Genetics Institute, Inc.

Wyeth

Yongchang Qiu

Rodney M Hewick

Jack H Wang

Lih-Ling Lin

Joseph Lawrence Wooters

Jun Kuai

Elliott Nickbarg

The invention relates to compositions and methods comprising lymphotoxin-beta receptor (LT&#x3b2;R) modulators, which activate or inhibit LT&#x3b2;R signaling. LT&#x3b2;R modulators are useful for treating lymphocyte mediated immunological diseases and cancer, and more particularly, for regulating mitochondrial-mediated apoptosis. This invention relates to soluble forms of the LT&#x3b2;R complex proteins that act as LT&#x3b2;R activating or inhibiting agents. This invention also relates to the use of soluble molecules, directed against either the LT&#x3b2;R, its ligands, LIGHT and LT&#x3b2;1&#x3b1;2, or its intracellular binding partners, that function to regulate LT&#x3b2;R signaling. A novel screening method for selecting soluble receptors, antibodies and other agents that modulate LT&#x3b2;R signaling is provided.

Method of identifying a modulator of a LT&#x3b2;R complex signaling pathway

The method of the invention provides novel compounds, termed acid-labile isotope-coded extractants (ALICE), for quantitative mass spectrometric analysis of protein mixtures. The compounds contain a thiol-reactive group that is used to capture cysteine-containing peptides from all peptide mixtures, an acid-labile linker, and a non-biological polymer. One of the two acid-labile linkers is isotopically labeled and therefore enables the direct quantitation of peptides/proteins through mass spectrometric analysis. Because no functional proteins are required to capture peptides, a higher percentage of organic solvent can be used to solubilize the peptides, particularly hydrophobic peptides, through the binding, washing and eluting steps, thus permitting much better recover of peptides. Moreover, since the peptides are covalently linked to the non-biological polymer (ALICE), more stringent washing is allowed in order to completely remove non-specifically bound species. Finally, peptides captured by ALICE are readily eluted from the polymer support under mild acidic condition with high yield and permit the direct down stream mass spectrometric analysis without any further sample manipulation. In combination with our novel dual column two dimensional liquid chromatography-mass spectrometry (2D-LC-MS/MS) design, the ALICE procedure proves to a general approach for quantitative mass spectrometric analysis of protein mixtures with better dynamic range and sensitivity.

Acid-labile isotope-coded extractant (ALICE) and its use in quantitative mass spectrometric analysis of protein mixtures

Arginine-containing cysteine-modifying compounds useful for MALDI-MS analysis of proteins are provided. These compounds termed isotope-coded ionization enhancement reagents (ICIER) can provide ionization enhancement in MALDI-MS, relative quantitation, and additional database searching constraints at the same time without any extra sample manipulation. More specifically, ICIER increase the ionization efficiency of cysteine-containing peptides by attachment of a guanidino functional group. ICIER also increase the overall hydrophilicity of these peptides due to the hydrophilic nature of ICIER and thus increase the percentage of recovery of these peptides during sample handling and processing such as in-gel digestion or liquid chromatography. Finally, a combination of both light and heavy ICIER provides an accurate way to obtain relative quantitation of proteins by MALDI-MS and additional database searching constraints (number of cysteine residues in every single peptide peak) to increase the confidence of protein identification by peptide mass mapping.

Isotope-coded ionization-enhancing reagents (ICIER) for high-throughput protein identification and quantitation using matrix-assisted laser desorption ionization mass spectrometry

The method of the invention provides novel compounds, termed acid-labile isotope-coded extractants (ALICE), for quantitative mass spectrometric analysis of protein mixtures. The compounds contain a thiol-reactive group that is used to capture cysteine-containing peptides from all peptide mixtures, an acid-labile linker, and a non-biological polymer. One of the two acid-labile linkers is isotopically labeled and therefore enables the direct quantitation of peptides/proteins through mass spectrometric analysis. Because no functional proteins are required to capture peptides, a higher percentage of organic solvent can be used to solubilize the peptides, particularly hydrophobic peptides, through the binding, washing and eluting steps, thus permitting much better recovery of peptides. Moreover, since the peptides are covalently linked to the non-biological polymer (ALICE), more stringent washing is allowed in order to completely remove non-specifically bound species. Finally, peptides captured by ALICE are readily eluted from the polymer support under mild acidic condition with high yield and permit the direct down stream mass spectrometric analysis without any further sample manipulation. In combination with our novel dual column two dimensional liquid chromatography-mass spectrometry (2D-LC-MS/MS) design, the ALICE procedure proves to a general approach for quantitative mass spectrometric analysis of protein mixtures with better dynamic range and sensitivity.

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Arlington, MA, United States of America

Yongchang Qiu