In-depth mass spectrometric mapping of the human vitreous proteome

Proteome Science

Jürgen Kopitz²  Martina Schnölzer³  Frank G Holz¹  Tore Kempf³  Boris V Stanzel¹  Marion Bergmann²  Agnes Hotz-Wagenblatt³  Uwe Warnken³  Kerstin Kammerer³  Tim U Krohne¹  Sebastian Aretz² 

[1] University Eye Hospital, University of Bonn, Bonn, Germany;Institute of Pathology, University of Heidelberg, Im Neuenheimer Feld 220, Heidelberg, D-69120, Germany;Functional Proteome Analysis, German Cancer Research Center (DKFZ), Heidelberg, Germany

关键词: Prefractionation strategy;    Eye disease;    Vitreous proteome;    Human eye;   

Others  :  816937
DOI  :  10.1186/1477-5956-11-22

 received in 2013-01-15, accepted in 2013-04-26,  发布年份 2013

【 摘 要 】

Mapping of proteins involved in normal eye functions is a prerequisite to identify pathological changes during eye disease processes. We therefore analysed the proteome of human vitreous by applying in-depth proteomic screening technologies. For ethical reasons human vitreous samples were obtained by vitrectomy from “surrogate normal patients” with epiretinal gliosis that is considered to constitute only negligible pathological vitreoretinal changes. We applied different protein prefractionation strategies including liquid phase isoelectric focussing, 1D SDS gel electrophoresis and a combination of both and compared the number of identified proteins obtained by the respective method. Liquid phase isoelectric focussing followed by SDS gel electrophoresis increased the number of identified proteins by a factor of five compared to the analysis of crude unseparated human vitreous. Depending on the prefractionation method proteins were subjected to trypsin digestion either in-gel or in solution and the resulting peptides were analysed on a UPLC system coupled online to an LTQ Orbitrap XL mass spectrometer. The obtained mass spectra were searched against the SwissProt database using the Mascot search engine. Bioinformatics tools were used to annotate known biological functions to the detected proteins. Following this strategy we examined the vitreous proteomes of three individuals and identified 1111 unique proteins. Besides structural, transport and binding proteins, we detected 261 proteins with known enzymatic activity, 51 proteases, 35 protease inhibitors, 35 members of complement and coagulation cascades, 15 peptide hormones, 5 growth factors, 11 cytokines, 47 receptors, 30 proteins of visual perception, 91 proteins involved in apoptosis regulation and 265 proteins with signalling activity. This highly complex mixture strikingly differs from the human plasma proteome. Thus human vitreous fluid seems to be a unique body fluid. 262 unique proteins were detected which are present in all three patient samples indicating that these might represent the constitutive protein pattern of human vitreous. The presented catalogue of human vitreous proteins will enhance our understanding of physiological processes in the eye and provides the groundwork for future studies on pathological vitreous proteome changes.

【 授权许可】

   
2013 Aretz et al.; licensee BioMed Central Ltd.

【 图 表 】

Figure 1.

Figure 2.

Figure 2.

Figure 4.

Figure 5.

Figure 6.

