the most complete description of the adult chicken lens proteome to date and identified alternate forms of beta A2 and beta B2.
    Investigative Ophthalmology &amp Visual Science 09/2004; 45(8):2705-15. · 3.44 Impact Factor
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    ABSTRACT: The purpose of this study is to compare the protein composition of the B-3 line of transformed human lens epithelial (HLE) cells to that of freshly dissected HLE cells. This provides baseline data on lens cell proteins from fresh lens cells and from the B-3 cell line, which is often used as a model system for the lens. Human lens epithelial cells adherent to the lens capsule were dissected into central (undifferentiated) and peripheral (partially differentiated) populations. Fully differentiated human lens fiber cells were isolated from the outer cortical layers of the lens. HLE B-3 cells were analyzed at several passage levels. Extracts were prepared from each cell type and the proteins resolved by two-dimensional polyacrylamide gel electrophoresis (2-DE). Representative gel patterns were visually compared, spots excised, and trypsin digests prepared. The peptide compositions of the digests were analyzed using either liquid chromatography electrospray ionization tandem mass spectrometry or atmospheric pressure-matrix-assisted laser desorption ionization mass spectrometry, using a liquid chromatography classic ion trap (LCQ) mass spectrometer. Two-DE patterns were obtained for fresh and cultured cell types. Similar patterns were observed between central and peripheral HLE cells, both of which contained high levels of alphaA-, alphaB-, and betaB2-crystallins; alpha-enolase; and aldehyde dehydrogenase. HLE B-3 cultured cells were characterized by a marked loss of crystallins and a relatively higher level of noncrystallin proteins--most notably, high molecular weight, acidic proteins. Whereas subunit d of adenosine triphosphate (ATP) synthase, alphaB-crystallin, galectin, glyceraldehyde-3-phosphate dehydrogenase, alpha-enolase, actin, peptidylprolyl isomerase A, phosphatidylethanolamine-binding protein, and vimentin were present in both fresh and cultured lens epithelium, only the high abundance of alpha-enolase, galectin-1, and vimentin suggested that B-3 cells were lens derived. Freshly dissected noncultured HLE cells from both central and peripheral regions contain a high concentration of crystallins that mask the detection of less abundant proteins by 2-DE. Transformation and culture of HLE cells causes a loss of these crystallins and an increase in the relative concentration of other proteins. However, most of these noncrystallin proteins were different from those observed in noncultured HLE cells. These results suggest that transformation markedly alters the protein expression pattern in immortalized HLE cells and that caution should be exercised when using them to study properties of HLE cells in vivo.
    Investigative Ophthalmology &amp Visual Science 11/2003; 44(11):4829-36. · 3.44 Impact Factor