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Sorting and zooming: Subcellular proteomics is booming!
JOU RN A L OF P ROT EO M IC S 7 2 ( 2 0 09 ) 1– 3
a v a i l a b l e a t w w w. s c i e n c e d i r e c t . c o m
w w w. e l s e v i e r. c o m / l o c a t e / j p r o t
Editorial
Sorting and zooming: Subcellular proteomics is booming! I am pleased to introduce to you this special issue of the Journal of Proteomics, dedicated to biochemical and proteomics insights into subcellular organelles and cell compartments. In the past decade, organelle proteomics has emerged as a powerful approach to provide information on the association of proteins with specific organelles along with implications for their structural organization and functions [1]. Classically, an organelle is one of several structures delimited by a lipid bilayer membrane with specialized functions, suspended in the cytoplasm of a eukaryotic cell. This special issue contains 7 reviews and 3 original articles on several organelles and also on two cell compartments not enclosed by a membrane, i.e., the nuclear matrix and the extracellular secretome. The organelles and (sub-)cellular compartments that are highlighted in this issue are indicated in Fig. 1. Many sub-cellular structures are covered in this issue. For reviews of the ER/Golgi system and mitochondria, the reader is referred to two recent reviews [2,3]. The first review of this special issue by Yan, Aebersold and Raines, provides an excellent historical overview of the technologies and strategies that have been used for organelle proteomics. They describe how proteomic analyses have matured from being simple catalogs to quantitative organelle signatures based on collections of marker proteins to which novel members can be assigned and (novel) functions inferred. Pros and cons of different approaches are discussed in the context of applications for selected organelles. They conclude that despite improved methods for organelle purification, complete removal of contaminant proteins is hard to achieve. Therefore, elimination of false-positives using subtractive or quantitative (differential) approaches is mandatory, and these strategies are discussed. Especially combining quantitative proteomics information on all purified fractions with advanced bioinformatics has allowed profiling multiple organelles simultaneously in only partially purified samples. Cellular signaling and communication takes place at the plasma membrane and membrane proteins represent drug targets for the majority of known drugs. Therefore, it is not surprising that many proteomics studies have focused on tackling the plasma membrane proteome. The second review of this issue, by Zheng and Foster, focuses on
biochemical approaches for the study of membrane microdomains. An extensive overview is provided of microdomain functions along with the biochemical methods to isolate them. Detergent-based and detergent-free methods as well as affinity-based methods are discussed for the isolation of detergent-resistant membranes, lipid rafts, caveolae, tetraspanin microdomains and GPI-anchored proteins. From the discussed examples, it is clear that the choice of detergent can have an enormous impact on the proteins recovered and further validation is needed before conclusions can be drawn. Lysosomes are single membrane-enclosed organelles that contain a multitude of enzymes capable of digestion of macromolecules in the cell. Callahan, Bagshaw and Mahuran review lysosome proteomics studies focused on the lysosome membrane, and discuss the role of the identified lysosomal proteins in vesicle trafficking. Lysosome biogenesis and associated storage disorders, as well as possible links to other pathologies such as cancer. The review of Xiao and Veenstra focuses on vesicles, including exosomes, membrane-enclosed vesicles with distinct functions, and matrix vesicles, important for formation of the extracellular matrix. Both pre-proteomic and proteomic studies are discussed in the context of known and novel functions as well as implications for developmental- and tumor biology and biomarker discovery. Importantly, results from proteomics indicate that both the extracellular matrix and vesicles play more specific and dynamic roles than simple structural support. Possible functions that are discussed include: intercellular communication, regulation of signal transduction, cell motility, chemotaxis and material exchange among cells. Neurons depend on trophic support from target cells for their differentiation and survival. The review on signaling endosomes by Wu and Mobley et al. summarizes current insights into the vesicular carriers of trophic signals that are picked up at axon terminals and are retrogradely transported back to the cell body to exert their trophic effect. Most evidence derives from an elegant series of hypothesis-driven immunochemical and molecular biological studies and points to signaling endosomes as the primary carrier of the trophic signal, though controversies still exist. The authors also
1874-3919/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.jprot.2009.01.016
2
JOUR NAL OF P ROT EOM IC S 7 2 ( 2009) 1– 3
Fig. 1 – Anatomy of the animal cell. Organelles and cellular components are indicated. THIS SPECIAL ISSUE CONTAINS REVIEWS ON THE PLASMA MEMBRANE AND MICRODOMAINS, THE NUCLEUS (I.E., NUCLEAR ENVELOPE, NUCLEAR PORE AND NUCLEAR MATRIX), LYSOSOME, SIGNALING ENDOSOMES, EXTRACELLULAR VESICLES AND AS WELL AS PRIMARY CONTRIBUTIONS ON SYNAPTIC PROTEIN COMPLEXES, SECRETED PROTEINS FROM PLATELETS AND BIOINFORMATICS APPROACHES APPLIED TO NUCLEAR FRACTIONS (Reproduced with permission from http://micro.magnet.fsu.edu/cells/animalcell.html, Acknowledgements Molecular Expressions).
