Harvard College

Dana-farber-cancer Institute Inc.

Whitehead Institute for Biomedical Research

Steven P Gygi

Wilhelm Haas

Martin Helmut Wuhr

Brian K Erickson

Loren D Walensky

Gregory H Bird

Craig Braun

Graeme C McAlister

Judit Villen

Scott Anthony Gerber

Junmin Peng

David Sabatini

Marc W Kirschner

Lawrence Licklider

Kazuishi Kubota

Robert A Saxton

Lynne Chantranupong

Christopher Michael Rose

Robert A Everley

Yonghao Yu

Leonid Peshkin

Ramin Rad

Melanie P Gygi

Sean Beausoleil

Carlos Augusto Gartner

Graeme Conrad McAlister

Graeme Conrad Mcalister

In some embodiments, a mass spectrometry tag may comprise a linker region, a mass balance region, and a reporter region. The mass spectrometry tag may be configured to fragment in a mass spectrometer via an energy dependent process to produce multiple reporter molecules. For example, the reporter region of the tag may be configured to produce at least two reporter molecules via fragmentation. In some embodiments, one or more regions of the tag may comprise at least one heavy isotope. In some such embodiments, the ability to fragment into multiple reporter molecules as well as the placement and/or number of heavy isotope(s) allows the mass spectrometry tag to be distinguished from other similar mass spectrometry tags. In some such embodiments, the ability to distinguish between tags having the same or substantially similar total mass to charge ratio and reporter region mass may allow the system to have a greater multiplexing capacity than conventional systems.

Reagents for quantitative mass spectrometry

The invention relates to methods of identifying compounds that modulate mTORC1 activity in a cell by modulating the activity of CASTOR1, as well as to the use of such identified compounds in the modulation of mTORC1 and the treatment of diseases and conditions characterized by aberrant mTORC1 activity.

Methods of identifying modulators of CASTOR1-GATOR2 interaction and use of same to modulate mTORC1

Described herein is a kit of materials prepared for assays that involve determining relative abundance and/or absolute abundance of various targeted peptides. The kit may comprise trigger versions of target peptides with masses offset from the respective target peptides by predetermined and known amounts. The trigger peptides may be present in amounts that may be readily detected via a mass spectrometry analysis. When mixed with samples that are analyzed, detection of the trigger peptides indicates where in the mass-spectrometer output the target peptide may be found. The kit may include a predetermined amount of synthetic versions of one or more of the target peptides. A measured relative abundance of this synthetic peptide relative to that of the target peptides yields an absolute quantitative value of the target peptide. Also disclosed is a method of preparing a plurality of samples to be submitted for mass spectrometer analysis in parallel.

Techniques for high throughput targeted proteomic analysis and related systems and methods

A mass-spectrometry-based method and substrates are provided herein for large scale kinome activity profiling directly from crude lysates using 90 chemically synthesized peptide substrates with amino acid sequences derived from known phosphoproteins. Quantification of peptide phosphorylation rates was achieved via the use of stable isotope labeled synthetic peptides. A method and substrates for obtaining 90 simultaneous activity measurements in a single-reaction format were developed and validated. The kinome activity profiling strategy was successfully applied with lysates of: cells manipulated by combination of mitogen stimulation, pharmacological perturbation and siRNA-directed kinase knockdown; seven different breast cancer cell lines treated with gefitinib; and each of normal and cancerous tissue samples from renal cell carcinoma patients. This method concurrently measures multiple peptide phosphorylation rates to provide a diagnostic fingerprint pattern for activated kinases, protein phosphatases, modulators of these enzymes, and pathways (kinome) from as little starting material as a few cells.

Methods, compositions and kits for high throughput kinase activity screening using mass spectrometry and stable isotopes

Embodiments are directed to a method, a computer readable medium encoded with instructions that, when executed, perform a method, and a system for performing mass spectrometry analysis. Molecules of different samples may be labeled with a chemical tag, allowing a multiplexed analysis of multiple samples. The labeled molecules may be fragmented, each fragmented molecule creating at least two separate ions. The relative abundance of each of the heavier ions, which may comprise the original molecule from the sample, may be measured. A relative abundance of the labeled molecules in each of the samples may be determined from the measured relative abundances of the heavier ions.

