Below are some of our lab's research highlights over the years:
Developing the quantitative proteomics approach, SILAC. The completion of the draft human genome in 2001 and advances in MS-based protein identification led to the emerging field of proteomics. In place of single proteins, proteomics examines biological systems by collective analyses of proteins by measuring changes in abundance, subcellular localization, interactions in multi-protein complexes, and post-translationally modified (PTM) forms. A global quantitative proteomics strategy is necessary for the unbiased quantitative analyses of these changes. Shao-En is the first author on the SILAC paper describing the approach to quantify protein and peptides, published in Molecular and Cellular Proteomics in 2002 (Refs 1 and 2). Although the ICAT chemical tagging strategy developed in 1999 was the prototypical and dominant quantitative proteomics approach at the time, it was limited to labeling and enriching cysteine-containing peptides. 15N-isotopic labeling for protein quantification had been described in 1999 but was practical only for organisms that could grow on 15N enriched media like yeast and bacteria. SILAC was optimized for use in mammalian cell culture and it is extremely simple and robust to use. It has since been extended to whole organisms like mice, worms, zebrafish and flies. Commercial kits were developed by multiple vendors (Pierce, Invitrogen, etc.) and the method is widely used by researchers worldwide. The original SILAC paper has been cited over 4000 times (Google Scholar, July 2016). Quantitative methods using isobaric chemical labels like iTRAQ and TMT have been developed but SILAC is still the dominant metabolic labeling approach and its user base continues to grow.
1. Ong SE, Blagoev B, Kratchmarova I, Kristensen DB, Steen H, Pandey A, Mann M. Stable isotope labeling by amino acids in cell culture, SILAC, as a simple and accurate approach to expression proteomics. Mol Cell Proteomics. 2002 May;1(5):376-86.
2. Ong SE, Kratchmarova I, Mann M. Properties of 13C-substituted arginine in stable isotope labeling by amino acids in cell culture (SILAC). J Proteome Res. 2003 Mar-Apr;2(2):173-81.
3. Ong SE. The expanding field of SILAC. Anal Bioanal Chem. 2012 Sep;404(4):967-76.
4. Lau HT, Suh HW, Golkowski M, Ong SE. Comparing SILAC- and stable isotope dimethyl-labeling approaches for quantitative proteomics. J Proteome Res. 2014 Sep 5;13(9):4164-74. PMCID: PMC4156256.
Temporal and systems-analyses of proteomes. Systems-level analyses of protein abundance changes in proteomics were impractical before global and unbiased quantitative methods like SILAC. I am a first author of a paper describing a triple labeling SILAC approach to study phosphotyrosine-dependent signaling in EGFR dependent signaling pathways (Ref. 5); this study first demonstrated the power of proteomics for unbiased analyses of cellular signaling pathways and was extended to peptide-based analyses of PTMs like phosphorylation, acetylation, ubiquitination, methylation etc.
1. Blagoev B*, Ong SE*, Kratchmarova I, Mann M. Temporal analysis of phosphotyrosine-dependent signaling networks by quantitative proteomics. Nat Biotechnol. 2004 Sep;22(9):1139-45. *First Authors
2. Golkowski M, Shimizu-Albergine M, Suh HW, Beavo JA, Ong SE. Studying mechanisms of cAMP and cyclic nucleotide phosphodiesterase signaling in Leydig cell function with phosphoproteomics. Cell Signal. 2016;28(7):764-78. doi: 10.1016/j.cellsig.2015.11.014. PubMed PMID: 26643407.
