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Protocols

Selected references from the Broad proteomics group that describe methods and tools we have developed, optimized and applied in our studies:

 

TMT Isobaric mass tag labeling of cells and tissues for deepscale proteome and phosphoproteome analysis

Reproducible workflow for multiplexed deep-scale proteome and phosphoproteome analysis of tumor tissues by liquid chromatography-mass spectrometry. Mertins et al. Nature Protocols. 2018 Jul;13(7):1632-1661. PMID: 29988108. PMCID: PMC6211289.

Streamlined Protocol for Deep Proteomic Profiling of FAC-sorted Cells and Its Application to Freshly Isol­ated Murine Immune Cells.  Myers et al. Mol Cell Proteomics. 2019 May;18(5):995-1009. PMID: 30792265. PMCID: PMC6495249.

TMT Labeling for the Masses: A Robust and Cost- efficient, In-solution Labeling Approach. Zecha et al. Mol Cell Proteomics. 2019 Jul;18(7):1468-1478. PMID: 30967486. PMCID: PMC6601210.

 

Methods and applications of proteogenomic analysis of human cancers

Connecting genomic alterations to cancer biology with proteomics: The NCI Clinical Proteomic Tumor Analysis Consortium. Ellis et al. Cancer Discovery. 2013 3(10) 1108-1112. PMCID: PMC3800055.

Microscaled proteogenomic methods for precision oncology. Satpathy et al. Nature Communications. 2020 Jan 27;11(1):532. PMID: 31988290. PMCID: PMC6985126.

Methods, Tools and Current Perspectives in Proteogenomics. Ruggles et al. Mol Cell Proteomics. 2017 Jun;16(6):959-981. PMID: 28456751. PMCID: PMC5461547.

Proteomics, Post-translational Modifications, and Integrative Analyses Reveal Molecular Heterogeneity within Medulloblastoma Subgroups. Archer et al. Cancer Cell. 2018 561(7721):132-136. PMCID: PMC6372116

Proteogenomic Characterization Reveals Therapeutic Vulnerabilities in Lung Adenocarcinoma. Gillette et al. Cell. 2020 182(1): 200-225.e35. PMCID: PMC7373300.

Proteogenomic Landscape of Breast Cancer Tumorigenesis and Targeted Therapy. Krug, et al. Cell. 2020 Nov 25;183(5):1436-1456.e31. PMID: 33212010.

Proteogenomics connects somatic mutations to signalling in breast cancer. Mertins, et al. Nature. 2016 Jun 2;534(7605):55-62. PMID: 27251275. PMCID: PMC5102256.

 

Global Analysis of Protein Posttranslational Modifications and Biotinylation

Serial enrichment of posttranslational modifications (phospho, acetyl, etc.)
Integrated proteomic analysis of post-translational modifications by serial enrichment. Nature Methods.et al. 2013 10(7): 634 – 637. PMCID: PMC3943163.

Proteomics, Post-translational Modifications, and Integrative Analyses Reveal Molecular Heterogeneity within Medulloblastoma Subgroups. Archer et al. Cancer Cell. 2018 561(7721):132-136. PMCID: PMC6372116

Microscaled proteogenomic methods for precision oncology. Satpathy et al. Nature Communications. 2020 Jan 27;11(1):532. PMID: 31988290. PMCID: PMC6985126.

Proteogenomic Characterization Reveals Therapeutic Vulnerabilities in Lung Adenocarcinoma. Gillette et al. Cell. 2020 182(1): 200-225.e35. PMCID: PMC7373300.

Ischemia in tumors induces early and sustained phosphorylation changes in stress kinase pathways but does not affect global protein levels. Mertins, et al. Mol Cell Proteomics. 2014 Jul;13(7):1690-704. PMID: 24719451. PMCID: PMC4083109.

 

Acetylation
Deep, quantitative coverage of the lysine acetylome using novel anti-acetyl-lysine antibodies and an optimized proteomic workflow. Svinkina et al. Mol Cell Proteomics. 2015: 14(9):2429-40. PMCID: PMC4563726.

 

Ubiquitylation
Large-scale identification of ubiquitination sites by mass spectrometry. Udeshi et al. Nature Protocols. 201) 8(10):1950-60. PMCID:  PMC4725055.

Rapid and deep-scale ubiquitylation profiling for biology and translational research. Udeshi et al. Nature Communications. 2020 Jan 17;11(1):359. doi: 10.1038/s41467-019-14175-1. PMID: 31953384. PMCID: PMC6969155.

 

Biotinylation
Antibodies to biotin enable large-scale detection of biotinylation sites on proteins. Nat Methods. 2017 Dec;14(12):1167-1170. PMID: 29039416.

 

Histone Modifications by Global Chromatin Profiling

Global chromatin profiling reveals NSD2 mutations in pediatric acute lymphoblastic leukemia. Jaffe et al. Nature Genetics 2013 45(11): 1386-1391. PMCID: PMC4262138.

Building the Connectivity Map of epigenetics: Chromatin profiling by quantitative targeted mass spectrometry. Creech et al. Methods 2014 15(72):57-64. PMCID: PMC4300274.