Accurate and interference-free multiplexed quantitative proteomics using mass spectrometry

A method of performing a mass spectrometry analysis includes labeling each of a plurality of samples with a corresponding chemical tag; forming a first plurality of ions from molecules in the samples; selecting a subset of the first plurality of ions, the subset being selected by isolating ions of the first plurality of ions in a plurality of ranges of mass-to-charge; forming a second plurality of ions by fragmenting ions in the subset; and measuring information indicative of a quantity of each of the plurality of chemical tags present in each of the plurality of samples.

Mass spectrometry for multiplexed quantitation using multiple frequency notches

The invention provides a method detecting and quantifying proteins by mass spectrophotometric analysis using peptide internal standards and provides a highly sensitive way of detecting protein modifications. In one aspect, the invention provides a method for determining a site of ubiquitination in a polypeptide and for evaluating ubiquitination targets in a population of polypeptides. In this way, a proteome ubiquitination map can be obtained which comprises information relating to the ubiquitination states of a plurality of cellular polypeptides. Maps can be obtained for a variety of different types of cells and cell states. For example, ubiquitination targets in normal and diseased cells can be evaluated. Preferably, the map is stored as data files in a database. Individual ubiquitinated polypeptides identified can be used to generate molecular probes diagnostic of a cell state and/or can serve as targets for agents that modulate one or more cellular processes.

Detection and quantification of modified proteins

The present invention relates to phosphopeptide compositions and anti-phosphopeptide antibody compositions. Also provided are methods of identifying phosphorylation sites in phosphorylated peptides and phosphorylation site motifs.

Phosphopeptide compositions and anti-phosphopeptide antibody compositions and methods of detecting phosphorylated peptides

The invention provides reagents, kits and methods for detecting and/or quantifying proteins in complex mixtures, such as a cell lysate. The methods can be used in high throughput assays to profile cellular proteomes. In one aspect, the invention provides a peptide internal standard labeled with a stable isotope and corresponding in amino acid sequence to the amino acid sequence of a subsequence of a target polypeptide. In another aspect, the peptide internal standard is labeled at a modified amino acid residue and is used to determine the presence of, and/or quantitate the amount of a particular modified form of a protein.

Absolute quantification of proteins and modified forms thereof by multistage mass spectrometry

The invention provides non-affinity based isotope tagged peptides, chemistries for making these peptides, and methods for using these peptides. In one aspect, tags comprise a reactive site (RS) for reacting with a molecule on a protein to form a stable association with the peptide (e.g., a covalent bond) and an anchoring site (AS) group for reversibly or removably anchoring the tag to a solid phase such as a resin support. Anchoring may be direct or indirect (e.g., through a linker molecule). Preferably, the anchoring site comprises a biotin compound. Preferably, the tag comprises a mass-altering label, such as a stable isotope, such that association of the tag with the peptide can be monitored by mass spectrometry. The reagents can be used for rapid and quantitative analysis of proteins or protein function in mixtures of proteins.

Capture and release based isotope tagged peptides and methods for using the same

A system for automatically performing liquid chromatography analysis of low volume liquid chemical samples at nanosecond flow rates using an analysis column that integrates a pre-concentration trapping column and a chromatography separation column terminating at an electrospray nozzle of an online mass spectrometer. The analysis column consists of a capillary having an inside diameter of between 75 and 125 microns packed throughout with a porous bed of micron particles. A branch outlet positioned 10 to 16 centimeters upstream from the nozzle divides the analysis column into an upstream pre-concentration trap and a downstream separation column. An autosampler delivers low volume liquid samples to the upstream inlet via a two-position valve. Feed connections couple the autosampler to upstream inlet when the valve is open to inject a liquid sample into the pre-concentration trap at a maximum loading flow rate in the range from 0.5 to 50 microliters/minute. Thereafter, when the valve closes, it terminates the further injection the sample, and a concentrated portion of the sample then passes though the chromatography separation column at a much slower flow rate between 10 and 1,000 nanoliters per minute. Throughput can be doubled by coupling two such analysis columns to a single autosampler using a ten-port, two position valve. A single column can be supplied through a six port two-position valve.

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Foxborough, MA, United States of America

Steven P Gygi