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Refined mass spec for proteomics
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than five nuclei. The total number of myonuclei showed a 50% decrease in the mutants. In addition, when mutants were infected with retrovirus expressing NFATC2, they could be rescued and the resulting myotubes were similar to those of wild-type animals.
TRENDS in Cell Biology Vol.11 No.7 July 2001
These results show that the mutant myoblasts could differentiate into multinucleated cells, and the reduced CSA of the mutant muscles appears to be related to the suppression of continuous myoblast fusion in the middle of the myofibers in a way that does not affect the morphology of the muscle cells.
283
1 Horsley, V. et al. (2001) Regulation of the growth of multinucleated muscle cells by an NFATC2-dependent pathway. J. Cell Biol. 153, 329–338
Chung L. Lau [email protected]
Cell hypertrophy might be necessary but is not sufficient for cell division The control of animal size necessitates a regulation of the size of each organ, the latter requiring fine-tuning of cell size and cell number1. To this end, one of the most puzzling present questions in cell biology is the apparent discrepancy between the very general and common activation of signal transduction (ST) pathways by hormones and growth factors and the very specific final effects of these extracellular signals. One glaring such discrepancy is the fact that growth factors with apparently similar effects on ST can induce in the same cells either cell hypertrophy or cell proliferation. This is the case of angiotensin II (AII) and PDGF.BB in rat aortic smooth muscle cells2 and insulin-like growth factor 1 (IGF1) and thyroid-stimulating hormone (TSH)/cyclic AMP in dog thyroid cells3. In the former case, Servant and colleagues show that initial events triggered by both AII and PDGF.BB are apparently similar up to the synthesis of cyclins D1, D2
more the case in cell biology, generalization could be unwarranted. For example, in the case of dog thyroid cells, the TSH cyclic AMP cascade, which triggers (unlike IGF1) DNA synthesis and cell division, also increases (unlike IGF1) accumulation of p27Kip1!
and D3 and activation of cyclin-dependent kinase 4 (CDK4). But, unlike PDGF.BB, AII has incomplete effects and fails to induce cyclin E and to phosphorylate and activate CDK2 (Ref. 2). This failure, the authors postulate, relates to a persistence of the cyclin CDK inhibitor p27Kip1, whose levels are decreased by PDGF.BB by inhibition of synthesis at the transcription level and increased degradation of the mRNA and protein. Thus, the failure of AII to drive progression from cell hypertrophy to DNA synthesis and cell division could be ascribed to a failure to remove the p27Kip1 block to cyclin–CDK function. The differences in the pattern of activation of early ST pathways that could explain this difference are unknown. They might be quantitative and kinetic rather than qualitative – or something could be missing from our current level of understanding. On the other hand, as is becoming more and
Jacques E. Dumont [email protected]
enrichment [Oda, Y. et al. (2001) Nat. Biotechnol. 19, 379–382]. In a similar chemistry-based approach, the lab of Ruedi Aebersold from the Institute for Systems Biology in Seattle replaced phosphate groups with a thiol-based linker to achieve essentially the same goal [Zhou, H. et al. (2001) Nat. Biotechnol. 19, 375–378]. These approaches now permit the identification of serine/threonine phosphorylation as well as phosphorylation on tyrosine. Antibodies against phosphotyrosine are suitable for affinity purification of target proteins for analysis, whereas antibodies to phosphoserine/threonine are not. The new methods both yield a mass of information from a single experiment as well as lending themselves to automation. Many classes of protein are typically lost when samples are analysed by conventional mass
spectrometric analysis. A further paper, from the group of John R. Yates III at the Scripps Research Institute in California, describes a new method for the large-scale analysis of protein samples by mass spectrometry [Washburn, M.P. et al. (2001) Nat. Biotechnol. 19, 242–247]. The wonderfully titled MudPIT technique (for ‘Multi-Dimensional Protein Identification Technology’) eliminates the need for 2D gel electrophoresis of samples and instead employs 2D liquid chromatography. This approach facilitates the inclusion of several classes of protein routinely missed in other analyses – large, peripheral and integral membrane proteins were all identified with the new method. Analysis of a yeast whole cell lysate identified 1484 different proteins, far more than have previously been identified in a single study. D.S.
1 Potter, C.J. et al. (2001) Drosophila tsc1 functions with tsc2 to antagonize insulin signaling in regulating cell growth, cell proliferation and organ size. Cell 105, 357–368 2 Servant, M.J. et al. (2000) Differential regulation of p27Kip1 expression by mitogenic and hypertrophic factors: involvement of transcriptional and posttranscriptional mechanisms. J. Cell Biol. 148, 543–556 3 Depoortere, F. et al. (2000) Transforming growth factor β1 selectively inhibits the cyclic AMPdependent proliferation of primary thyroid epithelial cells by preventing the association of cyclin D3–cdk4 with nuclear p27kip1. Mol. Biol. Cell 11, 1061–1076
In Brief
Refined mass spec for proteomics Three papers published in Nature Biotechnology earlier this year describe advanced methods for the identification of proteins using mass spectrometry. Two of the papers describe approaches to detect phosphorylated proteins using chemical approaches, while the third describes a method to capture those proteins that are routinely missed in screening. The detection of phosphorylated proteins by these approaches is aided by the simple method of enriching for phosphorylated proteins before analysis. The group of Brian T. Chait at Rockefeller University in New York used chemical replacement of phosphate groups with biotin followed by avidin-based capture to achieve this
http://tcb.trends.com 0962-8924/01/$ – see front matter © 2001 Elsevier Science Ltd. All rights reserved.
than five nuclei. The total number of myonuclei showed a 50% decrease in the mutants. In addition, when mutants were infected with retrovirus expressing NFATC2, they could be rescued and the resulting myotubes were similar to those of wild-type animals.