summarize a few proteomics studies done on signaling endosomes in the context of retrograde EGFR signaling and stress the need for quantitative proteomics to delineate the identity and primary carrier of the retrograde signal and the mechanisms involved. At the heart of every eukaryotic cell lies the cell nucleus, a complex structure consisting of multiple subnuclear structures and compartments. The nuclear envelope is a complex double membrane system that encloses the genome. On the outside the nuclear envelope is contiguous with the endoplasmic reticulum and connects to the cytoplasmic filament system, and on the inside, it connects to chromatin and the nuclear filament system. Batrakou, Kerr and Schirmer discuss pre-proteomic and proteomic analyses of the nuclear envelope and nuclear pore complexes. Recent MS-based proteomics has extended the existing parts lists and indicates besides the classical transport functions, multiple functions at the nuclear periphery, including signal transduction. The authors question whether all previously defined contaminant proteins are true contaminants (some of them are transiently interacting, and some of them may have multiple functions in the cell) and they stress the importance of using different purification procedures in combination with enrichment analysis (as opposed to subtractive analysis) to allow for identification of novel components and elucidation of associated functions.
The nuclear matrix has been postulated to be a determinant of nuclear structure, and to provide a dynamic protein scaffold with a central role in chromatin organization and chromosome function. Albrethsen, Knol and Jiménez review biochemical and a few proteomics studies that provide support for these functions. They discuss some of the controversies in the field, as well as proteomic evidence that the nuclear matrix and associated subnuclear domains are relevant for cancer biomarker discovery. The synapse is one of the most complex organelles, containing over 1500 proteins. The work of Klemmer, Smit and Li reported in this issue, combines organelle purification with analysis of immune-precipitated protein complexes to provide insight into the organization of the synapse. They focused on 8 known synaptic proteins involved in neuroplasticity. Known and novel interactions are reported, the latter awaiting validation in independent experiments. In addition, they constructed a protein–protein interaction map that highlights proteins shared by different complexes. Secreted proteins, proteins shed from the surface and exosomes together make up the so-called secretome. In this issue, Piersma, Broxterman and Jiménez et al report a highaccuracy activated platelet secretome along with extensive bioinformatics and annotation with information from previous platelet datasets, which represents the largest and most comprehensive analysis to date. This approach offers unique
3
JOU RN A L OF P ROT EO M IC S 7 2 ( 2 0 09 ) 1– 3
possibilities to analyse the role of platelet-secreted proteins in physiology and in diseases such as atherosclerosis and cancer. In the last original article of this issue, Mosley and Washburn et al. report a label-free quantitative proteomics approach, along with extensive bioinformatics, to characterize nuclear fractions isolated from yeast by sucrose gradient sedimentation. An interesting aspect is the integration with other large-scale data sets from yeast. A protein correlation profiling approach was employed based on normalized spectral counting. Cofractionation is shown for members of known protein complexes based on normalized spectral counting profiles. This study clearly shows the power of label-free quantitation based on a refined spectral counting method. From the papers in this special issue, it is clear that MSbased proteomics technologies have been powerful to confirm known organelle components and biology, and to generate new hypotheses that can be tested in targeted follow-up experiments. I envision that the coming few years will see a great increase in biologically-driven organelle and cell compartment studies that will assess dynamic changes in organelle constituents and shuttling between different compartments, following different stimuli and in different disease states. When all these analyses will be put together, a systems biology insight into cellular function may become a reality.
