Originally published In Press as doi:10.1074/mcp.R600005-MCP200 on June 19, 2006.
Molecular & Cellular Proteomics 5:1787-1798, 2006.
© 2006 by The American Society for Biochemistry and Molecular Biology, Inc.
Cancer
Mass Spectrometry-based Proteomic Studies of Human Anaplastic Large Cell Lymphoma*
Megan S. Lim
and
Kojo S. J. Elenitoba-Johnson
From the Department of Pathology and Associated Regional and University Pathologists Institute for Clinical and Experimental Pathology, University of Utah Health Sciences Center, Salt Lake City, Utah 84132
 |
ABSTRACT
|
|---|
Malignant lymphomas are a diverse group of malignant neoplasms that arise as a result of a complex interplay of multiple factors including genetic aberrations, immunosuppression, and exposure to noxious agents such as ionizing radiation and chemical agents. Anaplastic large cell lymphoma (ALCL) is an aggressive T-lineage lymphoma harboring chromosomal translocations involving the anaplastic lymphoma kinase (ALK) tyrosine kinase. The most common translocation in ALCL is the t(2;5)(p23;q35). This results in the formation of a chimeric fusion kinase, nucleophosmin/ALK. Nucleophosmin/ALK activates numerous downstream signaling pathways resulting in enhanced survival and proliferation. Using a variety of mass spectrometry-driven proteomic strategies, we have studied several aspects of the ALCL proteome. In this review, we provide a summary of mass spectrometry-based proteomic studies that expands the current understanding of the molecular pathogenesis of ALCL and provides the basis for the identification of biomarkers and targets for novel therapeutic agents.
Human malignant lymphomas are clonal proliferations of lymphocytes that arise from B- or T-cells and are heterogeneous with respect to their clinical presentation, pathogenesis, and biologic behavior. Hodgkin lymphoma and non-Hodgkin lymphoma (NHL)1 are the two major categories of malignant lymphomas recognized (1). The overall incidence of NHL increased by about 75% between 1973 and 1991 and now accounts for 
54,370 new malignancies diagnosed annually in the United States (1). Important advances in the understanding of the molecular biology and immunology of the lymphocytes and related cells have culminated in the World Health Organization classification scheme that incorporates the clinical, histopathologic, immunophenotypic, and molecular genetic characteristics of the lymphomas and defines distinct pathobiologic entities.
NHLs arise as a result of multiple factors, including genetic aberrations, congenital or acquired immunosuppression, exposure to ionizing radiation, and chemical agents. In many cases, the primary abnormality is a cytogenetic aberration that is consistently associated with a particular subtype of malignant lymphoma. The cytogenetic abnormality is frequently a nonrandom chromosomal translocation that leads to juxtaposition of a proto-oncogene (e.g. c-myc) to one of the antigen receptor genes (immunoglobulin heavy chain or T-cell receptor). Alternatively chromosomal translocations may lead to the formation of fusion proteins with oncogenic activity that is often central to the tumor pathogenesis. In addition to proto-oncogene activation, other mechanisms implicated in lymphoma genesis and progression include deregulation of antiapoptosis genes (e.g. bcl-2) and tumor suppressor gene inactivation (e.g. p53).
Anaplastic large cell lymphoma (ALCL) is a distinct subtype of aggressive peripheral T-cell lymphomas harboring chromosomal translocations involving the ALK tyrosine kinase (2, 3). Morphologically they are characterized by large cells with pleomorphic cytology with multilobated "horseshoe-shaped" nuclei (see Fig. 1). The t(2;5)(p23;q35) chromosomal aberration resulting in overexpression of a chimeric oncogene, nucleophosmin/anaplastic lymphoma kinase (NPM/ALK) (4, 5), is the most common translocation found in these tumors (see Fig. 1). Native NPM is a nucleolar phosphoprotein whose functions include ribosomal RNA assembly, chaperone activities, and nuclease activity (6). Anaplastic lymphoma kinase (CD236) is a receptor tyrosine kinase of the insulin receptor subfamily (7, 8) with homology to leukocyte tyrosine kinase. It is a highly conserved receptor tyrosine kinase and is critical for Drosophila muscle development (9, 10). In humans, its expression is highly tissue-specific and is normally restricted to cells of the nervous system where it is thought to play a role in neural development (11, 12) via its ligands pleiotrophin (13) and midkine (14). An N-terminal oligomerization domain within NPM facilitates the dimerization of NPM/ALK, leading to autophosphorylation and constitutive activation of the ALK tyrosine kinase (15), which is the causative oncogene in t(2;5)-positive ALCLs (8, 16) (see Fig. 1).
View larger version (37K):
[in this window]
[in a new window]
|
FIG. 1. Anaplastic large cell lymphomas are characterized by the t(2;5)(p23;q35) chromosomal translocation. A, ALCLs are composed of pleomorphic malignant lymphoma cells. B, nucleophosmin located on chromosome 5 is fused to the C-terminal end of anaplastic lymphoma kinase located on chromosome 2. The NPM portion in the fusion protein contains an oligomerization domain (OD), whereas the intracytoplasmic moiety of the fusion protein contains the tyrosine kinase domain (TKD) and tyrosine residues for recruitment of adaptor proteins. The dimerization domain within the NPM leads to the oligomerization of the fusion protein and to autoactivation of the kinase, which can be detected in the malignant cells. AD, activation domain; NLS, nuclear localization signal; TM, transmembrane domain; MB, molybdylate binding.
|
|
Activated ALK induces multiple downstream signaling molecules including phospholipase C
(PLC) (17), phosphatidylinositol 3-kinase (PI3K) (18), RAC-serine/threonine protein kinase (AKT) (19), Janus kinase 3 (JAK3) (20), signal transducer and activator of transcription 3 (STAT3) (21), and the non-receptor protein kinase (SRC) (22), which leads to enhanced cell proliferation, survival, and apoptosis inhibition (see Fig. 2). The NPM/ALK protein plays a key role in ALCL lymphomagenesis and has been shown to cause lymphoid malignancy in vitro and in vivo (23).
View larger version (29K):
[in this window]
[in a new window]
|
FIG.. 2. Multiple signaling proteins/pathways are activated by NPM/ALK. Constitutive activation of NPM/ALK tyrosine kinase leads to recruitment of signaling molecules via the SH2 and PTB motifs located within the cytoplasmic domain of ALK. These in turn activate downstream pathways including PI3K (PI 3-Kinase)/AKT, JAK2–3/STAT3–5, and PLC/Ras/ERK (extracellular signal-regulated kinase) pathways, which enhance cell survival, proliferation, and inhibition of apoptosis. FKHD, forkhead.
|
|
Advances in mass spectrometry-driven proteomics have shifted the paradigm of translational cancer research (for a review, see Lim and Elenitoba-Johnson (24)). Although the ultimate goals would be to identify biomarkers for diagnosis, prognosis, and development of novel agents for therapy, significant investments into understanding the basic protein building blocks and the proteomic circuitry at a global level need to be made. To this end, we have focused on mass spectrometry-based proteomic studies to accomplish the following: 1) global proteome analysis and annotation, 2) identification of interacting partners of ALK, 3) comprehensive analysis of deregulated signaling pathways, 4) phosphoproteome analysis, 5) quantitative profiling of proteomic consequences of targeted therapeutic agents, and 6) identification of ALK translocation partners. The approaches outlined in this review, although focused on NPM/ALK-positive ALCL, can be applicable to the study of malignant lymphoma in general. This review summarizes the recently obtained mass spectrometry-based proteomic studies that have enhanced not only the understanding of the molecular pathogenesis of NPM/ALK-positive ALCLs but also identified potential novel therapeutic targets.
|
GLOBAL PROTEOME PROFILING OF NPM/ALK-POSITIVE ANAPLASTIC LARGE CELL LYMPHOMA
|
|---|
The signaling pathways and biological mechanisms involved in the pathogenesis of NPM/ALK-positive ALCL are not completely understood, and a comprehensive profile of its proteome has yet to be performed. Large scale profiling of the proteome of ALCL will provide the initial information required to define the proteomic building blocks and will be of utility in the discovery of pathogenetic mechanisms, diagnostic biomarkers, and treatment strategies.