【 参考文献 】

• [1]Le Goff MM, Bishop PN: Adult vitreous structure and postnatal changes. Eye (Lond) 2008, 22:1214-1222.
• [2]Theocharis AD, Papageorgakopoulou N, Feretis E, Theocharis DA: Occurrence and structural characterization of versican-like proteoglycan in human vitreous. Biochimie 2002, 84:1237-1243.
• [3]Freddo TF, Bartels SP, Barsotti MF, Kamm RD: The source of proteins in the aqueous humor of the normal rabbit. Invest Ophthalmol Vis Sci 1990, 31:125-137.
• [4]Barsotti MF, Bartels SP, Freddo TF, Kamm RD: The source of protein in the aqueous humor of the normal monkey eye. Invest Ophthalmol Vis Sci 1992, 33:581-595.
• [5]Funatsu H, Yamashita T, Yamashita H: Vitreous fluid biomarkers. Adv Clin Chem 2006, 42:111-166.
• [6]Holekamp NM: The vitreous gel: more than meets the eye. Am J Ophthalmol 2010, 149:32-36.
• [7]Yamane K, Minamoto A, Yamashita H, Takamura H, Miyamoto-Myoken Y, Yoshizato K, Nabetani T, Tsugita A, Mishima HK: Proteome analysis of human vitreous proteins. Mol Cell Proteomics 2003, 2:1177-1187.
• [8]Koyama R, Nakanishi T, Ikeda T, Shimizu A: Catalogue of soluble proteins in human vitreous humor by one-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis and electrospray ionization mass spectrometry including seven angiogenesis-regulating factors. J Chromatogr B Analyt Technol Biomed Life Sci 2003, 792:5-21.
• [9]Kim T, Kim SJ, Kim K, Kang UB, Lee C, Park KS, Yu HG, Kim Y: Profiling of vitreous proteomes from proliferative diabetic retinopathy and nondiabetic patients. Proteomics 2007, 7:4203-4215.
• [10]Chowdhury UR, Madden BJ, Charlesworth MC, Fautsch MP: Proteome analysis of human aqueous humor. Invest Ophthalmol Vis Sci 2010, 51:4921-4931.
• [11]Bennett KL, Funk M, Tschernutter M, Breitwieser FP, Planyavsky M, Mohien CU, Muller A, Trajanoski Z, Colinge J, Superti-Furga G, Schmidt-Erfurth U: Proteomic analysis of human cataract aqueous humour: Comparison of one-dimensional gel LCMS with two-dimensional LCMS of unlabelled and iTRAQ(R)-labelled specimens. J Proteomics 2011, 74:151-166.
• [12]Lowry OH, Rosegrough NJ, Farr AL, Randall RJ: Protein measurement with the Folin phenol reagent. J Biol Chem 1951, 193:265-275.
• [13]Kang D, Gho M, Suh M, Kang C: Highly sensitive and fast protein detection with Coomassie Brilliant Blue in sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Bull Korean Chem Soc 2002, 11:1511-1512.
• [14]Lossner C, Meier J, Warnken U, Rogers MA, Lichter P, Pscherer A, Schnolzer M: Quantitative proteomics identify novel miR-155 target proteins. PLoS One 2011, 6:e22146.
• [15]Huang DW, Sherman BT, Lempicki RA: Systematic and integrative analysis of large gene lists using DAVID Bioinformatics Resources. Nature Protoc 2009, 4:44-57.
• [16]Huang DW, Sherman BT, Lempicki RA: Bioinformatics enrichment tools: paths toward the comprehensive functional analysis of large gene lists. Nucleic Acids Res 2009, 37:1-13.
• [17]Thomas PD, Kejariwal A, Campbell MJ, Mi H, Diemer K, Guo N, Ladunga I, Ulitsky-Lazareva B, Muruganujan A, Rabkin S, Vandergriff JA, Doremieux O: PANTHER: a browsable database of gene products organized by biological function, using curated protein family and subfamily classification. Nucleic Acids Res 2003, 31:334-341.
• [18]Mi H, Dong Q, Muruganujan A, Gaudet P, Lewis S, Thomas PD: PANTHER version 7: improved phylogenetic trees, orthologs and collaboration with the Gene Ontology Consortium. Nucleic Acids Res 2010, 38:D204-D210.
• [19]del Val C, Ernst P, Falkenhahn M, Fladerer C, Glatting KH, Suhai S, Hotz-Wagenblatt A: ProtSweep, 2DSweep and DomainSweep: protein analysis suite at DKFZ. Nucleic Acids Res 2007, 35:W444-450.
• [20]Shitama T, Hayashi H, Noge S, Uchio E, Oshima K, Haniu H, Takemori N, Komori N, Matsumoto H: Proteome profiling of vitreoretinal diseases by cluster analysis. Proteomics Clin Appl 2008, 2:1265-1280.
• [21]Gao BB, Chen X, Timothy N, Aiello LP, Feener EP: Characterization of the vitreous proteome in diabetes without diabetic retinopathy and diabetes with proliferative diabetic retinopathy. J Proteome Res 2008, 7:2516-2525.
• [22]Smiddy WE, Michels RG, Green WR: Morphology, pathology, and surgery of idiopathic vitreoretinal macular disorders. A review. Retina 1990, 10:288-296.
• [23]Anderson NL, Polanski M, Pieper R, Gatlin T, Tirumalai RS, Conrads TP, Veenstra TD, Adkins JN, Pounds JG, Fagan R, Lobley A: The human plasma proteome: a nonredundant list developed by combination of four separate sources. Mol Cell Proteomics 2004, 3:311-326.
• [24]Lyngholm M, Vorum H, Nielsen K, Ehlers N, Honoré B: Attempting to distiguish between endogenous and contaminating cytokeratins in a corneal proteomic study. BMC Ophthalmol 2011, 11:3. BioMed Central Full Text
• [25]Fort PE, Lampi KJ: New focus on alpha-crystallins in retinal neurodegenerative diseases. Exp Eye Res 2011, 92:98-103.
• [26]Wiesner J, Vilcinskas A: Antimicrobial peptides: the ancient arm of the human immune system. Virulence 2010, 1:440-464.
• [27]You J, Fitzgerald A, Cozzi PJ, Zhao Z, Graham P, Russell PJ, Walsh BJ, Willcox M, Zhong L, Wasinger V, Li Y: Post-translation modification of proteins in tears. Electrophoresis 2010, 31:1853-1861.
• [28]Schloetzer-Schrehardt U, André S, Janko C, Kaltner H, Kopitz J, Gabius HJ, Herrmann M: Adhesion/growth regulatory galectins in the human eye: localization profiles and tissue reactivities as standard to detect disease-associated alterations. Graefes Arch Clin Exp Ophthalmol 2012, 250:1169-1180.
• [29]Wyatt K, Gao C, Tsai JY, Fariss RN, Ray S, Wistow G: A role for lengsin, a recruited enzyme, in terminal differentiation in the vertebrate lens. J Biol Chem 2008, 283:6607-6615.
• [30]Hindson VJ, Gallagher JT, Halfter W, Bishop PN: Opticin binds to heparan and chondroitin sulfate proteoglycans. Invest Ophthalmol Vis Sci 2005, 46:4417-4423.
• [31]Grayson C, Reid SN, Ellis JA, Rutherford A, Sowden JC, Yates JR, Farber DB, Trump D: Retinoschisin, the X-linked retinoschisis protein, is a secreted photoreceptor protein, and is expressed and released by Weri-Rb1 cells. Hum Mol Genet 2000, 9:1873-1879.
• [32]Wistow G, Bernstein SL, Wyatt MK, Behal A, Touchman JW, Bouffard G, Smith D, Peterson K: Expressed sequence tag analysis of adult human lens for the NEIBank Project: over 2000 non-redundant transcripts, novel genes and splice variants. Mol Vis 2002, 8:171-184.
• [33]An E, Lu X, Flippin J, Devaney JM, Halligan B, Hoffman EP, Strunnikova N, Csaky K, Hathout Y: Secreted proteome profiling in human RPE cell cultures derived from donors with age related macular degeneration and age matched healthy donors. J Proteome Res 2006, 5:2599-261.