 

Targeted MS Assay Development and Application (MRM, SRM, PRM)

Targeted Peptide Measurements in Biology and Medicine: Best Practices for Mass Spectrometry-based Assay Development Using a Fit-for-Purpose Approach. Carr et al. Mol Cell Proteomics. (2014) 13(3): 907-917. PMCID: PMC3945918.

Simplified and Efficient Quantification of Low Abundance Proteins at Very High Multiplex by Targeted Mass Spectrometry. Burgess et al. Mol Cell Proteomics. 2014 13(4): 1137-1149. PMCID: PMC3977191.

Demonstrating the feasibility of large-scale development of standardized assays to quantify human proteins. Kennedy et al. Nature Methods.

CPTAC Assay Portal: a repository of targeted proteomic assays. Whiteaker et al. Nature Methods.

Large-Scale Interlaboratory Study to Develop, Analytically Validate and Apply Highly Multiplexed, Quantitative Peptide Assays to Measure Cancer-Relevant Proteins in Plasma. Abbatiello et al. Mol. Cell Proteomics. American Society for Biochemistry and Molecular Biology; 2015 Sep;14(9):2357–74. PMCID: PMC4563721

Recommendations for the Generation, Quantification, Storage, and Handling of Peptides Used for Mass Spectrometry-Based Assays. Hoofnagle et al. Clin Chem 2016 62(1):48–69. PMCID: PMC4830481.

Automated Micro-chromatography Enables Multiplexing of Immunoaffinity Enrichment of Peptides to Greater than 150 for Targeted MS-Based Assays. Ippoliti et al. Analytical Chem 2016 Aug 2;88(15):7548-55. PMID: 27321643.

New Guidelines for Publication of Manuscripts Describing Development and Application of Targeted Mass Spectrometry Measurements of Peptides and Proteins. Abbatiello et al. Mol Cell Proteomics. 2017 16(3): 327-328. PMCID: PMC5340997.

Design, implementation and multisite evaluation of a system suitability protocol for the quantitative assessment of instrument performance in liquid chromatography-multiple reaction monitoring-MS (LC-MRM-MS). Abbatiello, et al. Mol Cell Proteomics. 2013 Sep;12(9):2623-39. PMID: 23689285. PMCID: PMC3769335.

 

Plasma Proteomic and Biomarker discovery

Quantitative, multiplexed workflow for deep analysis of human blood plasma and biomarker discovery by mass spectrometry. Keshishian, et al. Nat Protoc. 2017 Aug;12(8):1683-1701. PMID: 28749931. PMCID: PMC6057147.

Rapid, deep and precise profiling of the plasma proteome with multi-nanoparticle protein corona. Blume, et al. Nat Commun (2020) 11(1): 3662. PMCID: PMC7376165.

A pipeline that integrates the discovery and verification of plasma protein biomarkers reveals candidate markers for cardiovascular disease. Addona, et al. Nat Biotechnol. 2011 Jun 19;29(7):635-43. PMID: 21685905. PMCID:
PMC3366591.

Multi-site assessment of the precision and reproducibility of multiple reaction monitoring-based measurements of proteins in plasma. Addona, et al. Nat Biotechnol. 2009 Jul;27(7):633-41. PMID: 19561596. PMCID: PMC2855883.

 

Extracellular Matrix Analysis Methods

The Extracellular Matrix: Tools and Insights for the “Omics” Era. Naba et al. Matrix Biology 2015  49: 10-24. PMCID: PMC5013529.

Proteomic Profiling of the ECM of Xenograft Breast Cancer Metastases in Different Organs Reveals Distinct Metastatic Niches. Hebert et al. Cancer Research 2020 Apr 1;80(7):1475-1485. PMID: 32019869. PMCID: PMC7127975.

 

Proximity Ligation Labeling of protein, DNA and RNA using APEX and TurboID

Spatially resolved proteomic mapping in living cells with the engineered peroxidase APEX2. Hung et al. Nature Protocols. 2016 Mar;11(3):456-75. PMID: 26866790. PMCID: PMC4863649.

Antibodies to biotin enable large-scale detection of biotinylation sites on proteins. Nat Methods. 2017 Dec;14(12):1167-1170. PMID: 29039416.

Discovery of proteins associated with a predefined genomic locus via dCas9-APEX-mediated proximity labeling. Myers et al. Nature Methods.

Efficient proximity labeling in living cells and organisms with TurboID. Branon et al. Nature Biotechnology 2018 Oct;36(9):880-887. Erratum in: Nature Biotechnology 2020 Jan;38(1):108. PMID: 30125270. PMCID: PMC6126969.

Split-TurboID enables contact-dependent proximity labeling in cells. Cho et al. Proc Natl Acad Sci U S A. 2020 Jun 2;117(22):12143-12154. PMID: 32424107. PMCID: PMC7275672.

Proximity labeling in mammalian cells with TurboID and split-TurboID. Cho et al. Nature Protocols. 2020 Dec;15(12):3971-3999. PMID: 33139955.