Encoding of cellular methylation by SILAC for confident PTM identification and quantification. Cellular protein methylation is best known in the context of histone biology and epigenetics. Despite clear evidence of methylation on non-histone substrates, its functional importance is largely underappreciated. Challenges of methylated peptide identification in proteomics include poor enrichment yields by antibodies targeting methylated amino acids and peptide misidentification owing to the combinatorial complexity of methylation analysis; as an example mono-, di-, tri-methylation can occur on lysines and many amino acids differ in mass by a CH2 group. Shao-En is the first author of the paper describing the heavy methyl SILAC labeling approach which uses 13C2H3-methionine to specifically encode the methyl-PTM with isotopic labels (Ref. 6). Incorporating heavy methyl labels throughout the proteome, identification of different methylated peptide forms can be determined by the mass difference between light and heavy labeled states. We described the confident identification and quantification of methylated sites, including novel substrates. Heavy methyl SILAC has been used by many of the leading labs in the methylation field to study the dynamics of histone turnover.
1. Ong SE, Mittler G, Mann M. Identifying and quantifying in vivo methylation sites by heavy methyl SILAC. Nat Methods. 2004 Nov;1(2):119-26.
Proteomics approaches to study ADP-ribosylation. ADP-ribosylation of protein substrates occurs post-translationally on structurally and chemically diverse amino acids including aspartate, glutamate, lysine, arginine and cysteines. ADP-ribose can be added singly as mono(ADP-ribose) or as a polymeric chain of varying length of 2-200 units. Due to the heterogeneity of the PTM and the 17-member family of enzymes, ADP-ribosyltransferases (aka PARPs), that can apply this PTM to cellular protein substrates, the biological roles and significance of this enigmatic PTM has become an intense area of research. We developed a proteomics approach to identify sites of ADP-ribosylation based on an enrichment of the phosphoribose group upon cleavage of the PTM by a phosphodiesterase. This method is generally applicable to study all ADP-ribosylated substrates, in contrast to other published methods that detect peptides modified on certain ADP-ribosylated residues.
1. Daniels CM, Ong SE*, Leung AKL*. Phosphoproteomic approach to characterize protein mono- and poly(ADP-ribosyl)ation sites from cells. J Proteome Res. 2014;13(8):3510–3522. PMCID: PMC4123941. *Corresponding authors
2. Daniels CM, Ong SE, Leung AKL. The Promise of Proteomics for the Study of ADP-Ribosylation. Mol Cell; 2015;58(6):911–924. PMCID: PMC4486045
3. Daniels CM, Ong SE, Leung AKL. ADP-Ribosylated Peptide Enrichment and Site Identification: The Phosphodiesterase-Based Method. Methods Mol Biol. 2017;1608:79-93. doi: 10.1007/978-1-4939-6993-7_7. PubMed PMID: 28695505.
4. Daniels CM, Thirawatananond P, Ong SE, Gabelli SB, Leung AK. Nudix hydrolases degrade protein-conjugated ADP-ribose. Sci Rep. 2015;5:18271. doi: 10.1038/srep18271. PubMed PMID: 26669448; PMCID: PMC4680915.
5. Palazzo L, Daniels CM, Nettleship JE, Rahman N, McPherson RL, Ong SE, Kato K, Nureki O, Leung AK, Ahel I. ENPP1 processes protein ADP-ribosylation in vitro. FEBS J. 2016;283(18):3371-88. doi: 10.1111/febs.13811. PubMed PMID: 27406238; PMCID: PMC5030157.
6. McPherson RL, Abraham R, Sreekumar E, Ong SE, Cheng SJ, Baxter VK, Kistemaker HA, Filippov DV, Griffin DE, Leung AK. ADP-ribosylhydrolase activity of Chikungunya virus macrodomain is critical for virus replication and virulence. Proc Natl Acad Sci U S A. 2017;114(7):1666-71. doi: 10.1073/pnas.1621485114. PubMed PMID: 28143925; PMCID: PMC5321000.
