Teck Yew Low

An affinity purification-mass spectrometry (AP-MS) method was
employed to identify novel substrates of the SCFβTrCP ubiquitin ligase.
A FLAG-HA tagged version of the F-box protein βTrCP2, the substrate
recognition subunit of SCFβTrCP, was used as bait. βTrCP2 wild type and
the two mutants βTrCP2-R447A and βTrCP2-ΔF were expressed and
purified from HEK293T cells to be able to discriminate between
potential substrates of SCFβTrCP and unspecific binders. Affinity-purified
samples were analyzed by mass spectrometry-based proteomics,
applying ultra-high performance liquid chromatography (UHPLC)
coupled to high-resolution tandem mass spectrometry. The raw mass
spectrometry data have been deposited to the PRIDE partner
repository with the identifiers PXD001088 and PXD001224. The
present dataset is associated with a research resource published in T.
Y. Low, M. Peng, R. Magliozzi, S. Mohammed, D. Guardavaccaro, A.J.R.
Heck, A systems-wide screen identifies substrates of the SCFβTrCP
ubiquitin ligase. Sci. Signal. 7 (2014) rs8–rs8,10.1126/scisignal.2005882.

I was brought up in a small town called Batu Pahat, working my way up to win a Malaysian government scholarship for undergraduate studies which allowed me to enrol in the Imperial College in Biotechnology. I was then offered a research scholarship by the National University of Singapore to work on my PhD project based on the 2-D gel technology, classical biochemical methods and MALDI-TOF MS. Not satisfied, I then acquired skills in mass spectrometry especially FT and Orbitrap, as well as in different types of chromatography which facilitates separation of proteins, peptides and post-translational modifications. I am actively involved in biological and biochemical research in the fields of stem cells and signal transduction. Since 2 years ago, I have also extended my reach to the integration of genomics, transcriptomics and proteomics. From time to time, I am also contacted for reviewing manuscripts submitted to international peer reviewed journals such as Proteomics and Nature Nanotechnology. I am open for all opportunities in bioanalytical research, especially in exploring possible future new directions, questions and applications in biodiversity and their conservation, renewable bio-energy resources, synthetic organisms and synthetic biology. My ORCID profile is available in http://orcid.org/0000-0001-7878-7534.

刘德耀，马来西亚华裔，毕业于伦敦帝国理工学院，又于2005在新加坡国立大学获取博士学位。从事蛋白质组研究逾十年，对飞行时间质谱（TOF）﹑ 离子阱（ion trap）﹑ 傅立叶变换静电场轨道阱（FT Orbitrap）与傅立叶变换离子回旋共振（FT-ICR）的操作以及流程优化有相当的经验，尤其善于利用各种气相离子裂解技术：如碰撞诱导解离（CAD）﹑ 更高能量碰撞诱导解离（HCD）与电子转移解离（ETD）来达到蛋白以及其修饰（磷酸化和泛素化）的测序和定量的目的。现任职于荷兰乌得勒支大学-荷兰国家蛋白质组中心，利用各种分析技术来解决生物及生化课题，尤其在干细胞研究，Wnt 信号转导及整合基因组﹑转录组﹑蛋白质组方面陆续于优质科学期刊如：自然（Nature）﹑ 细胞（Cell）和其子刊等发表论文。对将来的工作重心始终保持开的态度。不过希望能在蛋白质组技术的运用与合成生物学或合成基因组学结合，在环境与物种保育及再生能源等领域做出贡献。

Protein digestion using a dedicated protease represents a key element in a typical mass spectrometry (MS)-based shotgun proteomics experiment. Up to now, digestion has been predominantly performed with trypsin, mainly because of its high specificity, widespread availability and ease of use. Lately, it has become apparent that the sole use of trypsin in bottom-up proteomics may impose certain limits in our ability to grasp the full proteome, missing out particular sites of post-translational modifications, protein segments or even subsets of proteins. To overcome this problem, the proteomics community has begun to explore alternative proteases to complement trypsin. However, protocols, as well as expected results generated from these alternative proteases, have not been systematically documented. Therefore, here we provide an optimized protocol for six alternative proteases that have already shown promise in their applicability in proteomics, namely chymotrypsin, LysC, LysN, AspN, GluC and ArgC. This protocol is formulated to promote ease of use and robustness, which enable parallel digestion with each of the six tested proteases. We present data on protease availability and usage including recommendations for reagent preparation. We additionally describe the appropriate MS data analysis methods and the anticipated results in the case of the analysis of a single protein (BSA) and a more complex cellular lysate (Escherichia coli). The digestion protocol presented here is convenient and robust and can be completed in ~2 d.