RNA-protein interaction mapping via MS2- or Cas13-based APEX targeting. Han et al.  Proc Natl Acad Sci USA 2020 117(36): 22068-22079.

 

Affinity Proteomics for Protein-Protein, Protein-small Molecule and Protein-DNA/RNA Interaction

Protein-Protein Interaction
Integrated Genomics of Crohn's Disease Risk Variant Identifies a Role for CLEC12A in Antibacterial Autophagy. Begun et al. Cell Reports 2015  11(12):1905-18. PMCID: PMC4507440.

TMEM258 Is a Component of the Oligosaccharyltransferase Complex Controlling ER Stress and Intestinal Inflammation. Graham et al. Cell Reports 2016 17(11):2955-2965. PMID: 27974209

The C9orf72-interacting protein Smcr8 is a negative regulator of autoimmunity and lysosomal exocytosis. Zhang et al. Genes Development 2018  32(13-14):929-943. PMCID: PMC6075033.

C1orf106 is a colitis risk gene that regulates stability of epithelial adherens junctions. Mohanan  et al. Science 2018  359(6380): 1161-1166. PMCID: PMC6008784.

 

Protein-small molecule Interaction
Identifying the proteins to which small-molecule probes and drugs bind in cells. Ong et al. Proc Nat Acad Sci. 2009  106(12): 4617-4622. PMCID: PMC2649954

Empirical Bayes Analysis of Quantitative Proteomics Experiments. Margolin et al. PLoS One. 2009  4(10): 1-15. PMCID: PMC2759080

AAK1 Identified as an Inhibitor of Neuregulin-1/ErbB4-Dependent Neurotrophic Factor Signaling Using Integrative Chemical Genomics and Proteomics. Kuai et al. Chem Biol 2011  18(7): 891-906. PMCID: PMC3368601.

Selective killing of cancer cells by a small molecule targeting the stress response to ROS. Raj et al. Nature 2011.  475(7355): 231-234. PMCID: PMC3316487.

Identification of Regulators of Polyploidization Presents Therapeutic Targets for Treatment of AMKL. Wen et al. Cell 2012  150 (3): 575–589. PMCID: PMC3613864.

Identifying cellular targets of small-molecule probes and drugs with biochemical enrichment and SILAC. Ong et al. Methods Mol Biol 2012 803: 129-140.

Target Identification and mechanism of action in chemical biology and drug discovery. Schenone et al. Nature Chemical Biology 2013; 9; 232-240

BRG1 Loss Predisposes Lung Cancers to Replicative Stress and ATR Dependency. Gupta, et al. Cancer Res. 2020 Sep 15;80(18):3841-3854. PMID: 32690724. PMCID: PMC7501156.


Protein-RNA/DNA Interaction
The NORAD lncRNA assembles a topoisomerase complex critical for genome stability. Munschauer et al. Nature. 2018 Sep;561(7721):132-136. Erratum in:Nature. 2018 Oct 2. PMID: 30150775. The SARS-CoV-2 RNA-protein interactome in infected human cells. Schmidt, et al. Nat Microbiol. 2020 Dec 21. PMID: 33349665.

 

HLA Peptide Analysis and Prediction
Mass Spectrometry Profiling of HLA-Associated Peptidomes in Mono-allelic Cells Enables More Accurate Epitope Prediction. Abelin et al. Immunity 2017 Feb 21;46(2):315-326. PMID: 28228285. PMCID: PMC5405381.

A large peptidome dataset improves HLA class I epitope prediction across most of the human population. Sarkizova et al. Nature Biotechnology 2020 Feb;38(2):199-209. PMID: 31844290. PMCID: PMC7008090.

 

Software and Tools for data analysis

Phosphosite-specific Signature Analysis
A Curated Resource for Phosphosite-specific Signature Analysis. Krug, et al. Mol Cell Proteomics. 2019 Mar;18(3):576-593. PMID: 30563849. PMCID: PMC6398202.

 

Targeted MS Assays

Automated detection of inaccurate and imprecise transitions in peptide quantification by multiple reaction monitoring mass spectrometry. Abbatiello, et al. Clin Chem. 2010 Feb;56(2):291-305. PMID: 20022980. PMCID: PMC2851178.

Statistical characterization of multiple-reaction monitoring mass spectrometry (MRM-MS) assays for quantitative proteomics. Mani, et al. BMC Bioinformatics. 2012;13 Suppl 16(Suppl 16):S9. PMID: 23176545. PMCID: PMC3489552.

 

Predicting MS peptide fragmentation

Prediction of high-responding peptides for targeted protein assays by mass spectrometry. Fusaro, et al. Nat Biotechnol. 2009 Feb;27(2):190-8. PMID: 19169245. PMCID: PMC2753399.

 

DIA-MS data
Specter: linear deconvolution for targeted analysis of data-independent acquisition mass spectrometry proteomics. Peckner et al. Nature Methods.

Avant-garde: An automated data-driven DIA data curation tool. Vaca Jacome et al. Nature Methods (2020) 17(12):1237-1244 PMID: 33199889 PMCID: PMC7723322