Phosphorylation of small molecular weight polypeptides in the iris-ciliary complex, aqueous humor and vitreous humor

Banerjee, Amit Kumar; Richiert, Dawn M.; Bagchi, M.

doi:10.1016/0167-4838(91)90525-5

Cited by 5 publications

(4 citation statements)

References 9 publications

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“…The concentration of neurotransmitters and neuropeptides in the retina may be affected by retinal diseases. The concentrations of these agents may then be similarly affected in the adjacent vitreous [1,2]. Characterizing the chemical content of the vitreous, then, will advance our understanding of normal function and disease states of the retina.…”

Section: Introductionmentioning

confidence: 97%

Determination of amino acids in rat vitreous perfusates by capillary electrophoresis

Thongkhao-On¹,

Kottegoda²,

Pulido

et al. 2004

Electrophoresis

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In vivo determinations of amino acids are important for improving our understanding of physiological states of biological tissue function and dysfunction. However, the chemically complex matrix of different biological fluids complicates the assay of this important class of molecules. We introduce a method for characterizing the amino acid composition of submicroliter volumes of vitreous humor perfusates. Low-flow push-pull perfusion sampling is compatible with collecting small volume samples in a complicated matrix that are potentially difficult to separate. An efficient, sensitive, and rapid analysis of amino acids from in vivo perfusates of the vitreous is presented with 3-(4-carboxybenzoyl)-2-quinoline-carboxaldehyde (CBQCA) derivatitation and capillary electrophoresis (CE) separation with laser-induced fluorescence detection (LIF). Derivatization with CBQCA for up to 2 h provided high sensitivity and low detection limits at the nM level. Seventeen amino acids including D-serine (D-Ser) and D-aspartate (D-Asp) were resolved in less than 10 min. Importantly, D-Ser is separated from its enantiomeric pair. Characterization of vitreal amino acids with this assay technique will be useful for understanding ocular diseases and physiological mechanisms in vision.

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Section: Introductionmentioning

confidence: 97%

Determination of amino acids in rat vitreous perfusates by capillary electrophoresis

Thongkhao-On¹,

Kottegoda²,

Pulido

et al. 2004

Electrophoresis

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“…8 For example, low molecular weight phosphopolypeptides activated the differentiation and degradation of lens cell growth in the iris–ciliary complex, aqueous humour and VH derived from experimental animals. 8 In the retina, phosphorylations of proteins were accelerated under ischaemic conditions, such as in ischaemia–reperfusion injury of rats or branch vein occlusion in the cat. 9,10…”

Section: Introductionmentioning

confidence: 99%

“…7 In ocular tissues of experimental animals, some expressed proteins are either phosphorylated to promote their activity or phosphorylated by some other proteins for their biological effect. 8 For example, low molecular weight phosphopolypeptides activated the differentiation and degradation of lens cell growth in the iris -ciliary complex, aqueous humour and VH derived from experimental animals. 8 In the retina, phosphorylations of proteins were accelerated under ischaemic conditions, such as in ischaemia-reperfusion injury of rats or branch vein occlusion in the cat.…”

Section: Introductionmentioning

confidence: 99%

“…8 For example, low molecular weight phosphopolypeptides activated the differentiation and degradation of lens cell growth in the iris -ciliary complex, aqueous humour and VH derived from experimental animals. 8 In the retina, phosphorylations of proteins were accelerated under ischaemic conditions, such as in ischaemia-reperfusion injury of rats or branch vein occlusion in the cat. 9,10 Moreover, in the postmortem human retina, it was reported that the apoptosis-inducing phosphorylation of protein kinase C contributes to retinal ganglion cell loss in glaucoma, 11 and six isoforms of NRL (29 -35 kDa) were expressed specifically and generated by phosphorylation.…”

Section: Introductionmentioning

confidence: 99%

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Identification of phosphotyrosyl proteins in vitreous humours of patients with vitreoretinal diseases by sodium dodecyl sulphate–polyacrylamide gel electrophoresis/Western blotting/matrix-assisted laser desorption time-of-flight mass spectrometry

Mukai¹,

Nakanishi

Shimizu

et al. 2008

Ann Clin Biochem

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Background: It is important to explain target proteins for the understanding of the pathogenesis of vitreoretinal diseases. In a previous study, we identified more than 100 proteins including seven angiogenic-modulated factors in vitreous humours (VHs). Although there have been many reports of expressed protein profiles in VHs, only a few of these are modified proteins, such as those undergoing phosphorylation and oxidation. Methods: We applied Western blotting (WB), selective staining of phosphoproteins and mass spectrometry to detect and identify phosphoproteins in VHs of patients with vitreoretinal diseases. After the removal of albumin and immunoglobulins A/G in VHs, the proteins were separated by sodium dodecyl sulphate-polyacrylamide gel electrophoresis and reacted with anti-PY-20 monoclonal antibody on transfer membranes, and treated proteins were visualized with Phos-tag TM and

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Comparison of protein mixtures in aqueous humor by membrane preconcentration ‐ capillary electrophoresis ‐ mass spectrometry

et al. 1998

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The significance of proteomic research is coupled with the recent exponential growth of these investigations. Currently, the most popular techniques used for these studies include the coupling of 1- and 2-dimensional electrophoresis with mass spectrometric analysis of the extracted and digested proteins. However, detection limits of gel staining methods have led to a search for complimentary techniques that afford the detection of lower concentrations of biologically relevant proteins. In the present studies, we have evaluated the applicability of on-line capillary electrophoresis - mass spectrometry (CE-MS) for this application. Specifically, we used membrane preconcentration-CE-MS (mPC-CE-MS) to analyze 13 samples of human aqueous humor (AH) from patients with various ocular pathologies (cataract, cataract plus glaucoma, and cataract plus pseudoexfoliation syndrome). This approach enabled rapid analysis of a relatively large volume (1 microL of each specimen, and a protein map for each was created. Measured average molecular weights (Mr) were used to tentatively identify proteins after search of the SWISS-PROT database using TagIdent from ExPaSy. Among those proteins tentatively identified are beta-2 microglobulin (Mr 11731.2), apolipoprotein A1 (Mr 28078.6) and serum albumin (Mr 66400). Proteins with Mr of 4349 (unidentified), 11731.2 (beta-2 microglobulin), 13400-14100 (immunoglobulin fragments), 28078.2 (apolipoprotein A1) and approximately 68000 (serum albumin) were observed in the majority of specimens. Generally no significant differences were noted in the protein composition of aqueous humor samples from different pathologies. However, the absence of an Mr 13345 protein and its oxidized form (Mr 13361) in samples from patients with pseudoexfoliation syndrome was noted. Occasionally the alpha-and beta-chains of hemoglobin, a contaminant in aqueous humor introduced during sampling, were also detected. We conclude from these studies that mPC-CE-MS is an attractive complimentary technique for proteome research, as this approach enables direct mapping and characterization of low concentrations of proteins that are present in complex physiologically derived fluids.

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Phosphorylation of small molecular weight polypeptides in the iris-ciliary complex, aqueous humor and vitreous humor

Cited by 5 publications

References 9 publications

Determination of amino acids in rat vitreous perfusates by capillary electrophoresis

Determination of amino acids in rat vitreous perfusates by capillary electrophoresis

Identification of phosphotyrosyl proteins in vitreous humours of patients with vitreoretinal diseases by sodium dodecyl sulphate–polyacrylamide gel electrophoresis/Western blotting/matrix-assisted laser desorption time-of-flight mass spectrometry

Comparison of protein mixtures in aqueous humor by membrane preconcentration ‐ capillary electrophoresis ‐ mass spectrometry

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