We have annotated the largest database of proteins comprising the proteome of the NPM/ALK-positive ALCL (25). 2 To identify and catalog a comprehensive list of proteins expressed in the NPM/ALK-positive ALCL, lysates from the SUDHL-1 cell line were used to obtain three subcellular fractions: nuclear, cytoplasmic, and membrane. To enhance the separation of the proteome even further, the subcellular fractions were separated by size using 13% SDS-PAGE after which each of the lanes were cut into 32 slices, subjected to in-gel digestion, and analyzed by LC-MS/MS (see Fig. 3A for experimental design). The MS data were interpreted using SEQUESTTM to search the UniProt database and analyzed by ProteinProphetTM and INTERACTTM to assess error rates. This approach was simple, fast, and effective in simplification of sample complexity and allowed for the identification of proteins with molecular masses ranging from 7 to 620 kDa.
View larger version (25K):
[in this window]
[in a new window]
|
FIG.. 3. Experimental approaches utilized for mass spectrometry-based proteomic studies of human lymphoma. A, global proteome analysis using subcellular fraction. Identification of proteins within the protein network of NPM/ALK using immunoprecipitation (B) or affinity tagging (C) and quantitative proteomic analysis using cleavable ICAT (C-ICAT) (D). Subsequent to tandem MS identification of proteins, extensive data analysis is performed using SEQUEST, ProteinProphet, INTERACT, and PeptideProphetTM. The proteins are validated by Western blot analysis (WB) and immunohistochemistry using high density tissue microarrays (TMA). M, membrane; N, nuclear; C, cytosolic; 1D, one-dimensional.
|
|
A total of 623 proteins consisting of 210 membrane, 229 cytoplasm, and 184 nuclear proteins were identified with a 
5% error rate. Extensive annotation and PubMed investigation of a subset of 209 proteins indicated that 19.9% were reported previously to be expressed in T-cells, and 44.7% were reported to have important function in cancers. Notably only 2.4% of the proteins have been reported previously in the literature to be implicated in ALCL pathogenesis. GoMinerTM (27) protein function analysis on the ALCL proteome revealed a highly complex system and identified proteins involved in diverse cellular functions such as catalytic, enzyme, signaling, motor, translation, transcription, and structural functions. Fig. 4 summarizes the functional categories of proteins identified within the cytoplasmic, nuclear, and membrane fractions of ALCL. The results of this study demonstrate the utility of subcellular fractionation combined with one-dimensional SDS-PAGE and LC-MS/MS for the comprehensive, high throughput identification of proteins within a cellular proteome.
View larger version (24K):
[in this window]
[in a new window]
|
FIG.. 4. Functional categories of proteins expressed in the membrane, cytoplasmic, and nuclear fraction of ALCL. GoMiner analysis of proteins identified in the membrane, cytoplasmic, and nuclear fractions reveals proteins from diverse functional categories.
|
|
|
IDENTIFICATION OF ALK-INTERACTING PROTEINS BY TANDEM MS
|
|---|
The molecular mechanisms involved in NPM/ALK-mediated transformation are not fully understood. Tyrosine residues located within the cytoplasmic domain of ALK act as docking sites for recruitment of signaling molecules via the SRC homology 2 (SH2) and phosphotyrosine binding (PTB) protein motifs (8). The NPM/ALK fusion protein effects its transforming capacity via several key signaling pathways including the PI3K pathway leading to AKT activation (18) as well as activation of the JAK/STAT pathway (21, 28, 29). Indeed co-immunoprecipitation studies have demonstrated the interaction of ALK with PLC (17), IRS-1 (30), heat shock protein 90 (Hsp90) (31), Grb, ShcC (32), JAK3 (21), JAK2 (28), STAT5 (29), and STAT3 (21, 33). Because ALK plays a critical role in the activation of multiple signaling pathways, determination of its interacting proteins and those that form the complex of proteins within its "interactome" may provide novel insights as to its function as well as reveal potential targets for novel therapies.
A mass spectrometry-based proteomic strategy was used to determine the identity of proteins that interact with NPM/ALK tyrosine kinase (see Fig. 3B) (34). The ALK interactome was enriched using a monoclonal antibody against ALK, and the components were separated by one-dimensional SDS-PAGE. Distinct bands that were present in the ALK immunocomplex but not in the IgG control immunoprecipitation were digested and subjected to LC and ESI-MS/MS.
Among the over 40 proteins identified in the ALK interactome, nine proteins had been reported previously to be important mediators of the ALK signal pathway and interacted with ALK. These include PLC1 (17), PI3K (18, 19), JAK2 (28), JAK3 (21), STAT3 (21, 33), and IRS (8). More importantly, many proteins not recognized previously to be associated with NPM/ALK but with potential NPM/ALK-interacting protein domains were identified. These included adaptor molecules (suppressors of cytokine signaling, Rho-GTPase-activating protein, and RAB35), kinases (MEK kinases 1 and 4, protein kinase C, myosin light chain kinase, cyclin G-associated kinase, EphA1, EphB, JNK kinase, and mitogen-activated protein kinase 1), phosphatases (meprin, PTPK, and protein phosphatase 2 subunit), and heat shock proteins (Hsp60 precursor). A comprehensive interactome map of NPM/ALK is illustrated in Fig. 5 using MetaCore (GeneGo Inc.), an integrated systems level analysis tool for high throughput MS and genomic data. Importantly a subset of the proteins that were identified by MS was confirmed by Western blotting and reciprocal immunoprecipitation. Interestingly two of the proteins that were identified by MS but not reported in the final report due to their low scores for identification (NIPA (35) and SRC (22)) were subsequently reported by two independent studies to interact with ALK and have important biologic functions in cellular transformation in ALK-positive lymphoma. The significant role of STAT3 in the pathogenesis of ALK-mediated lymphomagenesis has been demonstrated in mice where tumor formation was inhibited by genetic ablation using antisense STAT3 oligonucleotides (36). This study demonstrates the utility of antibody immunoprecipitation and peptide identification by nanoflow ESI-LC/MS/MS for the high throughput identification of proteins within the ALK signaling complex and potential definition of its signaling pathways. Although the specific nature of the protein interactions needs to be functionally validated, these approaches represent a high throughput method for the identification of proteins (37) within a complex and provide insights into the fundamental basis of their biologic activities.