TRENDS in Cell Biology Vol.11 No.7 July 2001
These results show that the mutant myoblasts could differentiate into multinucleated cells, and the reduced CSA of the mutant muscles appears to be related to the suppression of continuous myoblast fusion in the middle of the myofibers in a way that does not affect the morphology of the muscle cells.
283
1 Horsley, V. et al. (2001) Regulation of the growth of multinucleated muscle cells by an NFATC2-dependent pathway. J. Cell Biol. 153, 329–338
Chung L. Lau [email protected]
Cell hypertrophy might be necessary but is not sufficient for cell division The control of animal size necessitates a regulation of the size of each organ, the latter requiring fine-tuning of cell size and cell number1. To this end, one of the most puzzling present questions in cell biology is the apparent discrepancy between the very general and common activation of signal transduction (ST) pathways by hormones and growth factors and the very specific final effects of these extracellular signals. One glaring such discrepancy is the fact that growth factors with apparently similar effects on ST can induce in the same cells either cell hypertrophy or cell proliferation. This is the case of angiotensin II (AII) and PDGF.BB in rat aortic smooth muscle cells2 and insulin-like growth factor 1 (IGF1) and thyroid-stimulating hormone (TSH)/cyclic AMP in dog thyroid cells3. In the former case, Servant and colleagues show that initial events triggered by both AII and PDGF.BB are apparently similar up to the synthesis of cyclins D1, D2
more the case in cell biology, generalization could be unwarranted. For example, in the case of dog thyroid cells, the TSH cyclic AMP cascade, which triggers (unlike IGF1) DNA synthesis and cell division, also increases (unlike IGF1) accumulation of p27Kip1!
and D3 and activation of cyclin-dependent kinase 4 (CDK4). But, unlike PDGF.BB, AII has incomplete effects and fails to induce cyclin E and to phosphorylate and activate CDK2 (Ref. 2). This failure, the authors postulate, relates to a persistence of the cyclin CDK inhibitor p27Kip1, whose levels are decreased by PDGF.BB by inhibition of synthesis at the transcription level and increased degradation of the mRNA and protein. Thus, the failure of AII to drive progression from cell hypertrophy to DNA synthesis and cell division could be ascribed to a failure to remove the p27Kip1 block to cyclin–CDK function. The differences in the pattern of activation of early ST pathways that could explain this difference are unknown. They might be quantitative and kinetic rather than qualitative – or something could be missing from our current level of understanding. On the other hand, as is becoming more and
Jacques E. Dumont [email protected]
enrichment [Oda, Y. et al. (2001) Nat. Biotechnol. 19, 379–382]. In a similar chemistry-based approach, the lab of Ruedi Aebersold from the Institute for Systems Biology in Seattle replaced phosphate groups with a thiol-based linker to achieve essentially the same goal [Zhou, H. et al. (2001) Nat. Biotechnol. 19, 375–378]. These approaches now permit the identification of serine/threonine phosphorylation as well as phosphorylation on tyrosine. Antibodies against phosphotyrosine are suitable for affinity purification of target proteins for analysis, whereas antibodies to phosphoserine/threonine are not. The new methods both yield a mass of information from a single experiment as well as lending themselves to automation. Many classes of protein are typically lost when samples are analysed by conventional mass
spectrometric analysis. A further paper, from the group of John R. Yates III at the Scripps Research Institute in California, describes a new method for the large-scale analysis of protein samples by mass spectrometry [Washburn, M.P. et al. (2001) Nat. Biotechnol. 19, 242–247]. The wonderfully titled MudPIT technique (for ‘Multi-Dimensional Protein Identification Technology’) eliminates the need for 2D gel electrophoresis of samples and instead employs 2D liquid chromatography. This approach facilitates the inclusion of several classes of protein routinely missed in other analyses – large, peripheral and integral membrane proteins were all identified with the new method. Analysis of a yeast whole cell lysate identified 1484 different proteins, far more than have previously been identified in a single study. D.S.
1 Potter, C.J. et al. (2001) Drosophila tsc1 functions with tsc2 to antagonize insulin signaling in regulating cell growth, cell proliferation and organ size. Cell 105, 357–368 2 Servant, M.J. et al. (2000) Differential regulation of p27Kip1 expression by mitogenic and hypertrophic factors: involvement of transcriptional and posttranscriptional mechanisms. J. Cell Biol. 148, 543–556 3 Depoortere, F. et al. (2000) Transforming growth factor β1 selectively inhibits the cyclic AMPdependent proliferation of primary thyroid epithelial cells by preventing the association of cyclin D3–cdk4 with nuclear p27kip1. Mol. Biol. Cell 11, 1061–1076
In Brief
Refined mass spec for proteomics Three papers published in Nature Biotechnology earlier this year describe advanced methods for the identification of proteins using mass spectrometry. Two of the papers describe approaches to detect phosphorylated proteins using chemical approaches, while the third describes a method to capture those proteins that are routinely missed in screening. The detection of phosphorylated proteins by these approaches is aided by the simple method of enriching for phosphorylated proteins before analysis. The group of Brian T. Chait at Rockefeller University in New York used chemical replacement of phosphate groups with biotin followed by avidin-based capture to achieve this
http://tcb.trends.com 0962-8924/01/$ – see front matter © 2001 Elsevier Science Ltd. All rights reserved.