REFERENCES [1] Andersen JS, Mann M. Organellar proteomics: turning inventories into insights. EMBO Rep Sep 2006;7(9):874–9. [2] Au CE, Bell AW, Gilchrist A, Hiding J, Nilsson T, Bergeron JJ. Organellar proteomics to create the cell map. Curr Opin Cell Biol Aug 2007;19(4):376–85.
[3] Dimmer KS, Rapaport D. Proteomic view of mitochondrial function. Genome Biol 2008;9(2):209. Connie R. Jiménez, Ph.D. is a biologist and proteomics researcher with an interest in subcellular organelles and compartments in relation to function in health and disease. She is head of the OncoProteomics Laboratory in the VUmc-Cancer Center Amsterdam research building at the VU University Medical Center. From 1993–1997, during her graduate studies at the Vrije Universiteit in Amsterdam, she pioneered the use of MALDI-TOF mass spectrometry for semi-quantitative endogenous peptide profiling of single cells and tissue homogenates. Since her post-doc at UCSF (1997–1999, San Francisco, USA), she has used proteomics as a tool to solve a range of neuroscience-related questions. A current focus is on targeted biomarker discovery in cancer and neurodegenerate disease and on connecting disease-related proteins to altered protein networks and pathways (see www.oncoproteomics.nl ). In 2000, Dr. Jimenez initiated and since then coordinates the Netherlands Proteomics Platform. Dr. Jimenez participates in several international networks (European Proteomics Association, biomarker discovery committee of the HUPO-Brain project, and the International Cancer Biomarker Consortium). She is an editorial board member of the Journal of Proteomics.
Connie R. Jiménez OncoProteomics Laboratory, Department of Medical Oncology, VU University Medical Center, Amsterdam, The Netherlands
a v a i l a b l e a t w w w. s c i e n c e d i r e c t . c o m
w w w. e l s e v i e r. c o m / l o c a t e / j p r o t
Editorial
Sorting and zooming: Subcellular proteomics is booming! I am pleased to introduce to you this special issue of the Journal of Proteomics, dedicated to biochemical and proteomics insights into subcellular organelles and cell compartments. In the past decade, organelle proteomics has emerged as a powerful approach to provide information on the association of proteins with specific organelles along with implications for their structural organization and functions [1]. Classically, an organelle is one of several structures delimited by a lipid bilayer membrane with specialized functions, suspended in the cytoplasm of a eukaryotic cell. This special issue contains 7 reviews and 3 original articles on several organelles and also on two cell compartments not enclosed by a membrane, i.e., the nuclear matrix and the extracellular secretome. The organelles and (sub-)cellular compartments that are highlighted in this issue are indicated in Fig. 1. Many sub-cellular structures are covered in this issue. For reviews of the ER/Golgi system and mitochondria, the reader is referred to two recent reviews [2,3]. The first review of this special issue by Yan, Aebersold and Raines, provides an excellent historical overview of the technologies and strategies that have been used for organelle proteomics. They describe how proteomic analyses have matured from being simple catalogs to quantitative organelle signatures based on collections of marker proteins to which novel members can be assigned and (novel) functions inferred. Pros and cons of different approaches are discussed in the context of applications for selected organelles. They conclude that despite improved methods for organelle purification, complete removal of contaminant proteins is hard to achieve. Therefore, elimination of false-positives using subtractive or quantitative (differential) approaches is mandatory, and these strategies are discussed. Especially combining quantitative proteomics information on all purified fractions with advanced bioinformatics has allowed profiling multiple organelles simultaneously in only partially purified samples. Cellular signaling and communication takes place at the plasma membrane and membrane proteins represent drug targets for the majority of known drugs. Therefore, it is not surprising that many proteomics studies have focused on tackling the plasma membrane proteome. The second review of this issue, by Zheng and Foster, focuses on