View larger version (31K):
[in this window]
[in a new window]
|
FIG.. 5. Proteins identified within the NPM/ALK protein network. The proteins identified within the NPM/ALK immunocomplex are visualized using GeneGO software analysis. reg, regulated; PKC, protein kinase C; GAK, cyclin G-associated kinase.
|
|
Using an affinity pulldown and MS/MS strategy we analyzed the interactome of the phosphatase and tensin homolog (PTEN) tumor suppressor, a multifunctional protein deregulated in many types of cancer (38) (Fig. 3C). Wild type PTEN cDNA was inserted into pTRC-His2 vector to create a hexahistidine (His6)-tagged protein that was expressed in Escherichia coli. Lysate from SUDHL-1 cells was used in pulldown assays, utilizing affinity for nickel-agarose beads. Bound proteins were eluted with imidazole, digested, and analyzed by LC-MS/MS. Acquired data were searched against the National Center for Biotechnology Information (NCBI) nr.fasta non-redundant protein database using the SEQUEST algorithm (39) and screened using INTERACT and ProteinProphet. All experiments were performed in duplicate with His6-lacZ serving as control.
A total of 79 proteins were identified in the wild type His6-PTEN pulldown by MS/MS. In silico analysis using four publicly available databases (Online Mendelian Inheritance in Man (OMIM), Human Protein Reference Database (HPRD), The IntAct Project Database, and PubMed) revealed 251 documented primary and secondary protein interactions with the PTEN tumor suppressor. This in silico analysis confirmed 42 of 79 (53%) of the proteins identified by MS/MS. The remaining 37 proteins represent probable PTEN interactions not documented previously in public databases or reported in the literature. The proteins that interact with PTEN are annotated in the Biomolecular Interaction Network Database. These results highlight the value of combining both in vitro biochemical approaches with in silico analyses for a comprehensive study of protein-protein interactions (38).
|
PROTEOMIC ANALYSIS OF NPM/ALK-POSITIVE AND NPM/ALK-NEGATIVE CELLS
|
|---|
Although the majority of ALCLs carry chromosomal translocations involving ALK, the remaining 10–20% do not show evidence of ALK gene deregulation. The NPM/ALK chimeric tyrosine kinase modulates key signaling pathways important for its survival including PI3K/AKT (18) and JAK/STAT (21, 28, 29); however, the global impact of constitutive NPM/ALK expression in tumor cells is unknown. The proteomic differences between ALK-positive and ALK-negative ALCLs can be analyzed by a variety of approaches that are complementary in their overall efficiency and utility.
Although the most widely used proteomic approach is the two-dimensional gel-based method followed by mass spectrometry (40), the recent development of LC-LC-MS/MS permits the sensitive detection of low abundance proteins, membrane proteins, and proteins with extreme pI (41–43). Furthermore the ability to perform global quantitative proteomics has been significantly enhanced by the advent of ICATTM, which is efficient in simplifying the proteome and in combination with three-dimensional LC-MS/MS permits detection and quantification of proteins and peptides from very complex samples (44, 45).
To determine the comprehensive proteomic changes that occur in response to the singular molecular abnormality of NPM/ALK expression, we have performed quantitative proteomic analysis of NPM/ALK-positive and NPM/ALK-negative lymphoid cells using cleavable ICAT and electrospray ionization tandem mass spectrometry (Fig. 3D).
NPM/ALK was ectopically expressed in Jurkat T-cells, and the proteomic expression profile was compared with that of vector-transfected cells. Equivalent quantities of total cell lysates obtained from the NPM-ALK-transfected cells and the vector control cells were labeled with either a light cleavable ICAT reagent (12C9) or a heavy cleavable ICAT reagent (13C9). The cleavable ICAT reagent contains a thiol-reactive group and biotin moiety that is separated by a linker molecule that is either isotopically labeled with nine atoms of 13C or not labeled. After covalent labeling, the protein mixtures were combined and subjected to avidin affinity chromatography to recover ICAT-labeled proteins. Off-line fractions were collected, digested with trypsin, and analyzed by automated reverse phase nanospray LC coupled on line with ESI-MS/MS. Relative protein expression levels were determined by analysis of ion chromatograms for each of the ICAT-labeled peptides and determining the expression ratios using XPRESSTM software.
Overall 110 proteins showed a 1.5-fold or greater change in the NPM/ALK-positive cells as compared with the vector control cells. Of these, 73 proteins were up-regulated in the NPM/ALK-positive cells, whereas 37 proteins were down-regulated. Proteins from nearly every cellular compartment including nucleus, cytoplasm, membrane, and secretome were identified to be differentially expressed. Proteins of both high abundance (cytoskeletal proteins such as myosin heavy polypeptide) and low abundance (transcription factors and growth factor receptors) were observed. Analysis of differentially expressed proteins revealed them to belong to a variety of functional groups. The most common functional group of proteins that were up-regulated were those that belong to kinases (14 of 79); those associated with cell adhesion, migration, and cytoskeletal function (14 of 79); and those associated with proliferation and protein synthesis (10 of 79). The others represent phosphatases (3 of 79), small GTPases (5 of 79), and proteins associated with centrosome assembly (7 of 79). Importantly many were hypothetical proteins and represent ideal targets for functional studies. As shown in Fig. 6, our quantitative proteomic studies reveal that NPM/ALK deregulates the expression of numerous proteins within previously described and novel signaling modules important for cell proliferation, survival, apoptosis evasion, and tumor dissemination. These data show that comprehensive analysis of the proteomic changes produced by a singular molecular alteration such as NPM/ALK facilitates understanding of tumor pathobiology and enables the identification of novel biomarkers and therapeutic targets.
View larger version (41K):
[in this window]
[in a new window]
|
FIG.. 6. NPM/ALK induces the expression of proteins that are involved in diverse cellular functions. The expression of NPM/ALK induced the expression of proteins implicated in multiple signaling pathways and cellular functions such as cell motility, microtubule assembly, protein synthesis, invasion, and proliferation. MAPK, mitogen-activated protein kinase; PI, phosphatidylinositol.
|
|
Cussac et al. (46) utilized two-dimensional gel electrophoresis followed by MALDI-TOF MS to compare the proteome of the cytoplasmic soluble proteins of an NPM/ALK-positive cell line (SUDHL-1) with that of NPM/ALK-negative cell line (FE-PD). The spot patterns of the two-dimensional electrophoresis gels were analyzed with an image analysis software that detected 82 proteins that were differentially expressed between the two cell lines. Identification of the proteins demonstrated that they belonged to five broad functional categories including signaling, cytoskeleton and motility, chaperones, control of protein expression and degradation, and homeostasis and metabolism. A comparison of cell lines representative of two morphologic subtypes of NPM/ALK-positive ALCLs, namely the common type (SUDHL-1) and the small cell variant (COST), revealed distinct set of proteins that were selectively differentially expressed within each category. Furthermore the relative overexpression of one protein, carbonic anhydrase II, in the small cell variant was validated in tissue biopsy samples of small cell variant of ALCL. The differential proteomic analysis was extended by performing two-dimensional liquid chromatography, which allowed the separation of a larger panel of proteins and increased the number of differentially expressed proteins to 175.
