Proteomics
The emerging field of proteomics provides a powerful avenue to carry out functional studies of protein-protein interactions and characterization of signal transduction pathways. This is in large part due to advances in protein sample preparation, analytical sensitivity of mass spectrometers and improvements in instrument software and protein databases ) . Until recently, assessment of interacting proteins has only been feasible via laborious and time-consuming molecular biologic techniques such as the yeast-two-hybrid system. Using a variety of approaches including protein complex purification, immunoprecipitation, affinity chromatography followed by high performance liquid chromatography (HPLC) and electrospray ionization and tandem mass spectrometry (ESI-MS/MS), interacting proteins of CD4 receptor complex and protein kinase Ce signaling complex have been identified.

The recent development of multidimensional liquid chromatographic methods combined with tandem mass spectrometry (LC-LC-MS/MS) has permitted sensitive detection of low abundance proteins, membrane proteins and proteins with extreme isoelectric points (pI). The ability to perform global quantitative proteomics has been significantly enhanced by the advent of the isotope-coded affinity tag-based technology (ICAT™ ) which is efficient in simplifying the proteome, and in combination with 3-D LC-MS/MS permits detection and quantification of proteins and peptides from very complex samples.

Our laboratory has utilized a functional proteomic approach to study anaplastic large cell lymphomas.

Identification of NPM-ALK interacting proteins by tandem mass spectrometry
Proteins that interact with ALK tyrosine kinase play important roles in mediating downstream cellular signals, and are potential targets for novel therapies. Using a functional proteomic approach, we determined the identity of proteins that interact with the ALK tyrosine kinase by co-immunoprecipitation with anti-ALK antibody followed by electrospray ionization (ESI) and tandem mass spectrometry (MS/MS). A total of 46 proteins were identified as unique to the ALK immunocomplex using monoclonal and polyclonal antibodies while 11 proteins were identified in the NPM immunocomplex. Previously reported proteins in the ALK signal pathway were identified including PI3-K, Jak2, Jak3, Stat3, Grb2, IRS and PLCγ1. More importantly, many proteins previously not recognized to be associated with NPM-ALK, but with potential NPM-ALK interacting protein domains were identified. These include adaptor molecules (SOCS, Rho-GTPase activating protein, RAB35), kinases (MEK kinase 1 and 4, PKC, MLCK, cyclin G-associated kinase, EphA1, JNK kinase, MAP kinase 1), phosphatases (meprin, PTPK, protein phosphatase 2 subunit) and heat shock proteins (Hsp60 precursor). Proteins identified by MS were confirmed by western blotting and reciprocal immunoprecipitation. 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.

Quantitative proteomic analysis of
differentially expressed proteins induced by NPM-ALK overexpression
The global molecular and cellular consequences of NPM-ALK overexpression are largely unknown. In addition, the identity and function of only a limited number of downstream molecules important for its oncogenic activity are currently understood. We have used a functional quantitative proteomic approach to determine the global effects of NPM-ALK expression on cell function. Jurkat cells were transfected with a plasmid containing the NPM-ALK chimeric gene. The NPM-ALK overexpressing Jurkat cells were compared to those expressing the vector only. Quantitative analyses of differentially expressed proteins were determined by isotope-coded affinity tagging (ICAT™) followed by liquid chromatography (LC) and tandem mass spectrometry (MS/MS). Equivalent quantities of total cell lysates obtained from the NPM-ALK transfected cells and the vector control cells were ICAT™ labeled, and subjected to avidin affinity chromatography. Offline fractions were collected, digested with trypsin, and analyzed by automated reverse phase nanospray LC-MS/MS. Some 124 proteins showed a 1.5 fold or greater change in the NPM-ALK positive cells as compared to the vector control cells. Of these, 79 proteins were unregulated by greater than 1.5-fold while 45 proteins were downregulated by greater than 1.5-fold. Differential expression of selected proteins was validated by western blot analyses. Analysis of functional groups of proteins demonstrated upregulation of protein kinases, cytoskeletal proteins and proteins associated with proliferation and translation. In addition, upregulation of proteins previously reported to be important mediators of the ALK signaling pathway including, PLCγ1, Ki-67, GRB2, Jak2, and PI3-K were observed. This study, for the first time, reveals the global proteomic consequences of NPM-ALK overexpression as a singular molecular abnormality and provides novel insight into the signal transduction pathways influenced by induced cellular transformation of NPM-ALK.

References

1. Aebersold R, Mann M. Mass spectrometry-based proteomics. Nature. 2003 Mar 13;422(6928):198-207.
2. Crockett DK, Lin Z, Elenitoba-Johnson KS and Lim MS Identification of NPM-ALK interacting proteins by tandem mass spectrometry. Oncogene 2004 In press.
3. Duyster J, Bai RY, Morris SW. Translocations involving anaplastic lymphoma kinase (ALK). Oncogene. 2001 Sep 10;20(40):5623-37.
4. Edmondson RD, Vondriska TM, Biederman KJ, Zhang J, Jones RC, Zheng Y, Allen DL, Xiu JX, Cardwell EM, Pisano MR, Ping P. Protein kinase C epsilon signaling complexes include metabolism- and transcription/translation-related proteins: complimentary separation techniques with LC/MS/MS. Mol Cell Proteomics. 2002 Jun;1(6):421-33.
5. Figeys D, McBroom LD, Moran MF.Mass spectrometry for the study of protein-protein interactions. Methods. 2001 Jul;24(3):230-9.
6. Gygi SP, Rist B, Gerber SA, Turecek F, Gelb MH, Aebersold R. Quantitative analysis of complex protein mixtures using isotope-coded affinity tags Nat Biotechnol. 1999 Oct;17(10):994-9.
7. Kadin ME, Carpenter C. Systemic and primary cutaneous anaplastic large cell lymphomas. Semin Hematol. 2003 Jul;40(3):244-56.
8. Morris SW, Xue L, Ma Z, Kinney MC. Alk+ CD30+ lymphomas: a distinct molecular genetic subtype of non-Hodgkin's lymphoma. Br J Haematol. 2001 May;113(2):275-95.
9. Morris SW, Kirstein MN, Valentine MB, Dittmer KG, Shapiro DN, Saltman DL and Look AT (1994) Fusion of a kinase gene, ALK, to a nucleolar protein gene, NPM, in non- Hodgkin's lymphoma Science, 263, 1281-4.
10. Shiio Y, Donohoe S, Yi EC, Goodlett DR, Aebersold R, Eisenman RN.Quantitative proteomic analysis of Myc oncoprotein function. EMBO J. 2002 Oct 1;21(19):5088-96.