University of Virginia Patent Foundation

Rijksuniversiteit Te Leiden

Thermo Finnigan LLC

The University of Birmingham

University of Pittsburgh

The University of Oklahoma

Agenus Inc.

Johns Hopkins University

Albert Einstein College of Medicine

University Technologies International, Inc.

Rijsuniversiteit Te Leiden

Donald F Hunt

Jeffrey Shabanowitz

Victor H Engelhard

John Edward Philip Syka

Els A J M Goulmy

Joshua J Coon

Mark Cobbold

George C Stafford, Jr

Craig L Slingluff, Jr

Jarrod A Marto

Olivera J Finn

Rebecca C Obeng

Kara L Cummings

Angela Lee Zarling

Henry Kao

Andrea L Cox

William H Hilderbrand

David Kittlesen

Ann M English

Scott A Ugrin

Dina L Bai

Robert E Settlage

Ann Michelle English

Robert E Christian

Philip D Compton

Andrew Norris

Stacy Alyse Malaker

Oriana E Hawkins

John Edward Syka

John E P Syka

Suzanne L Topalian

Christopher Mullen

Lee Earley

Teresa P DiLorenzo

Scott M Lieberman

Angela L Ambakhutwala

Forest M White

Weihan Wang

Scott Ficarro

Lichao Zhang

Jonathan Skipper

Ronald C Hendrikson

Florence A Depontieu

Jie Qian

Victor M Engelhard

Els A Goulmy

Jennifer G Abelin

Sarah Amy Penny

Anne M Evans

Stanley G Nathenson

Pere Santamaria

Jarod A Marto

Scott A Urgin

The present disclosure relates to novel and improved methods of analyzing proteins, peptides and polypeptides by mass spectrometry using ion-ion reactions. More specifically the disclosure relates to improved methods for implementing the m/z selective arresting of ion-ion reactions within the ion-ion reaction cell of a mass spectrometer system during a period where ion-ion reactions are performed.

Rapid identification and sequence analysis of intact proteins in complex mixtures

A set of target peptides are presented by HLA A*0101, A*0201, A*0301, B*4402, B*2705, B*1402, and B*0702 on the surface of disease cells. They are envisioned to among other things (a) stimulate an immune response to the proliferative disease, e.g., cancer, (b) to function as immunotherapeutics in adoptive T cell therapy or as a vaccine, (c) facilitate antibody recognition of tumor boundaries in surgical pathology samples, (d) act as biomarkers for early detection and/or diagnosis of the disease, and (e) act as targets in the generation antibody-like molecules which recognize the target-peptide/MHC complex.

Target peptides for immunotherapy and diagnostics

The present invention describes novel tumor-specific phosphorylated peptides, nucleic acids encoding those peptides, and antibodies generated against said peptides. The genes, peptides, and antibodies described herein may be used as diagnostic indicators of the presence of breast cancer and/or used in therapeutics to treat breast cancer.

Identification of MHC class I phospho-peptide antigens from breast cancer utilizing SHLA technology and complementary enrichment strategies

A set of target peptides are presented by HLA A*0201, B*0301, B*0702 and B*2705 on the surface of disease cells. They are envisioned to, among other things, stimulate an immune response to the proliferative disease, e.g., colorectal cancer, to function as immunotherapeutics in adoptive T cell therapy or as a vaccine, facilitate antibody recognition of tumor boundaries in surgical pathology samples, act as biomarkers for early detection and/or diagnosis of the disease, and/or act as targets in the generation antibody-like molecules which recognize the target-peptide/MHC complex.

Target peptides for colorectal cancer therapy and diagnostics

The application discloses compositions, methods, systems, and apparatuses for rapid sequence analysis of proteins, including location of post-translational modifications and disulfide bonds. Limited digestion of fully denatured antibody occurs in seconds by flowing sample in 8M urea at constant pressure through a micro column reactor containing immobilized aspergillopepsin I, resulting in a product mixture containing 3-10 kDa peptides, which is then fractionated by capillary column chromatography and analyzed by both electron transfer dissociation (ETD) and collision activated dissociation mass spectrometry. This method provides 95% sequence coverage of a mAb and detects numerous post-translational modifications. For disulfide bond location, native mAb is subjected to longer digestion times. Release of disulfide containing peptides from accessible regions of the folded protein occurs with short digestion times. The identity of peptides connected by a disulfide bond is determined using ETD and ion-ion proton transfer chemistry.