|
PHOSPHOPROTEOME ANALYSIS OF NPM/ALK-POSITIVE LYMPHOMAS
|
|---|
The constitutive activation of NPM/ALK provides a critical signal in downstream signaling proteins/pathways that ultimately enhances the survival and growth of ALCLs. Analysis of the phosphotyrosine proteome in ALK-positive ALCL cells may lead to identification of phosphotyrosine-containing proteins that associate with ALK and play a role in tumor cell survival as well as potential drug targets that may reduce ALK signaling. Because of their low abundance, analysis of the phosphotyrosine proteome has been challenging. IMAC has been used to purify phosphopeptides from cell lysate (47), but this technique isolates all phosphorylated residues, including phosphoserine and phosphothreonine as well as the least abundant phosphotyrosine (48). Enrichment of the phosphotyrosine proteome with anti-phosphotyrosine antibodies is essential given the low stoichiometry of tyrosine phosphorylation and sample complexity (49). Furthermore enrichment of the phosphotyrosine proteome may enhance detection of proteins by LC-MS/MS in a more expedient and sensitive manner than IMAC (50).
In an effort to carry out global analysis of the phosphotyrosine proteins in NPM/ALK-positive ALCLs, we have optimized strategies for enrichment of the phosphotyrosine proteome of SUDHL-1 cell lines using antibody-mediated enrichment techniques.3 We compared the phosphoproteome using immunoaffinity (IA) enrichment with that of immunoprecipitation (IP) in ALK-positive ALCL cells for subsequent detection by immunoblot and LC-MS/MS. Immunoprecipitation was more effective at enrichment than immunoaffinity chromatography as determined by the number of phosphotyrosine-immunoreactive bands on Western blot analysis and by subsequent LC-MS/MS. Investigation of immunoprecipitation enrichment using two commercially available antibodies demonstrated improved enrichment and detection of phosphotyrosines using 4G10 over sc-18182. Examination of IP enriched proteins by LC-MS/MS demonstrated a 2.5-fold increase in the numbers of proteins identified over IA enriched proteins. Proteins identified by LC-MS/MS in both IP and IA enriched samples were subjected to GoMiner analysis for cellular localization and molecular functions.
Alternatively peptides containing phosphotyrosines have been directly isolated from multienzyme protease-digested protein extracts with phosphotyrosine-specific antibody and identified by tandem mass spectrometry (52). Using this strategy, 278 phosphopeptides representing 180 phosphotyrosine sites were identified from the SUDHL-1 cell line. Because peptides are analyzed, this approach allows the identification of not only proteins but specific phosphorylation sites. Furthermore nine sites of tyrosine phosphorylation within ALK were identified, five of which were novel. The functional significance of these novel sites of tyrosine phosphorylation remains unknown and may represent novel SH2 or PTB sites through which adaptor proteins may interact with ALK in a phosphospecific manner and pose exciting targets for future research. The phosphorylation sites are annotated in the PhosphoSite database, which is a comprehensive resource of human and mouse in vivo phosphorylation sites (53). In addition, the data have been utilized to determine the protein phosphorylation motifs of cells constitutively expressing activated NPM/ALK by analyzing the distribution of amino acid residues surrounding phosphotyrosine sites using an iterative statistical approach (54). Their analysis was able to extract four novel motif classes; this is of particular interest as they may represent candidate consensus sequences for NPM/ALK.
|
PROTEOMIC CONSEQUENCES OF TARGETED INHIBITION OF ABERRANT SURVIVAL SIGNALS
|
|---|
Comparative quantitative analysis of cells in response to selective small molecular inhibitors of deregulated signaling proteins/pathways can aid in understanding the biologic mechanisms of its action. Furthermore many small molecule inhibitors have pleiotropic effects and may inhibit multiple proteins and pathways. Many of the drugs are in Phase II/III clinical trials, and thus knowledge of the proteomic consequences of these drugs may provide important clinically relevant information. One such chemical is the benzoquinone ansamycin geldanamycin (GA) and derivative 17-allylamino-17-demethoxygeldanamycin that elicit antitumor activity in ALCL (31, 55). This group of ansamycins was initially thought to target tyrosine kinases and was therefore used as tyrosine kinase inhibitors. Subsequent studies identified Hsp90 as the primary target of GA (56). Hsp90 is a highly conserved, ubiquitous molecular chaperone that is required for the stability and conformational maturation of a diverse group of client proteins, including components of signaling pathways exploited by cancer cells for survival and proliferation (57).
Hsp90 client proteins include receptor tyrosine kinases such as human epidermal growth factor receptor family kinases, breakpoint cluster region-Abelson (BCR-ABL), and NPM-ALK; cytosolic signaling proteins such as AKT, v-Raf-1 murine leukemia viral oncogene homolog 1 (RAF-1), and inhibitor of nuclear factor 
B kinase (IKK); and cell cycle regulators including cyclin-dependent kinase 4, polo-like kinase 1, and survivin (56–58). Inhibition of Hsp90 by ansamycins in ALK-positive ALCL cells results in down-regulation of NPM/ALK protein kinase activity (55) leading to cellular apoptosis (31). Clearly there are multiple cellular targets of Hsp90, yet the comprehensive effects of Hsp90 inhibition in ALK-positive ALCL cells are unknown.
To assess the quantitative proteomic changes in ALK-positive ALCL cells due to Hsp90 inhibition by GA, equal mounts of lysates from vehicle-treated and cells exposed to 10 µM GA were subjected to ICAT labeling after 12 h of incubation followed by LC-MS/MS analysis of proteins. Examination of the data from 35 individual strong cationic exchange fractions resulted in a total of 2921 peptides identified that were matched to 1129 known database entries and 314 unique proteins.
The results demonstrated that GA induced G2/M cell cycle arrest followed by caspase-3-mediated apoptosis. Moreover from the 170 ICAT-labeled peptides, 49 proteins were up-regulated 1.5-fold or greater, and 70 proteins were down-regulated 1.5-fold or greater in GA-treated cells. Importantly several proteins involved in diverse cellular functions, including signal transduction, DNA metabolism, nucleic acid metabolism, protein metabolism, cell growth and maintenance, and energy pathways, were identified. Some of the down-regulated proteins are known to be involved in described signaling cascades such as JAK/STAT and MAPK (mitogen-activated protein kinase) as well as pathways previously unreported in ALK-positive ALCL including WNT, NF-B, and transforming growth factor ß. These studies demonstrate some of the molecular mechanisms by which Hsp90 inhibition reduced viability of ALK-positive ALCL cells and illustrate the diverse proteins whose expression is changed due to GA inhibition of Hsp90.
|
IDENTIFICATION OF ALK TRANSLOCATION PARTNERS BY TANDEM MASS SPECTROMETRY
|
|---|
The ALK gene at 2p23 is rearranged with a number of other fusion partner genes in ALCLs including clathrin heavy polypeptide-like gene (t(2,22)(p23;q11.2)) (59), nonmuscle tropomyosin 3 (TPM3) gene (t(1;2)(q35;p23)) (60), Trk fusion gene (t(2;3)(p23;q21)) (61), 5-aminoimidazole-4-carboxamide ribonucleotide formyltransferase/IMP cyclohydrolase gene (inv(2)(p23;q35)) (62), and moesin (63). The expression of full-length ALK receptor protein is normally limited to cells of neuronal origin.
