Analysis of T-Cell Leukemia/Lymphoma (TCL1) Oncoprotein Interacting Proteins by Immunoprecipitation and Tandem Mass Spectrometry.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 3566-3566
Author(s):  
Kojo S.J. Elenitoba-Johnson ◽  
Venkatesha Basrur ◽  
Adam Kronk ◽  
Charles Seiler ◽  
Damian Fermin ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Cell Adhesion ◽  
T Cell ◽  
Tandem Mass Spectrometry ◽  
B Cell ◽  
Cell Line ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
B Cell Lymphoma ◽  
Tandem Mass

Abstract TCL1 is a proto-oncogene whose deregulation has been implicated in the pathogenesis of T- and B-cell lymphoproliferative disorders. TCL1 expression is predominantly observed in developing B lymphocytes, whereas its overexpression in T cells is associated with mature T-cell lymphoproliferation in transgenic mice. Although recent studies indicate that dysregulated expression of TCL1 is important in mature B-cell transformation, very little is currently known about the function or interactions of TCL1, specifically in the B-cell context. In this study, we hypothesized that identification of proteins that interact with TCL1 may facilitate our understanding of the functional properties of TCL1. Proteomic analysis provides an opportunity to carry out functional studies of protein-protein interactions and characterization of functional interactomes. Using a functional proteomic approach, we determined the identity of proteins that interact with TCL1 by co-immunoprecipitation with anti-TCL1 antibody followed by liquid chromatography (LC), electrospray ionization (ESI) and tandem mass spectrometry (MS/MS). Immunoprecipitates of the TCL1 expressing SUDHL-16 B-cell lymphoma derived cell line were compared to that of hyperimmune rabbit immunoglobulin and a cell line that does not express TCL1 (SUDHL-1) by 1-dimensional sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE). Silver-stained protein bands from both immunocomplexes were excised and analyzed by ESI-LC-MS/MS. Proteins which were unique to the TCL1 immunocomplex included transcription factors (Jun B), phosphatases (protein phosphatase 2), ubiquitin-associated proteins (ubiquitin activating enzyme), cell adhesion proteins (ADP ribosylation factor 3) and proteins associated with apoptosis regulation (peroxidoxin 1 and scaffold attachment factor). Analysis of subcellular fractions demonstrated that TCL1 and complexes were localized to both the nuclear and cytoplasmic compartments. Importantly, known TCL1 interactors such as histone 3 were uniquely identified in the TCL1 complex. Proteins identified by MS were confirmed by western blotting and reciprocal immunoprecipitation. This study reveals novel TCL1 interactors and indicates that the protein may exert diverse cellular effects impacting apoptosis, cell survival, cytoskeletal organization and cell adhesion. Our interactome analysis provides unique insights into the cellular function of TCL1, a protein whose deregulation is increasingly implicated in the pathogenesis of a variety of hematopoietic neoplasms.

scholarly journals A Cysteine-Reactive Small Photo-Crosslinker Possessing Caged-Fluorescence Properties: Binding-Site Determination of a Combinatorially-Selected Peptide by Fluorescence Imaging/Tandem Mass Spectrometry

2018 ◽  
Vol 19 (11) ◽  
pp. 3682
Author(s):  
Kazuki Yatabe ◽  
Masaru Hisada ◽  
Yudai Tabuchi ◽  
Masumi Taki
Keyword(s):  
Mass Spectrometry ◽  
Tandem Mass Spectrometry ◽  
Binding Site ◽  
Fluorescence Imaging ◽  
Polyacrylamide Gel Electrophoresis ◽  
Stokes Shift ◽  
Sodium Dodecyl ◽  
Tandem Mass ◽  
Sds Page ◽  
Sh Group

To determine the binding-site of a combinatorially-selected peptide possessing a fluoroprobe, a novel cysteine reactive small photo-crosslinker that can be excited by a conventional long-wavelength ultraviolet handlamp (365 nm) was synthesized via Suzuki coupling with three steps. The crosslinker is rationally designed, not only as a bioisostere of the fluoroprobe, but as a caged-fluorophore, and the photo-crosslinked target protein became fluorescent with a large Stokes-shift. By introducing the crosslinker to a designated sulfhydryl (SH) group of a combinatorially-selected peptide, the protein-binding site of the targeted peptide was deduced by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE)/fluorescence imaging followed by matrix-assisted laser desorption ionization-time of flight tandem mass spectrometry (MALDI-TOF-MS/MS) analysis.

scholarly journals Quantification of Gly m 5.0101 in Soybean and Soy Products by Liquid Chromatography-Tandem Mass Spectrometry

Molecules ◽  
2018 ◽  
Vol 24 (1) ◽  
pp. 68 ◽  
Author(s):  
Hongmin Jia ◽  
Tianjiao Zhou ◽  
Hong Zhu ◽  
Li Shen ◽  
Pingli He
Keyword(s):  
Mass Spectrometry ◽  
Liquid Chromatography ◽  
Tandem Mass Spectrometry ◽  
Protein Extraction ◽  
Multiple Reaction Monitoring ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Tandem Mass ◽  
Chromatography Tandem Mass Spectrometry ◽  
Liquid Chromatography Tandem Mass

