Global Proteomic Profiling Research Articles

Statistical and Computational Methods for Comparative Proteomic Profiling Using Liquid Chromatography-Tandem Mass Spectrometry

The combined method of LC-MS/MS is increasingly being used to explore differences in the proteomic composition of complex biological systems. The reliability and utility of such comparative protein expression profiling studies is critically dependent on an accurate and rigorous assessment of quantitative changes in the relative abundance of the myriad of proteins typically present in a biological sample such as blood or tissue. In this review, we provide an overview of key statistical and computational issues relevant to bottom-up shotgun global proteomic analysis, with an emphasis on methods that can be applied to improve the dependability of biological inferences drawn from large proteomic datasets. Focusing on a start-to-finish approach, we address the following topics: 1) low-level data processing steps, such as formation of a data matrix, filtering, and baseline subtraction to minimize noise, 2) mid-level processing steps, such as data normalization, alignment in time, peak detection, peak quantification, peak matching, and error models, to facilitate profile comparisons; and, 3) high-level processing steps such as sample classification and biomarker discovery, and related topics such as significance testing, multiple testing, and choice of feature space. We report on approaches that have recently been developed for these steps, discussing their merits and limitations, and propose areas deserving of further research.

Multidimensional protein identification technology: current status and future prospects

Protein profiling using high-throughput tandem mass spectrometry has become a powerful method for analyzing changes in global protein expression patterns in cells and tissues as a function of developmental, physiologic and disease processes. This review summarizes the utility and practical application of multidimensional protein identification technology as a platform for comprehensive proteomic profiling of complex biologic samples. The strengths and potential problems and limitations associated with this powerful technology are discussed, with an emphasis placed on one of the biggest challenges currently facing large-scale expression profiling projects – namely, data analysis. Complementary bioinformatic computational data mining strategies, such as clustering, functional annotation and statistical inference, are also discussed as these are increasingly necessary for interpreting the results of global proteomic profiling studies.

Proteome Changes Induced by Rituximab: Significance for Monoclonal Antibody Therapy of B-Cell Lymphoproliferative Disorders.

Rituximab is a human chimeric monoclonal antibody that targets the CD20 receptor uniquely expressed on normal and malignant B-cells. While it is particularly useful for follicular lymphoma, Rituximab has recently been used for other types of B-cell proliferative disorders. Four major mechanisms have been proposed to account for the activity of Rituximab in B-cell lymphoproliferative disorders: 1) apoptosis and proliferation inhibition via intracellular signaling, 2) increased sensitivity to other chemotherapeutic drugs, 3) complement-dependent cytotoxicity (CDC), and 4) antibody-dependent cellular cytotoxicity (ADCC). A combination of all four mechanisms has also been proposed as the mechanism of action. However, the intracellular events that occur in B-cells in response to Rituximab are largely unknown.We have employed a global quantitative proteomic profiling approach to study the effects of Rituximab on a t(14;18)+ B cell lymphoma (SUDHL-4). A decrease in viability of 31.3 ± 2.9% was observed after treatment with 10μg/mL of Rituximab for 48 hours. We used isotope-coded affinity tagging (ICAT™) in combination with microcapillary liquid chromatography and tandem mass spectrometry (μLC-MS/MS) to quantitatively determine the proteomic differences between equal amounts of untreated and treated total cell lystates. Protein lysates were labeled with either heavy or light cleavable ICAT™ reagents, digested with trypsin, and purified using avidin affinity chromatography. The labeled peptides were then fractionated using off-line strong cation exchange (SCX) and each fraction was subjected to μLC-MS/MS analysis. Several proteins previously unknown to be expressed in B-cells were identified using this approach. Of the 123 positively identified proteins, 59 proteins were differentially expressed by greater than or less than 1.5 fold (25 upregulated, 34 downregulated). Analysis of differentially expressed proteins identified several functional groups, including integral membrane proteins (Integrin alpha-7 precursor), transcription factors (Zinc finger proteins 445 and 20), proteins involved in migration and adhesion (Astrotactin1), proteins associated with degradation and turnover (26S proteasome non-ATPase regulatory subunit 9), proteins affecting calcium-induced signaling (Tumor-associated calcium signal transducer 1 precursor), proteins associated with lipid rafts (Gemin7), and components of the phosphoinositol (PI4K-alpha) and NF-κB pathways (IKK-E). Our studies reveal the proteomic consequences of Rituximab administration to neoplastic B-cells, and provide novel insights into the mechanism of the drug's efficacy in the treatment of susceptible B-cell lymphoproliferative disorders.

Inhibition of the insulin-like growth factor receptor-1 tyrosine kinase activity as a therapeutic strategy for multiple myeloma, other hematologic malignancies, and solid tumors

Insulin-like growth factors and their receptor (IGF-1R) have been implicated in cancer pathophysiology. We demonstrate that IGF-1R is universally expressed in various hematologic (multiple myeloma, lymphoma, leukemia) and solid tumor (breast, prostate, lung, colon, thyroid, renal, adrenal cancer, retinoblastoma, and sarcoma) cells. Specific IGF-1R inhibition with neutralizing antibody, antagonistic peptide, or the selective kinase inhibitor NVP-ADW742 has in vitro activity against diverse tumor cell types (particularly multiple myeloma), even those resistant to conventional therapies, and triggers pleiotropic antiproliferative/proapoptotic molecular sequelae, delineated by global transcriptional and proteomic profiling. NVP-ADW742 monotherapy or its combination with cytotoxic chemotherapy had significant antitumor activity in an orthotopic xenograft MM model, providing in vivo proof of principle for therapeutic use of selective IGF-1R inhibitors in cancer.