ALK is also involved in rearrangement with other partner genes in inflammatory myofibroblastic tumors (IMTs) such as Ran-binding protein 2 (64) and TPM3 and TPM4 (65). The chimeric proteins consist of the intracytoplasmic portion of the ALK protein, which includes the catalytically active kinase domain, although in most instances the fusion partners encode ubiquitously expressed proteins with active promoters that promote elevated transcription of the ALK fusion transcript and protein.
Existing methods for the identification of chromosomal translocation partners are nucleic acid-based. These include direct cloning and sequencing following screening of cDNA libraries using probes complementary to the known translocation partner and PCR-based technologies such as rapid amplification of cDNA ends (66, 67), inverse PCR (68), inverse panhandle PCR (69, 70), and long distance PCR (71). The development of proteomics-based methods is desirable and would complement nucleic acid-based methods because of the ability of the peptide sequencing-based approach to distinguish fusion peptides from splice variants.
Recently we described a proteomic strategy to identify translocation partners encoding oncogenic chimeric fusion proteins using the human ALK oncogene product as a model system (72). We performed immunoaffinity enrichment of chimeric ALK fusion proteins from total cell lysates of an ALCL-derived cell line and a primary tissue biopsy of IMT. These tumor samples exhibited the chromosomal aberrations t(2;5)(p23;q35) and t(1;2)q25;2p23), respectively, by conventional cytogenetics and aberrant expression of the ALK protein by tissue immunohistochemistry. The candidate proteins were visualized by immunoblotting with anti-ALK antibodies, excised from the SDS-PAGE gel, and subjected to parallel digestion using four enzymes with different proteolytic cleavage specificities (72). For each sample, the aliquots from each specific enzyme digestion were processed and subjected to microelectrospray LC-MS/MS separately with the redundant data from each analysis superimposed to generate a composite sequence map of the chimeric fusion peptides. The combination of data from multiple digests was advantageous in that it produced simpler peptide mixtures than combinations of multiple enzyme digests in one tube. Thus, whereas individual digests yielded between 25% and 41% coverage for both NPM/ALK and TPM3/ALK, the combination of all four enzymes produced an overall coverage of 82.6 and 84.8%, respectively. Tandem mass spectrometry revealed multiple overlapping peptides, identifying the NPM and ALK proteins in the ALCL cell line and the TPM3 and ALK proteins in the biopsy specimen of the IMT. In each case, fusion peptides representing hybrid sequences from both translocation partners were identified that conclusively established the presence of aberrant fusions of NPM and TPM3 to the truncated ALK protein. The appropriate ALK chimeric fusions in both samples were confirmed by bidirectional DNA sequencing. This report demonstrates the utility of a mass spectrometry-based proteomic approach for the identification of a fusion partner of a chimeric protein, and the proteomic approach is applicable to the identification of the participating members of any fusion protein encoded from chromosomal translocation wherein one of the partners is known and an antibody is available to the known partner.
|
CONCLUSIONS/FUTURE DIRECTIONS
|
|---|
Over 11 variant translocations involving the ALK gene have been described in lymphomas and a subset of pediatric tumors including inflammatory myofibroblastic tumor and rhabdomyosarcoma indicating that the transforming potential of ALK tyrosine kinase is manifest in more than one cell type. Furthermore the full-length ALK receptor protein, normally limited to cells of neuronal origin, is expressed in a variety of non-hematopoietic tumors including rhabdomyosarcoma, neuroblastoma, glioblastoma, breast carcinoma, and melanomas. The signaling cascades induced by native full-length ALK receptor and variant ALK chimeric proteins commonly found in ALCLs and IMTs remain largely unknown. It will be important to characterize the specific signaling pathways that are activated by stimulation of the full-length ALK receptor. In this regard, quantitative proteomic approaches analyzing global phosphoproteomic changes will facilitate comprehensive identification of signaling pathways that are affected by full-length ALK stimulation.
Although studies have assessed inhibition of ALK by both small interfering RNA (73), short hairpin RNA (74), tyrosine kinase inhibitors (75), and more recently small molecule inhibitors with selective activity against ALK (76, 77), the proteomic effects of these agents have yet to be evaluated. By determining changes in protein expression in response to the specific inhibition of ALK, we may uncover novel proteins required for NPM/ALK survival as well as discover potential therapeutic targets that may be used in combination with ALK inhibitors. Chemical proteomic approaches using affinity columns with covalently linked drugs (78) may provide a broad view of potential substrates/targets of "specific" inhibitors of ALK.
Although the advances in biotechnology have permitted the global analysis of proteins expressed in various cellular tissues, the limited capacity to extract intact proteins has largely precluded the large scale proteomic analysis of formalin-fixed and paraffin-embedded tissues. Recent work from our laboratory (26) and others (51) has demonstrated the ability to utilize archived formalin-fixed and paraffin-embedded tissues to identify hundreds to thousands of proteins from whole and laser capture microdissected tissues from as little as 105 cells from blocks stored for over 10 years. The ability to utilize formalin-fixed, paraffin-embedded tissue material for mass spectrometry-based proteomics will provide significant advances in analysis of human lymphoma samples for diagnostic and therapeutic protein discovery.
The most critical but challenging step in mass spectrometry-based proteomic studies for human malignant lymphoma as for other disease models remains the functional validation of proteins and signaling pathways using serum and clinical tissue samples. Recent developments in protein microarrays that allow for high throughput analysis of a nanoliter volume of serum against tens of thousands of peptides/proteins provide an important approach for identification of secreted biomarkers and potential secretomic signature of patients with ALCL. Tissue microarrays constructed from biopsy samples obtained from well characterized clinical cohorts/clinical trials are also a critical component in the translation of protein identification to validation of biomarkers for diagnosis and prognosis.
|
ACKNOWLEDGMENTS
|
|---|
We thank all of the members of our research group for contributions to the work presented in this review. We thank Haminat Elenitoba-Johnson for assistance with the preparation of the figures.
|
FOOTNOTES |
|---|
Received, May 2, 2006, and in revised form, June 16, 2006.
Published, MCP Papers in Press, June 19, 2006, DOI 10.1074/mcp.R600005-MCP200
1 The abbreviations used are: NHL, non-Hodgkin lymphoma; ALCL, anaplastic large cell lymphoma; NPM, nucleophosmin; ALK, anaplastic lymphoma kinase; PLC, phospholipase C; JAK, Janus kinase; STAT, signal transducer and activator of transcription; PI3K, phosphatidylinositol 3-kinase; SH2, SRC homology 2; PTB, phosphotyrosine binding; IRS, insulin receptor substrate; JNK, c-Jun N-terminal kinase; MEK, mitogen-activated protein kinase/extracellular signal-regulated kinase kinase; PTEN, phosphatase and tensin homolog; IA, immunoaffinity; IP, immunoprecipitation; GA, geldanamycin; Hsp, heat shock protein; TPM, tropomyosin; IMT, inflammatory myofibroblastic tumor. 