Gly m 5.0101, the alpha subunit of β-conglycinin, is one of the major allergens found in soybeans that has been identified as causing an allergic reaction. Here, we developed a quantification method of Gly m 5.0101 with multiple reaction monitoring using the synthetic peptide 194NPFLFGSNR202 as the external standard. Firstly, the ground soybean was defatted and extracted with a protein extraction buffer. Then the crude extract was on-filter digested by trypsin and analyzed by liquid chromatography-tandem mass spectrometry. The selected peptide exhibited a detection limit of 0.48 ng/mL and a linear relationship in a concentration range from 1.6 to 500 ng/mL (r2 > 0.99). The developed method was successfully applied to quantify the Gly m 5.0101 level in dozens of soybean varieties from different sources and soybean products derived from different processing techniques. The developed method could be used to further analyze β-conglycinin in soybean seeds combined with sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis.

Distinct Interaction Networks of the MUM1/IRF4 in B- and T-Cell Lymphomas Identified by Tandem Mass Spectrometry.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 3925-3925
Author(s):  
Ali Gabali ◽  
Kevin P Conlon ◽  
Damian Fermin ◽  
Venkatesha Basrur ◽  
Megan S Lim ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
T Cell ◽  
Tandem Mass Spectrometry ◽  
B Cell ◽  
Interaction Networks ◽  
Tandem Mass ◽  
Interacting Proteins ◽  
Specific Context ◽  
T Cell Lymphomas ◽  
Multiple Myelomas

Abstract Abstract 3925 Poster Board III-861 Introduction Multiple myeloma oncogene 1/interferon regulatory factor 4 (MUM1/IRF4) is a 52-kDa transcriptional activator protein which plays an important role in interferon-stimulated response element (ISRE)-regulated signal transduction mechanisms important in lymphoid cell development and differentiation. MUM1/IRF4 is also critical for the generation of immunoglobulin-secreting plasma cells. MUM1/IRF4 is expressed in the activated B-cell (ABC)-like subset of diffuse large B-cell lymphomas (DLBCL) and is targeted by chromosomal translocations in a subset of multiple myelomas and peripheral T-cell lymphomas. Despite its importance in lymphocyte and lymphoma biology, and its addiction demonstrated in multiple myelomas, the proteomic network of MUM1/IRF4 interacting proteins has not been determined. Methods We determined the proteomic interaction networks of MUM1/IRF4 in B-cell and T-cell lymphoma contexts, by using a functional proteomic approach. Total cell lysates were prepared from MUM1/IRF4 expressing cell lines including OCI-LY10 ((ABC)-like DLBCL) and HH (mycosis fungoides derived-T-cell) and, as a negative control, from non-MUM1/IRF4 expressing SUDHL4 cell line. Immunoprecipitates of the MUM1/IRF4 expressing B- and T-cell line were compared to that of hyperimmune mouse immunoglobulin by 1-dimensional sodium dodecyl sulphate polyacrylamide gel electrophoresis. Coomassie-stained protein bands from both immunocomplexes were excised and analyzed by electrospray liquid chromatography tandem mass spectrometry (ESI-LC/MS/MS). Peptide sequences were identified by searching the MS/MS data against human IPI protein using database X!Tandem with k-score plug-in. Proteins found in the two control sets (SUDHL4 and IgG isotype) were manually subtracted from the proteins found in the experimental set. MS/MS results were validated using immunoprecipitation and Western blotting and some of the interacting proteins were further confirmed by reciprocal immunoprecipitation. Results A total of 163 and 94 proteins were identified from 12 Coomassie-stained bands which were unique to the MUM1/IRF4 immunocomplex in OCI-Ly10 and HH cell lines, respectively. Previously reported proteins in the MUM1/IRF4 signal pathway were identified including FK506 and TRAF family protein. More importantly, many proteins previously not associated with MUM1/IRF4 were identified. In common to both subsets were proteins such as ARHGDIA-involved in RhoA-mediated signaling. In the ABC-like B-cell (OCI-LY10) specific context, these included proteins known to be critical regulators of lymphocyte proliferation (RAB2A), motility, trafficking and cell adhesion. In the T-cell specific context, proteins known to play important roles in endocytosis (Reggies) and T-cell activation (GDI2, Guanine nucleotide exchange factors) were identified in the MUM1/IRF4 interactome. A subset of the proteins identified by MS were confirmed by western blotting and reciprocal immunoprecipitation. Conclusions Our studies reveal that although a minor subset of MUM1/IRF4 interacting proteins are common in B- and T-cells, MUM1/IRF4 exhibits distinct interaction partners dependent of specific cellular contexts. The diverse interaction networks implicate MUM1/IRF4 in previously undescribed functional roles in lymphocyte-specific subsets. Comprehensive elucidation of the protein-protein interaction networks in the specific cellular contexts will provide opportunities for exploitation of the knowledge for design of rational interventions targeting the critical nodes and modules in MUM1/IRF4-deregulation in B- and T-cell malignancies. Disclosures: No relevant conflicts of interest to declare.