Compositions and methods for analysis of protein sequences and post-translational modifications

We characterized a total of 175 HLA-DR-associated phosphopeptides using sequential affinity isolation, biochemical enrichment, mass spectrometric sequencing and comparative analysis. Many were derived from source proteins which may have roles in cancer development, growth and metastasis. Most were expressed exclusively by either melanomas or transformed B cells, suggesting the potential to define cell type-specific phosphatome 'fingerprints'. We generated HLA-DR&#x3b2;1*0101-restricted CD4T cells specific for a phospho-MART-1 peptide identified in two melanoma cell lines. These T cells showed specificity for phosphopeptide-pulsed antigen presenting cells as well as for intact melanoma cells. MHC II-restricted phosphopeptides recognizable by human CD4T cells are potential targets for cancer immunotherapy.

Phosphopeptides as melanoma vaccines

The invention provides improvements in reagents for use in electron transfer dissociation ionization techniques for use in mass spectrometry, particularly for sequencing peptides and proteins using mass spectrometric techniques involving electrospray ionization and MS/MS characterization of fragment ions. The novel reagents used in the inventive methods allow for more effective determination of protein sequences, especially of long peptides or post-translationally modified protein fragments. Use of the polycyclic aromatic hydrocarbons azulene, homoazulene, and acenaphthylene, and homodimers and heterodimers thereof, are described.

Reagents for electron transfer dissociation in mass spectrometry analysis

The present invention is based on the identification of a predominant ligand of CD8T cells that are responsible for type 1 diabetes. That ligand is islet-specific glucose-6-phosphatase catalytic subunit-related protein (IGRP). Several CD8T cell-binding peptides from IGRP are identified, including the peptide comprising amino acids 206-214 of the IGRP sequence, which has high avidity to the most prevalent T cell receptor of pathogenic CD8T cells in autoimmune diabetes. The invention thus provides oligopeptide and polypeptide compositions comprising YLKTN(A/I/L/V)FL (SEQ ID NO:3), FLWSVFWLI (SEQ ID NO:4), (T/A)YY(G/T)FLNFM (SEQ ID NO:5), LR(L/V)(F/L)(G/N)IDLL (SEQ ID NO:6), KWCANPDWI (SEQ ID NO:7), and SFCKSASIP (SEQ ID NO:8). Also provided are oligopeptide compositions 8-10 amino acids in length and completely homologous with a mammalian IGRP, where the oligopeptide is capable of binding a human MHC class I molecule. Additionally, various methods of treating a mammal using the above compositions are provided, where the mammal is at risk for or has type 1 diabetes. Also provided are methods of preventing a CD8T cell that is cytotoxic to pancreatic islet &#x3b2;-cells from destroying a mammalian &#x3b2;-cell, where the methods also use the above compositions. Further provided are methods for determining whether a mammal is at risk for or has type 1 diabetes, where the methods use the above compositions.

Antigens targeted by prevalent pathogennic T cells in type 1 diabetes and uses thereof

The present invention relates to a new method for fragmenting ions in a mass spectrometer through the use of electron transfer dissociation, and for performing sequence analysis of peptides and proteins by mass spectrometry. In the case of peptides, the invention promotes fragmentation along the peptide backbone and makes it possible to deduce the amino acid sequence of the sample, including modified amino acid residues, through the use of an RF field device.

Electron transfer dissociation for biopolymer sequence analysis

The invention provides a method for vaccinating a patient against a malignancy comprising introducing a protein or peptide comprising of all or an immunogenic fragment of a cyclin protein into the patient. The invention further provides a method of identifying tumor antigens.

Anticancer vaccine and diagnostic methods and reagents

A front-end reagent ion source for a mass spectrometer is disclosed. Reagent vapor is supplied to a reagent ionization volume located within a chamber of the mass spectrometer and maintained at a low vacuum pressure. Reagent ions are formed by interaction of the reagent vapor molecules with an electrical discharge (e.g., a glow discharge) within the ionization volume, and pass into the chamber of the mass spectrometer. At least one ion optical element located along the analyte ion path transports the reagent ions to successive chambers of the mass spectrometer. The reagent ions may be combined with the analyte ions to perform ion-ion studies such as electron transfer dissociation (ETD).

Method and apparatus for generation of reagent ions in a mass spectrometer

The present invention relates to a new method for identifying polypeptides by deducing the amino acid sequence of the carboxy and amino termini by a mass spectrometer analysis. The method comprises the steps of dissociating highly charged peptide precursor ions (e.g., z&#x3e;4) using electron transfer dissociation inducing anions followed by removal of those reagents and introduction of a second, proton transfer inducing anion type. The second PTR reaction duration is adjusted to convert the ETD products to primarily the +1 charge-state to reduce the highly charged c and z-type fragments, producing an m/z spectrum containing a series of c and z-type fragment ions that are easily interpreted to reveal the sequence of the amino and carboxy terminus, respectively.

Simultaneous sequence analysis of amino- and carboxy-termini

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Charlottesville, VA, United States of America

Donald F Hunt