2 C. Sjostrom, C. Seiler, D. Crockett, S. Tripp, K. Elenitoba-Johnson, and M. Lim, manuscript submitted.
3 J. C. Schumacher, D. K. Crockett, Z. Lin, K. S. J. Elenitoba-Johnson, and M. S. Lim, manuscript submitted.
* The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
To whom correspondence should be addressed: Div. of Anatomic Pathology, University of Utah Health Sciences Center, 50 North Medical Dr., Salt Lake City, UT 84132. Tel.: 801-581-5854; Fax: 801-585-3831; E-mail: megan.lim{at}path.utah.edu
|
REFERENCES
|
|---|
- Jaffe, E. S., Harris, N. L., Stein, H., and Vardiman, J. W. (eds)
(2001)
Pathology and Genetics of Tumours of Hematopoietic and Lymphoid Tissues, pp.230
–235, International Agency for Research on Cancer (IARC) Press, Lyon, France
- Mason, D. Y., Bastard, C., Rimokh, R., Dastugue, N., Huret, J. L., Kristoffersson, U., Magaud, J. P., Nezelof, C., Tilly, H., and Vannier, J. P.
(1990) CD30-positive large cell lymphomas (‘Ki-1 lymphoma’) are associated with a chromosomal translocation involving 5q35.
Br. J. Haematol.
74, 161
–168[Medline]
- Stein, H., Mason, D. Y., Gerdes, J., O’Connor, N., Wainscoat, J., Pallesen, G., Gatter, K., Falini, B., Delsol, G., Lemke, H., Schwarting, R., and Lennert, K.
(1985) The expression of the Hodgkin’s disease associated antigen Ki-1 in reactive and neoplastic lymphoid tissue: evidence that Reed-Sternberg cells and histiocytic malignancies are derived from activated lymphoid cells.
Blood
66, 848
–858[Abstract/Free Full Text]
- Morris, S. W., Kirstein, M. N., Valentine, M. B., Dittmer, K. G., Shapiro, D. N., Saltman, D. L., and Look, A. T.
(1994) Fusion of a kinase gene, ALK, to a nucleolar protein gene, NPM, in non- Hodgkin’s lymphoma.
Science
263, 1281
–1284; Correction
(1995)
Science
267, 316
–317[Free Full Text]
- Shiota, M., Fujimoto, J., Semba, T., Satoh, H., Yamamoto, T., and Mori, S.
(1994) Hyperphosphorylation of a novel 80 kDa protein-tyrosine kinase similar to Ltk in a human Ki-1 lymphoma cell line, AMS3.
Oncogene
9, 1567
–1574[Medline]
- Borer, R. A., Lehner, C. F., Eppenberger, H. M., and Nigg, E. A.
(1989) Major nucleolar proteins shuttle between nucleus and cytoplasm.
Cell
56, 379
–390[CrossRef][Medline]
- Iwahara, T., Fujimoto, J., Wen, D., Cupples, R., Bucay, N., Arakawa, T., Mori, S., Ratzkin, B., and Yamamoto, T.
(1997) Molecular characterization of ALK, a receptor tyrosine kinase expressed specifically in the nervous system.
Oncogene
14, 439
–449[CrossRef][Medline]
- Morris, S. W., Naeve, C., Mathew, P., James, P. L., Kirstein, M. N., Cui, X., and Witte, D. P.
(1997) ALK, the chromosome 2 gene locus altered by the t(2;5) in non-Hodgkin’s lymphoma, encodes a novel neural receptor tyrosine kinase that is highly related to leukocyte tyrosine kinase (LTK).
Oncogene
14, 2175
–2188; Correction
(1997)
Oncogene
15, 2883[CrossRef]
- Loren, C. E., Englund, C., Grabbe, C., Hallberg, B., Hunter, T., and Palmer, R. H.
(2003) A crucial role for the anaplastic lymphoma kinase receptor tyrosine kinase in gut development in Drosophila melanogaster.
EMBO Rep.
4, 781
–786[CrossRef][Medline]
- Stute, C., Schimmelpfeng, K., Renkawitz-Pohl, R., Palmer, R. H., and Holz, A.
(2004) Myoblast determination in the somatic and visceral mesoderm depends on Notch signalling as well as on milliways(mili(Alk)) as receptor for Jeb signalling.
Development
131, 743
–754[Abstract/Free Full Text]
- Souttou, B., Carvalho, N. B., Raulais, D., and Vigny, M.
(2001) Activation of anaplastic lymphoma kinase receptor tyrosine kinase induces neuronal differentiation through the mitogen-activated protein kinase pathway.
J. Biol. Chem.
276, 9526
–9531[Abstract/Free Full Text]
- Motegi, A., Fujimoto, J., Kotani, M., Sakuraba, H., and Yamamoto, T.
(2004) ALK receptor tyrosine kinase promotes cell growth and neurite outgrowth.
J. Cell Sci.
117, 3319
–3329[Abstract/Free Full Text]
- Powers, C., Aigner, A., Stoica, G. E., McDonnell, K., and Wellstein, A.
(2002) Pleiotrophin signaling through anaplastic lymphoma kinase is rate-limiting for glioblastoma growth.
J. Biol. Chem.
277, 14153
–14158[Abstract/Free Full Text]
- Stoica, G. E., Kuo, A., Powers, C., Bowden, E. T., Sale, E. B., Riegel, A. T., and Wellstein, A.
(2002) Midkine binds to anaplastic lymphoma kinase (ALK) and acts as a growth factor for different cell types.
J. Biol. Chem.
277, 35990
–35998[Abstract/Free Full Text]
- Bischof, D., Pulford, K., Mason, D. Y., and Morris, S. W.
(1997) Role of the nucleophosmin (NPM) portion of the non-Hodgkin’s lymphoma-associated NPM-anaplastic lymphoma kinase fusion protein in oncogenesis.
Mol. Cell. Biol.
17, 2312
–2325[Abstract]
- Shiota, M., and Mori, S.
(1997) Anaplastic large cell lymphomas expressing the novel chimeric protein p80NPM/ALK: a distinct clinicopathologic entity.
Leukemia
11, Suppl. 3,538
–540[Medline]
- Bai, R. Y., Dieter, P., Peschel, C., Morris, S. W., and Duyster, J.
(1998) Nucleophosmin-anaplastic lymphoma kinase of large-cell anaplastic lymphoma is a constitutively active tyrosine kinase that utilizes phospholipase C- to mediate its mitogenicity.
Mol. Cell. Biol.
18, 6951
–6961[Abstract/Free Full Text]
- Slupianek, A., Nieborowska-Skorska, M., Hoser, G., Morrione, A., Majewski, M., Xue, L., Morris, S. W., Wasik, M. A., and Skorski, T.
(2001) Role of phosphatidylinositol 3-kinase-Akt pathway in nucleophosmin/anaplastic lymphoma kinase-mediated lymphomagenesis.
Cancer Res.
61, 2194
–2199[Abstract/Free Full Text]
- Bai, R. Y., Ouyang, T., Miething, C., Morris, S. W., Peschel, C., and Duyster, J.
(2000) Nucleophosmin-anaplastic lymphoma kinase associated with anaplastic large-cell lymphoma activates the phosphatidylinositol 3-kinase/Akt antiapoptotic signaling pathway.