biochemical approaches for the study of membrane microdomains. An extensive overview is provided of microdomain functions along with the biochemical methods to isolate them. Detergent-based and detergent-free methods as well as affinity-based methods are discussed for the isolation of detergent-resistant membranes, lipid rafts, caveolae, tetraspanin microdomains and GPI-anchored proteins. From the discussed examples, it is clear that the choice of detergent can have an enormous impact on the proteins recovered and further validation is needed before conclusions can be drawn. Lysosomes are single membrane-enclosed organelles that contain a multitude of enzymes capable of digestion of macromolecules in the cell. Callahan, Bagshaw and Mahuran review lysosome proteomics studies focused on the lysosome membrane, and discuss the role of the identified lysosomal proteins in vesicle trafficking. Lysosome biogenesis and associated storage disorders, as well as possible links to other pathologies such as cancer. The review of Xiao and Veenstra focuses on vesicles, including exosomes, membrane-enclosed vesicles with distinct functions, and matrix vesicles, important for formation of the extracellular matrix. Both pre-proteomic and proteomic studies are discussed in the context of known and novel functions as well as implications for developmental- and tumor biology and biomarker discovery. Importantly, results from proteomics indicate that both the extracellular matrix and vesicles play more specific and dynamic roles than simple structural support. Possible functions that are discussed include: intercellular communication, regulation of signal transduction, cell motility, chemotaxis and material exchange among cells. Neurons depend on trophic support from target cells for their differentiation and survival. The review on signaling endosomes by Wu and Mobley et al. summarizes current insights into the vesicular carriers of trophic signals that are picked up at axon terminals and are retrogradely transported back to the cell body to exert their trophic effect. Most evidence derives from an elegant series of hypothesis-driven immunochemical and molecular biological studies and points to signaling endosomes as the primary carrier of the trophic signal, though controversies still exist. The authors also
1874-3919/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.jprot.2009.01.016
2
JOUR NAL OF P ROT EOM IC S 7 2 ( 2009) 1– 3
Fig. 1 – Anatomy of the animal cell. Organelles and cellular components are indicated. THIS SPECIAL ISSUE CONTAINS REVIEWS ON THE PLASMA MEMBRANE AND MICRODOMAINS, THE NUCLEUS (I.E., NUCLEAR ENVELOPE, NUCLEAR PORE AND NUCLEAR MATRIX), LYSOSOME, SIGNALING ENDOSOMES, EXTRACELLULAR VESICLES AND AS WELL AS PRIMARY CONTRIBUTIONS ON SYNAPTIC PROTEIN COMPLEXES, SECRETED PROTEINS FROM PLATELETS AND BIOINFORMATICS APPROACHES APPLIED TO NUCLEAR FRACTIONS (Reproduced with permission from http://micro.magnet.fsu.edu/cells/animalcell.html, Acknowledgements Molecular Expressions).
summarize a few proteomics studies done on signaling endosomes in the context of retrograde EGFR signaling and stress the need for quantitative proteomics to delineate the identity and primary carrier of the retrograde signal and the mechanisms involved. At the heart of every eukaryotic cell lies the cell nucleus, a complex structure consisting of multiple subnuclear structures and compartments. The nuclear envelope is a complex double membrane system that encloses the genome. On the outside the nuclear envelope is contiguous with the endoplasmic reticulum and connects to the cytoplasmic filament system, and on the inside, it connects to chromatin and the nuclear filament system. Batrakou, Kerr and Schirmer discuss pre-proteomic and proteomic analyses of the nuclear envelope and nuclear pore complexes. Recent MS-based proteomics has extended the existing parts lists and indicates besides the classical transport functions, multiple functions at the nuclear periphery, including signal transduction. The authors question whether all previously defined contaminant proteins are true contaminants (some of them are transiently interacting, and some of them may have multiple functions in the cell) and they stress the importance of using different purification procedures in combination with enrichment analysis (as opposed to subtractive analysis) to allow for identification of novel components and elucidation of associated functions.
