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- Bergström Lind, Sara, et al.
(författare)
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Immunoaffinity Enrichments Followed by Mass Spectrometric Detection for Studying Global Protein Tyrosine Phosphorylation
- 2008
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Ingår i: Journal of Proteome Research. - : American Chemical Society (ACS). - 1535-3893 .- 1535-3907. ; 7:7, s. 2897-2910
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Tidskriftsartikel (refereegranskat)abstract
- Phosphorylation of protein tyrosine residues regulates important cell functions and is, when dysregulated, often crucially involved in oncogenesis. It is therefore important to develop and evaluate methods for identifying and studying tyrosine phosphorylated (P-Tyr) proteins. P-Tyr proteins are present at very low concentrations within cells, requiring highly selective enrichment methods to be detected. In this study, we applied immunoaffinity as enrichment step for P-Tyr proteins. Five selected anti-phosphotyrosine antibodies (monoclonal antibodies 4G10, PY100, PYKD1, 13F9 and one polyclonal antiserum) were evaluated with respect to their capability to enrich P-Tyr proteins from cell extracts of the K562 leukemia cell line. The enrichment resulted in the detection of a group of proteins that potentially were tyrosine-phosphorylated (putative P-Tyr proteins). High accuracy identification of actual P-Tyr sites were performed using a highly selective and sensitive liquid chromatography Fourier transform mass spectrometer (LC-FTMS) setup with complementary collision activated dissociation (CAD) and electron capture dissociation (ECD) fragmentations. 4G10 and PY100 antibodies recognized the greatest number of putative P-Tyr proteins in initial screening experiments and were therefore further evaluated and compared in immunoaffinity enrichment of both P-Tyr proteins and peptides. Using the 4G10 antibody for enrichment of proteins, we identified 459 putative P-Tyr proteins by MS. Out of these proteins, 12 were directly verified as P-Tyr proteins by MS analysis of the actual site. Using the PY100 antibody for enrichment of peptides, we detected 67 P-Tyr peptides (sites) and 89 putative P-Tyr proteins. Generally, enrichment at the peptide level made it difficult to reliably determine the identity of the proteins. In contrast, protein identification following immunoaffinity enrichment at the protein level gave greater sequence coverage and thus a higher confidence in the protein identification. By combining all available information, 40 proteins were identified as true P-Tyr proteins from the K562 cell line. In conclusion, this study showed that a combination of immunoaffinity enrichment using multiple antibodies of both intact and digested proteins in parallel experiments is required for best possible coverage of all possible P-Tyr proteins in a sample.
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| 2. |
- Sjöberg, Lina, 1973-
(författare)
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Genesis och Jernet : ett möte mellan Sara Lidmans Jernbaneepos och bibelns berättelser
- 2006
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- The subject of this dissertation is intertextuality between the Hebrew Bible and a piece of twentieth century Swedish fiction. The main purpose is to create analytical tools and to find a way to deal with fragmentary intertextuality between bible and literature. The material dealt with is a modern suite of novels, Jernbarneeposet (“The Railroad Epic”) by Sara Lidman, on the one hand, and the stories about Abraham, Sarah and Hagar in Genesis 16 and 21 on the other. I suggest a model for analyzing fragmentary references, arguing that the process of interpretation becomes more transparent, and thus more open to critical discussion, if the interpreter deals with each separate reference on three levels – “marker”, “means” and “effect”. It is assumed that the reader of fragmentary intertextuality becomes engaged in a number of small hermeneutical operations. The purpose of systematization is to organize and articulate each step in such an operation. The second half of the dissertation takes its point of departure from an observation that the failure to interpret the significance of the personal relationships indicated in several Genesis stories is common to both traditional and postmodern biblical scholarship. Therefore, I investigate the possibility of using modern literary fiction as a tool in interpreting the emotional aspects of the Abraham, Sarah and Hagar stories. I propose that it is possible to make use of modern literary works in biblical scholarship, not as commentaries, but as inspiration. The question discussed is: How then, can this inspiration be integrated in serious biblical scholarship?
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| 3. |
- Weischenfeldt, Joachim, et al.
(författare)
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NMD is essential for hematopoietic stem and progenitor cells and for eliminating by-products of programmed DNA rearrangements
- 2008
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Ingår i: Genes & Development. - : Cold Spring Harbor Laboratory. - 1549-5477 .- 0890-9369. ; 22:10, s. 1381-1396
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Tidskriftsartikel (refereegranskat)abstract
- Nonsense-mediated mRNA decay (NMD) is a post-transcriptional surveillance process that eliminates mRNAs containing premature termination codons (PTCs). NMD has been hypothesized to impact on several aspects of cellular function; however, its importance in the context of a mammalian organism has not been addressed in detail. Here we use mouse genetics to demonstrate that hematopoietic-specific deletion of Upf2, a core NMD factor, led to the rapid, complete, and lasting cell-autonomous extinction of all hematopoietic stem and progenitor populations. In contrast, more differentiated cells were only mildly affected in Upf2-null mice, suggesting that NMD is mainly essential for proliferating cells. Furthermore, we show that UPF2 loss resulted in the accumulation of nonproductive rearrangement by-products from the Tcrb locus and that this, as opposed to the general loss of NMD, was particularly detrimental to developing T-cells. At the molecular level, gene expression analysis showed that Upf2 deletion led to a profound skewing toward up-regulated mRNAs, highly enriched in transcripts derived from processed pseudogenes, and that NMD impacts on regulated alternative splicing events. Collectively, our data demonstrate a unique requirement of NMD for organismal survival.
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