Confident identification of small-molecule and nucleic acid interactions by quantitative proteomics. The affinity enrichment experiment with a molecular bait, such as a protein, peptide, small-molecule or nucleic acid, is a widely used experimental design to discover functionally important protein-bait interactions. With the increased sensitivity and improved identification coverage of newer mass spectrometers, such experiments are difficult to interpret without quantitative proteomics, as hundreds of background binding proteins can be identified from “clean” and “specific” affinity pull-downs. We have published numerous primary research articles describing the use of quantitative proteomics to identify specific bait-protein interactions over the last decade. This has been especially important in the identification of proteins that interact with small-molecule probes and drugs (Refs. 1, 2) as well as protein-DNA interactions (Refs. 3, 4). Research in the Ong laboratory currently focuses on target identification for bioactive small molecules and the characterization of regulatory elements in the genome.
1. Ong SE, Schenone M, Margolin AA, Li X, Do K, Doud MK, Mani DR, Kuai L, Wang X, Wood JL, Tolliday NJ, Koehler AN, Marcaurelle LA, Golub TR, Gould RJ, Schreiber SL, Carr SA. Identifying the proteins to which small-molecule probes and drugs bind in cells. Proc Natl Acad Sci U S A. 2009 Mar 24;106(12):4617-22. PMCID: PMC2649954.
2. Golkowski M, Brigham JL, Perera GK, Romano GE, Maly DJ, Ong SE. Rapid profiling of protein kinase inhibitors by quantitative proteomics. Medchemcomm. 2014 Mar;5(3):363-369. PMCID: PMC3955727.
3. Wrann CD, Eguchi J, Bozec A, Xu Z, Mikkelsen T, Gimble J, Nave H, Wagner EF, Ong SE*, Rosen ED*. FOSL2 promotes leptin gene expression in human and mouse adipocytes. J Clin Invest. 2012 Mar;122(3):1010-21. doi: 10.1172/JCI58431. PMCID: PMC3322535. *Corresponding authors
4. Lee MN, Roy M, Ong SE, Mertins P, Villani AC, Li W, Dotiwala F, Sen J, Doench JG, Orzalli MH, Kramnik I, Knipe DM, Lieberman J, Carr SA, Hacohen N. Identification of regulators of the innate immune response to cytosolic DNA and retroviral infection by an integrative approach. Nat Immunol. 2013 Feb;14(2):179-85. PMCID: PMC3838897.
5. Golkowski M, Vidadala RS, Lombard CK, Suh HW, Maly DJ, Ong SE. Kinobead and Single-Shot LC-MS Profiling Identifies Selective PKD Inhibitors. J Proteome Res. 2017;16(3):1216-27. doi: 10.1021/acs.jproteome.6b00817. PubMed PMID: 28102076.
Kinome activity mapping to study cellular signaling. We use the kinobead approach to enrich kinases, their interacting partners, and associated phosphorylation sites to measure activities of signaling pathways in cells and tissue. Using a panel of cell lines, we have observed ~70% of the human kinome in our assays, providing good coverage of many signaling pathways. This approach was applied to study luteinizing hormone dependent signaling (Ref 1) and in a pharmacoproteomics platform to study drug response in liver cancer cell lines (Ref 2).
1. Golkowski M, Vidadala VN, Lau HT, Shoemaker A, Shimizu-Albergine M, Beavo J, Maly DJ, Ong SE. Kinobead/LC-MS Phosphokinome Profiling Enables Rapid Analyses of Kinase-Dependent Cell Signaling Networks. J Proteome Res. 2020;19(3):1235-47. doi: 10.1021/acs.jproteome.9b00742. PubMed PMID: 32037842.
2. Golkowski M, Lau HT, Chan M, Kenerson H, Vidadala VN, Shoemaker A, Maly DJ, Yeung RS, Gujral TS, Ong SE. Pharmacoproteomics Identifies Kinase Pathways that Drive the Epithelial-Mesenchymal Transition and Drug Resistance in Hepatocellular Carcinoma. Cell Syst. 2020;11(2):196-207 e7. doi: 10.1016/j.cels.2020.07.006. PubMed PMID: 32755597; PMCID: PMC7484106.