The nuclear matrix has been postulated to be a determinant of nuclear structure, and to provide a dynamic protein scaffold with a central role in chromatin organization and chromosome function. Albrethsen, Knol and Jiménez review biochemical and a few proteomics studies that provide support for these functions. They discuss some of the controversies in the field, as well as proteomic evidence that the nuclear matrix and associated subnuclear domains are relevant for cancer biomarker discovery. The synapse is one of the most complex organelles, containing over 1500 proteins. The work of Klemmer, Smit and Li reported in this issue, combines organelle purification with analysis of immune-precipitated protein complexes to provide insight into the organization of the synapse. They focused on 8 known synaptic proteins involved in neuroplasticity. Known and novel interactions are reported, the latter awaiting validation in independent experiments. In addition, they constructed a protein–protein interaction map that highlights proteins shared by different complexes. Secreted proteins, proteins shed from the surface and exosomes together make up the so-called secretome. In this issue, Piersma, Broxterman and Jiménez et al report a highaccuracy activated platelet secretome along with extensive bioinformatics and annotation with information from previous platelet datasets, which represents the largest and most comprehensive analysis to date. This approach offers unique
3
JOU RN A L OF P ROT EO M IC S 7 2 ( 2 0 09 ) 1– 3
possibilities to analyse the role of platelet-secreted proteins in physiology and in diseases such as atherosclerosis and cancer. In the last original article of this issue, Mosley and Washburn et al. report a label-free quantitative proteomics approach, along with extensive bioinformatics, to characterize nuclear fractions isolated from yeast by sucrose gradient sedimentation. An interesting aspect is the integration with other large-scale data sets from yeast. A protein correlation profiling approach was employed based on normalized spectral counting. Cofractionation is shown for members of known protein complexes based on normalized spectral counting profiles. This study clearly shows the power of label-free quantitation based on a refined spectral counting method. From the papers in this special issue, it is clear that MSbased proteomics technologies have been powerful to confirm known organelle components and biology, and to generate new hypotheses that can be tested in targeted follow-up experiments. I envision that the coming few years will see a great increase in biologically-driven organelle and cell compartment studies that will assess dynamic changes in organelle constituents and shuttling between different compartments, following different stimuli and in different disease states. When all these analyses will be put together, a systems biology insight into cellular function may become a reality.
REFERENCES [1] Andersen JS, Mann M. Organellar proteomics: turning inventories into insights. EMBO Rep Sep 2006;7(9):874–9. [2] Au CE, Bell AW, Gilchrist A, Hiding J, Nilsson T, Bergeron JJ. Organellar proteomics to create the cell map. Curr Opin Cell Biol Aug 2007;19(4):376–85.
[3] Dimmer KS, Rapaport D. Proteomic view of mitochondrial function. Genome Biol 2008;9(2):209. Connie R. Jiménez, Ph.D. is a biologist and proteomics researcher with an interest in subcellular organelles and compartments in relation to function in health and disease. She is head of the OncoProteomics Laboratory in the VUmc-Cancer Center Amsterdam research building at the VU University Medical Center. From 1993–1997, during her graduate studies at the Vrije Universiteit in Amsterdam, she pioneered the use of MALDI-TOF mass spectrometry for semi-quantitative endogenous peptide profiling of single cells and tissue homogenates. Since her post-doc at UCSF (1997–1999, San Francisco, USA), she has used proteomics as a tool to solve a range of neuroscience-related questions. A current focus is on targeted biomarker discovery in cancer and neurodegenerate disease and on connecting disease-related proteins to altered protein networks and pathways (see www.oncoproteomics.nl ). In 2000, Dr. Jimenez initiated and since then coordinates the Netherlands Proteomics Platform. Dr. Jimenez participates in several international networks (European Proteomics Association, biomarker discovery committee of the HUPO-Brain project, and the International Cancer Biomarker Consortium). She is an editorial board member of the Journal of Proteomics.
Connie R. Jiménez OncoProteomics Laboratory, Department of Medical Oncology, VU University Medical Center, Amsterdam, The Netherlands