Blood
96, 4319
–4327[Abstract/Free Full Text]
- Amin, H. M., Medeiros, L. J., Ma, Y., Feretzaki, M., Das, P., Leventaki, V., Rassidakis, G. Z., O’Connor, S. L., McDonnell, T. J., and Lai, R.
(2003) Inhibition of JAK3 induces apoptosis and decreases anaplastic lymphoma kinase activity in anaplastic large cell lymphoma.
Oncogene
22, 5399
–5407[CrossRef][Medline]
- Zamo, A., Chiarle, R., Piva, R., Howes, J., Fan, Y., Chilosi, M., Levy, D. E., and Inghirami, G.
(2002) Anaplastic lymphoma kinase (ALK) activates Stat3 and protects hematopoietic cells from cell death.
Oncogene
21, 1038
–1047[CrossRef][Medline]
- Cussac, D., Greenland, C., Roche, S., Bai, R. Y., Duyster, J., Morris, S. W., Delsol, G., Allouche, M., and Payrastre, B.
(2004) Nucleophosmin-anaplastic lymphoma kinase of anaplastic large-cell lymphoma recruits, activates, and uses pp60c-src to mediate its mitogenicity.
Blood
103, 1464
–1471[Abstract/Free Full Text]
- Kuefer, M. U., Look, A. T., Pulford, K., Behm, F. G., Pattengale, P. K., Mason, D. Y., and Morris, S. W.
(1997) Retrovirus-mediated gene transfer of NPM-ALK causes lymphoid malignancy in mice.
Blood
90, 2901
–2910[Abstract/Free Full Text]
- Lim, M. S., and Elenitoba-Johnson, K. S.
(2004) Proteomics in pathology research.
Lab. Investig.
84, 1227
–1244[CrossRef][Medline]
- Crockett, D. K., Seiler, C. E., III, Elenitoba-Johnson, K. S., and Lim, M. S.
(2005) Annotated proteome of a human T-cell lymphoma.
J. Biomol. Tech.
16, 341
–346[Medline]
- Crockett, D. K., Lin, Z., Vaughn, C. P., Lim, M. S., and Elenitoba-Johnson, K. S.
(2005) Identification of proteins from formalin-fixed paraffin-embedded cells by LC-MS/MS.
Lab. Investig.
85, 1405
–1415[CrossRef][Medline]
- Zeeberg, B. R., Feng, W., Wang, G., Wang, M. D., Fojo, A. T., Sunshine, M., Narasimhan, S., Kane, D. W., Reinhold, W. C., Lababidi, S., Bussey, K. J., Riss, J., Barrett, J. C., and Weinstein, J. N.
(2003) GoMiner: a resource for biological interpretation of genomic and proteomic data.
Genome Biol.
4, R28[CrossRef][Medline]
- Ruchatz, H., Coluccia, A. M., Stano, P., Marchesi, E., and Gambacorti-Passerini, C.
(2003) Constitutive activation of Jak2 contributes to proliferation and resistance to apoptosis in NPM/ALK-transformed cells.
Exp. Hematol.
31, 309
–315[CrossRef][Medline]
- Nieborowska-Skorska, M., Slupianek, A., Xue, L., Zhang, Q., Raghunath, P. N., Hoser, G., Wasik, M. A., Morris, S. W., and Skorski, T.
(2001) Role of signal transducer and activator of transcription 5 in nucleophosmin/ anaplastic lymphoma kinase-mediated malignant transformation of lymphoid cells.
Cancer Res.
61, 6517
–6523[Abstract/Free Full Text]
- Fujimoto, J., Shiota, M., Iwahara, T., Seki, N., Satoh, H., Mori, S., and Yamamoto, T.
(1996) Characterization of the transforming activity of p80, a hyperphosphorylated protein in a Ki-1 lymphoma cell line with chromosomal translocation t(2;5).
Proc. Natl. Acad. Sci. U. S. A.
93, 4181
–4186[Abstract/Free Full Text]
- Bonvini, P., Gastaldi, T., Falini, B., and Rosolen, A.
(2002) Nucleophosmin-anaplastic lymphoma kinase (NPM-ALK), a novel Hsp90-client tyrosine kinase: down-regulation of NPM-ALK expression and tyrosine phosphorylation in ALK+ CD30+ lymphoma cells by the Hsp90 antagonist 17-allylamino,17-demethoxygeldanamycin.
Cancer Res.
62, 1559
–1566[Abstract/Free Full Text]
- Miyake, I., Hakomori, Y., Shinohara, A., Gamou, T., Saito, M., Iwamatsu, A., and Sakai, R.
(2002) Activation of anaplastic lymphoma kinase is responsible for hyperphosphorylation of ShcC in neuroblastoma cell lines.
Oncogene
21, 5823
–5834[CrossRef][Medline]
- Zhang, Q., Raghunath, P. N., Xue, L., Majewski, M., Carpentieri, D. F., Odum, N., Morris, S., Skorski, T., and Wasik, M. A.
(2002) Multilevel dysregulation of STAT3 activation in anaplastic lymphoma kinase-positive T/null-cell lymphoma.
J. Immunol.
168, 466
–474[Abstract/Free Full Text]
- Crockett, D. K., Lin, Z., Elenitoba-Johnson, K. S., and Lim, M. S.
(2004) Identification of NPM-ALK interacting proteins by tandem mass spectrometry.
Oncogene
23, 2617
–2629[CrossRef][Medline]
- Bassermann, F., von Klitzing, C., Munch, S., Bai, R. Y., Kawaguchi, H., Morris, S. W., Peschel, C., and Duyster, J.
(2005) NIPA defines an SCF-type mammalian E3 ligase that regulates mitotic entry.
Cell
122, 45
–57[CrossRef][Medline]
- Chiarle, R., Simmons, W. J., Cai, H., Dhall, G., Zamo, A., Raz, R., Karras, J. G., Levy, D. E., and Inghirami, G.
(2005) Stat3 is required for ALK-mediated lymphomagenesis and provides a possible therapeutic target.
Nat. Med.
11, 623
–629[CrossRef][Medline]
- Lin, Z., Crockett, D. K., Lim, M. S., and Elenitoba-Johnson, K. S.
(2003) High-throughput analysis of protein/peptide complexes by immunoprecipitation and automated LC-MS/MS.
J. Biomol. Tech.
14, 149
–155[Medline]
- Crockett, D. K., Fillmore, G. C., Elenitoba-Johnson, K. S., and Lim, M. S.
(2005) Analysis of phosphatase and tensin homolog tumor suppressor interacting proteins by in vitro and in silico proteomics.
Proteomics
5, 1250
–1262[CrossRef][Medline]
- Yates, J. R., III, Eng, J. K., McCormack, A. L., and Schieltz, D.
(1995) Method to correlate tandem mass spectra of modified peptides to amino acid sequences in the protein database.
Anal. Chem.
67, 1426
–1436[Medline]
- Grolleau, A., Bowman, J., Pradet-Balade, B., Puravs, E., Hanash, S., Garcia-Sanz, J. A., and Beretta, L.
(2002) Global and specific translational control by rapamycin in T cells uncovered by microarrays and proteomics.
J. Biol. Chem.
277, 22175
–22184[Abstract/Free Full Text]
- Han, D. K., Eng, J., Zhou, H., and Aebersold, R.
