Development of a two‐dimensional gel electrophoresis database of human lysosomal proteins

Chataway, Tim; Whittle, Alison M.; Lewis, Martin; Bindloss, Colleen A.; Moritz, Robert L.; Simpson, Richard J.; Hopwood, John J.; Meikle, Peter J.

doi:10.1002/elps.1150190538

Cited by 11 publications

(6 citation statements)

References 18 publications

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“…The intracellular localization of the last two proteins (leukocystatin and human cellular repressor of E1A-stimulated genes (hCREG)) has not been previously reported [38±41]. To our knowledge, one group only recently attempted to systematically identify lysosomal proteins from human placenta [21]. N-terminal sequences pointed to five luminal lysosomal proteins, among them cathepsin D and b-hexosaminidase b (see our results, Table 1), and seven nonlysosomal proteins.…”

Section: Intracellular Localization Of the Identified Proteinsmentioning

confidence: 87%

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Towards a human repertoire of monocytic lysosomal proteins

et al. 2000

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The lysosomal compartment of human monocytic cells has never been investigated by a proteomic approach. By a combination of one-dimensional (1-D) and two-dimensional (2-D) gel electrophoresis, protein identification by N-terminal sequencing, matrix assisted laser desorption/ionization-mass spectrometry (MALDI-MS) peptide mass fingerprinting and tandem mass spectrometry (MS/MS) peptide sequence analysis, we initiated an exhaustive study of the human lyososomal proteome, which aims at establishing a 2-D reference map of human soluble lyososomal proteins. Human monocytic U937 cells were induced to secrete lysosomal soluble hydrolases by addition of NH4Cl in the culture medium. Since lysosomal soluble proteins are characterized by the presence of mannose-6-phosphate, they were purified on an affinity support bearing mannose-6-phosphate receptor. Analysis of the purified fraction led to the preliminary identification of fifteen proteins, among which twelve are well-known lysosomal hydrolases, one is assumed to be lysosomal on the basis of sequence homology to cysteine proteinases of the papain family, and two (leukocystatin and the human cellular repressor of E1A-stimulated genes) are described here for the first time as mannose-6-phosphate-containing proteins.

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Section: Intracellular Localization Of the Identified Proteinsmentioning

confidence: 87%

“…However, the isolation procedures were never validated by systematic protein identification, except once. In that case, N-terminal sequencing of the 2-DE-resolved proteins (a Percoll-generated fraction) resulted in the identification of several lysosomal hydrolases as well as numerous nonlysosomal major proteins [21].…”

Section: Introductionmentioning

confidence: 99%

Towards a human repertoire of monocytic lysosomal proteins

et al. 2000

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“…In eukaryotic cells, lysosomes serve as the terminal sites for delivery of material targeted for removal (42)(43)(44). For this purpose, lysosomes are equipped with a variety of more than 50 soluble acid-dependent hydrolases engaged in final degradation of both exogenous and endogenous macromolecules.…”