Development of B Cell Lymphoma in Eμ-Cyclin D1 X Mutant p53 Transgenic Mice.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 1907-1907
Author(s):  
Mitchell R. Smith ◽  
Indira J. Joshi ◽  
Fang Jin ◽  
Tahseen Al-Saleem
Keyword(s):  
T Cell ◽  
Transgenic Mice ◽  
B Cell ◽  
Cyclin D1 ◽  
Cell Line ◽  
Cell Lymphoma ◽  
B Cell Lymphoma ◽  
Mutant P53 ◽  
B Cell Lymphomas ◽  
T Cell Lymphomas

Abstract Background: Mantle cell lymphoma (MCL) is characterized by t(11;14) which dysregulates cyclin D1 expression. Eμ-cyclinD1 transgenic mice, however, are healthy. Additional genetic events must be necessary for lymphomagenesis, and knowledge of these would enhance understanding and therapy of MCL. In addition, a mouse model of MCL would be helpful in drug development. Alterations in p53 have been described in MCL, often associated with the blastic variant. Objectives and Methods: To determine whether p53 and cyclin D1 can cooperate in lymphomagenesis, we cross bred Eμ-cyclinD1 transgenic mice (Bodrug et al EMBO J, 1996, courtesy of Alan Harris) with mice transgenic for mutant p53 (Jackson Labs, Jacks et al Curr Biol, 1994). Progeny mice were monitored for presence of the transgenes by PCR of tail vein DNA and observed for development of disease. Results: Of mice carrying both aberrant genes, 24 of 38 developed B cell lymphoma. Mice did not become visibly ill until at least 12 months of age, with median age at sacrifice 15.5 (range 12–23) months. The lymphoma was generally disseminated, involving spleen, liver, diffuse adenopathy and marrow with occasional extranodal sites. Histology varied between small and large cell, with some having a vaguely follicular growth pattern. T cell lymphomas occurred in 2 other mice, while 5 developed osteosarcoma (1 of these in a mouse that also had B cell lymphoma). The B cell lymphomas were clonal by Cμ-VH PCR. Cyclin D1 expression was documented by Western analysis. A cell line has also been developed from one of the B cell lymphomas and this line rapidly grows into disseminated lymphoma in syngeneic mice. These B cell lymphomas differ from the thymic T cell lymphomas seen in heterozygous p53 mutant mice that do not co-express cyclin D1. The latency period differs from cyclin D1 x myc double transgenic mice. Conclusions: This model demonstrates cooperation between p53 and cyclin D1 pathways in B cell lymphomagenesis and should prove useful in delineating how these signals interact. The cell line may prove useful in pre-clinical testing of new agents for MCL.

scholarly journals Histopathological and immunophenotypical characterization of canine multicentric lymphoma in Brazil: a study of 203 cases

2020 ◽  
Vol 72 (3) ◽  
pp. 787-793
Author(s):  
P.C. Jark ◽  
C.P. Fracacio ◽  
L.A. Anai ◽  
M.C.L. Silva ◽  
S.G. Calazans ◽  
...  
Keyword(s):  
T Cell ◽  
B Cell ◽  
Cell Line ◽  
B Cell Lymphoma ◽  
High Grade ◽  
Canine Lymphoma ◽  
The Usa ◽  
T Cell Lines ◽  
Multicentric Lymphoma

ABSTRACT The immunophenotype is regarded as an independent prognostic factor in high-grade lymphomas, seeing that lymphomas of T-cell origin are associated with shorter survival time. Although a number of studies have evaluated the immunophenotypical profile of lymphoma in the USA and Europe, Brazilian research on the matter remains scarce. Exact characterization of the histopathological type is crucial to establish proper treatment and prognosis. This study evaluated the database of immunohistochemistry laboratories that perform immunophenotyping of canine lymphoma in Brazil. A total of 203 cases of multicentric lymphoma were classified according to the WHO classification. Immunophenotyping was able to identify 71.4% lymphomas of B-cell line, 27.1% of T-cell line and 1.5% of non-B cells and non-T cell lines. Diffuse large B-cell lymphoma was the most common with 59.1% of the cases. Among T-cell lymphomas, lymphoblastic was the most common (11.33% of the cases). Even though canine lymphomas tend to be high-grade, indolent lymphomas comprised 11.82% of the cases and T-zone lymphoma was the most prevalent (8.86%). The immunophenotype of multicentric lymphoma in Brazil is similar to those in other parts of the world, which suggests similar etiologic factors to the development of this disease.

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