(2001) Quantitative profiling of differentiation-induced microsomal proteins using isotope-coded affinity tags and mass spectrometry.
Nat. Biotechnol.
19, 946
–951[CrossRef][Medline]
- Gygi, S. P., Rist, B., Griffin, T. J., Eng, J., and Aebersold, R.
(2002) Proteome analysis of low-abundance proteins using multidimensional chromatography and isotope-coded affinity tags.
J. Proteome Res.
1, 47
–54[CrossRef][Medline]
- MacCoss, M. J., McDonald, W. H., Saraf, A., Sadygov, R., Clark, J. M., Tasto, J. J., Gould, K. L., Wolters, D., Washburn, M., Weiss, A., Clark, J. I., and Yates, J. R., III
(2002) Shotgun identification of protein modifications from protein complexes and lens tissue.
Proc. Natl. Acad. Sci. U. S. A.
99, 7900
–7905[Abstract/Free Full Text]
- Gygi, S. P., Rist, B., Gerber, S. A., Turecek, F., Gelb, M. H., and Aebersold, R.
(1999) Quantitative analysis of complex protein mixtures using isotope-coded affinity tags.
Nat. Biotechnol.
17, 994
–999[CrossRef][Medline]
- Shiio, Y., Donohoe, S., Yi, E. C., Goodlett, D. R., Aebersold, R., and Eisenman, R. N.
(2002) Quantitative proteomic analysis of Myc oncoprotein function.
EMBO J.
21, 5088
–5096[CrossRef][Medline]
- Cussac, D., Pichereaux, C., Colomba, A., Capilla, F., Pont, F., Gaits-Iacovoni, F., Lamant, L., Espinos, E., Burlet-Schiltz, O., Monsarrat, B., Delsol, G., and Payrastre, B.
(2006) Proteomic analysis of anaplastic lymphoma cell lines: Identification of potential tumour markers.
Proteomics
6, 3210
–3222[CrossRef][Medline]
- Kange, R., Selditz, U., Granberg, M., Lindberg, U., Ekstrand, G., Ek, B., and Gustafsson, M.
(2005) Comparison of different IMAC techniques used for enrichment of phosphorylated peptides.
J. Biomol. Tech.
16, 91
–103[Medline]
- Stensballe, A., Andersen, S., and Jensen, O. N.
(2001) Characterization of phosphoproteins from electrophoretic gels by nanoscale Fe(III) affinity chromatography with off-line mass spectrometry analysis.
Proteomics
1, 207
–222[CrossRef][Medline]
- Ibarrola, N., Molina, H., Iwahori, A., and Pandey, A.
(2004) A novel proteomic approach for specific identification of tyrosine kinase substrates using [13C]tyrosine.
J. Biol. Chem.
279, 15805
–15813[Abstract/Free Full Text]
- Machida, K., Mayer, B. J., and Nollau, P.
(2003) Profiling the global tyrosine phosphorylation state.
Mol. Cell. Proteomics
2, 215
–233[Abstract/Free Full Text]
- Hood, B. L., Darfler, M. M., Guiel, T. G., Furusato, B., Lucas, D. A., Ringeisen, B. R., Sesterhenn, I. A., Conrads, T. P., Veenstra, T. D., and Krizman, D. B.
(2005) Proteomic analysis of formalin-fixed prostate cancer tissue.
Mol. Cell. Proteomics
4, 1741
–1753[Abstract/Free Full Text]
- Rush, J., Moritz, A., Lee, K. A., Guo, A., Goss, V. L., Spek, E. J., Zhang, H., Zha, X. M., Polakiewicz, R. D., and Comb, M. J.
(2005) Immunoaffinity profiling of tyrosine phosphorylation in cancer cells.
Nat. Biotechnol.
23, 94
–101[CrossRef][Medline]
- Hornbeck, P. V., Chabra, I., Kornhauser, J. M., Skrzypek, E., and Zhang, B.
(2004) PhosphoSite: a bioinformatics resource dedicated to physiological protein phosphorylation.
Proteomics
4, 1551
–1561[CrossRef][Medline]
- Schwartz, D., and Gygi, S. P.
(2005) An iterative statistical approach to the identification of protein phosphorylation motifs from large-scale data sets.
Nat. Biotechnol.
23, 1391
–1398[CrossRef][Medline]
- Bonvini, P., Dalla Rosa, H., Vignes, N., and Rosolen, A.
(2004) Ubiquitination and proteasomal degradation of nucleophosmin-anaplastic lymphoma kinase induced by 17-allylamino-demethoxygeldanamycin: role of the co-chaperone carboxyl heat shock protein 70-interacting protein.
Cancer Res.
64, 3256
–3264[Abstract/Free Full Text]
- Neckers, L., Mimnaugh, E., and Schulte, T. W.
(1999) Hsp90 as an anti-cancer target.
Drug Resist. Updat.
2, 165
–172[CrossRef][Medline]
- Picard, D.
(2002) Heat-shock protein 90, a chaperone for folding and regulation.
Cell. Mol. Life Sci.
59, 1640
–1648[CrossRef][Medline]
- Caplan, A. J., Jackson, S., and Smith, D.
(2003) Hsp90 reaches new heights. Conference on the Hsp90 chaperone machine.
EMBO Rep.
4, 126
–130[CrossRef][Medline]
- Touriol, C., Greenland, C., Lamant, L., Pulford, K., Bernard, F., Rousset, T., Mason, D. Y., and Delsol, G.
(2000) Further demonstration of the diversity of chromosomal changes involving 2p23 in ALK-positive lymphoma: 2 cases expressing ALK kinase fused to CLTCL (clathrin chain polypeptide-like).
Blood
95, 3204
–3207[Abstract/Free Full Text]
- Lamant, L., Dastugue, N., Pulford, K., Delsol, G., and Mariame, B.
(1999) A new fusion gene TPM3-ALK in anaplastic large cell lymphoma created by a (1;2)(q25;p23) translocation.
Blood
93, 3088
–3095[Abstract/Free Full Text]
- Hernandez, L., Pinyol, M., Hernandez, S., Bea, S., Pulford, K., Rosenwald, A., Lamant, L., Falini, B., Ott, G., Mason, D. Y., Delsol, G., and Campo, E.
(1999) TRK-fused gene (TFG) is a new partner of ALK in anaplastic large cell lymphoma producing two structurally different TFG-ALK translocations.
Blood
94, 3265
–3268[Abstract/Free Full Text]
- Trinei, M., Lanfrancone, L., Campo, E., Pulford, K., Mason, D. Y., Pelicci, P. G., and Falini, B.
(2000) A new variant anaplastic lymphoma kinase (ALK)-fusion protein (ATIC-ALK) in a case of ALK-positive anaplastic large cell lymphoma.
Cancer Res.
60, 793
–798[Abstract/Free Full Text]
- Tort, F., Pinyol, M., Pulford, K., Roncador, G., Hernandez, L., Nayach, I., Kluin-Nelemans, H. C., Kluin, P., Touriol, C., Delsol, G., Mason, D., and Campo, E.
(2001) Molecular characterization of a new ALK translocation involving moesin (MSN-ALK) in anaplastic large cell lymphoma
TOP
ABSTRACT
GLOBAL PROTEOME PROFILING OF...