Section: Discussionmentioning

confidence: 99%

“Subcellular Proteomics” of Neuromelanin Granules Isolated from the Human Brain

Tribl

Gerlach

Marcus

et al. 2005

Molecular & Cellular Proteomics

113

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"Subcellular proteomics" is currently the most effective approach to characterize subcellular compartments. Based on the powerful combination of subcellular fractionation and protein identification by LC-MS/MS we were able for the first time to 1) isolate intact neuromelanin granules from the human brain and 2) establish the first protein profile of these granules. This compartment containing neuromelanin (NM) is primarily located in the primate's substantia nigra, one of the main brain regions that severely degenerates in Parkinson disease. We used mechanic tissue disaggregation, discontinuous sucrose gradient centrifugation, cell disruption, and organelle separation to isolate NM granules from human substantia nigra. Using transmission electron microscopy we demonstrated that the morphological characteristics of the isolated NM granules are similar to those described in human brain tissue. Fundamentally we found numerous proteins definitely demonstrating a close relationship of NM-containing granules with lysosomes or lysosomerelated organelles originating from the endosome-lysosome lineage. Intriguingly we further revealed the presence of endoplasmic reticulum-derived chaperones, especially the transmembrane protein calnexin, which recently has been located in lysosome-related melanosomes and has been suggested to be a melanogenic chaperone. Molecular & Cellular Proteomics 4:945-957, 2005.Melanins are widely distributed throughout the plant and animal kingdoms. In humans, these heterogeneous, complex polymer pigments occur naturally in the hair, the skin, the inner ear, the iris, and the choroid of the eye (1). Melanin in the brain has an appearance and structure similar to cutaneous melanins and has thus been named neuromelanin (NM) 1 (2). NM is found inter alia in dopaminergic neurons of a small area in the human midbrain important for the control of movement that is known as the substantia nigra pars compacta (SN; from the Latin meaning "black body"). The loss of this dark pigment and the resulting pallor of the SN is one of the most striking features of the common movement disorder Parkinson disease. A relationship between the loss of the dopaminergic SN cells and their NM content (3), a specific affinity to iron (4), and a significant binding of ␣-synuclein to NM in the diseased state (5) suggest a functional role for NM in neurodegeneration in Parkinson disease (6).Although much is known about the peripheral melanins to which NM is thought to be related, many basic questions remain to be answered about NM in the brain. Thus it is unclear why only some human dopamine neurons produce NM within their cytoplasm (7). Little is also known about the structure of NM, and the understanding of its genesis and function within the cell remains speculative.Nuclear magnetic resonance spectroscopic studies have shown that NM resembles synthetic cysteinyldopamine melanin more closely than the more simple dopamine melanin; however, human NM appears to be a structurally more complex chemical structure than any of the syn...

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“…In this regard, quantitative Western immunoblot analysis of specific organelle-marker proteins and morphological observation under electron microscopy can be used to evaluate the mechanical and functional integrity of organelles (Huber et al, 2003). The combination of 2DE with organelle separation approaches has led to the establishment of proteomic database for mitochondria (Fountoulakis et al, 2002;Rabilloud et al, 1998;Scharfe et al, 2000), lysosome (Chataway et al, 1998), endoplasmic reticulum (Garin et al, 2001), macromolecular structures as well as multiprotein complexes (for review article see Jung et al, 2000). Organelle preseparation has also been demonstrated to be useful for confirming the subcellular distribution of proteins and metabolites, monitoring the compartmental redistribution of target compounds and enhancing the detectability of low-abundant species.…”

Section: Organelle Prefractionation Methodsmentioning

confidence: 97%

Current Progress in Sample Preparation for Two-Dimensional Electrophoresis in Proteomics

Cai¹,

Chiu²,

2005

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Two dimensional polyacrylamide gel electrophoresis (2DE) has been a core technology of current proteomics for its high resolution and ability to detect proteins with post-translational modifications. This technology has been widely applied in numerous biomedical research projects including biomarker discovery and drug development. The application of narrow-range immobilized pH gradient strips (IPGs) and advanced detection methodologies have increased the resolution of 2DE technology. However, 2DE still suffers from its limitation in unambiguous detection of proteins of lowabundance. To improve this, sample preparation is the first and key step for successful 2DE analysis. This article summarizes current progresses in sample handling and prefractionation aiming to enrich proteins of interest according to their special physical and chemical properties so that the application of 2DE can be greatly extended.

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Development of a two‐dimensional gel electrophoresis database of human lysosomal proteins

Cited by 11 publications

References 18 publications

Towards a human repertoire of monocytic lysosomal proteins

Towards a human repertoire of monocytic lysosomal proteins

“Subcellular Proteomics” of Neuromelanin Granules Isolated from the Human Brain

Current Progress in Sample Preparation for Two-Dimensional Electrophoresis in Proteomics

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