T-cell lymphoma invasion and metastasis 1
(Tiam1) is a guanine nucleotide exchange factor
that specifically controls the activity of the small
GTPase Rac, a key regulator of cell adhesion,
proliferation and survival. Here, we report that in
response to mitogens, Tiam1 is degraded by the
ubiquitin-proteasome system via the SCFβTrCP
ubiquitin ligase. Mitogenic stimulation triggers
the binding of Tiam1 to the F-box protein βTrCP
via its degron sequence and subsequent Tiam1
ubiquitylation and proteasomal degradation. The
proteolysis of Tiam1 is prevented by βTrCP
silencing, inhibition of CK1 and MEK, or
mutation of the Tiam1 degron site. Expression of
a stable Tiam1 mutant that is unable to interact
with βTrCP results in sustained activation of the
mTOR/S6K signaling and increased apoptotic
cell death. We propose that the SCFβTrCPmediated
degradation of Tiam1 controls the
duration of the mTOR-S6K signaling pathway in
response to mitogenic stimuli.

Epithelial cell migration is crucial for the development and regeneration of epithelial tissues. Aberrant regulation of epithelial cell migration has a major role in pathological processes such as the development of cancer metastasis and tissue fibrosis. Here, we report that in response to factors that promote cell motility, the Rap guanine exchange factor RAPGEF2 is rapidly phosphorylated by I-kappa-B-kinase-β and casein kinase-1α and consequently degraded by the proteasome via the SCF(βTrCP) ubiquitin ligase. Failure to degrade RAPGEF2 in epithelial cells results in sustained activity of Rap1 and inhibition of cell migration induced by HGF, a potent metastatic factor. Furthermore, expression of a degradation-resistant RAPGEF2 mutant greatly suppresses dissemination and metastasis of human breast cancer cells. These findings reveal a molecular mechanism regulating migration and invasion of epithelial cells and establish a key direct link between IKKβ and cell motility controlled by Rap-integrin signaling.

Affinity purification coupled with mass spectrometry (AP-MS) is a widely used approach for the identification of protein-protein interactions. However, for any given protein of interest, determining which of the identified polypeptides represent bona fide interactors versus those that are background contaminants (for example, proteins that interact with the solid-phase support, affinity reagent or epitope tag) is a challenging task. The standard approach is to identify nonspecific interactions using one or more negative-control purifications, but many small-scale AP-MS studies do not capture a complete, accurate background protein set when available controls are limited. Fortunately, negative controls are largely bait independent. Hence, aggregating negative controls from multiple AP-MS studies can increase coverage and improve the characterization of background associated with a given experimental protocol. Here we present the contaminant repository for affinity purification (the CRAPome) and describe its use for scoring protein-protein interactions. The repository (currently available for Homo sapiens and Saccharomyces cerevisiae) and computational tools are freely accessible at http://www.crapome.org/.

Ubiquitin (Ub) is a small protein modifier that is covalently attached to the ε-amino group of lysine residues of protein substrates, generally targeting them for degradation. Due to the emergence of specific anti-diglycine (-GG) antibodies and the improvement in MS, it is now possible to identify more than 10 000 ubiquitylated sites in a single proteomics study. Besides cataloging ubiquitylated sites, it is equally important to unravel the biological relationship between ubiquitylated substrates and the ubiquitin conjugation machinery. Relevant to this, we discuss the role of affinity purification-MS (AP-MS), in characterizing E3 ligase-substrate complexes. Recently, such strategies have also been adapted to screen for binding partners of both deubiquitylating enzymes (DUBs) and ubiquitin-binding domains (UBDs). The complexity of the "ubiquitome" is further expanded by the fact that Ub itself can be ubiquitylated at any of its seven lysine residues forming polyubiquitin (polyUb), thus diversifying its lengths and topologies to suit a variety of molecular recognition processes. Therefore, applying MS to study polyUb linkages is also becoming an emerging and important area. Finally, we discuss the future of MS-based proteomics in answering important questions with respect to ubiquitylation.

LGR51 stem cells reside at crypt bottoms, intermingled with
Paneth cells that provide Wnt, Notch and epidermal growth factor
signals1. Here we find that the related RNF43 and ZNRF3 transmembrane
E3 ubiquitin ligases are uniquely expressed in LGR51
stem cells. Simultaneous deletion of the two genes encoding these
proteins in the intestinal epithelium of mice induces rapidly growing
adenomas containing high numbers of Paneth and LGR51
stem cells. In vitro, growth of organoids derived from these
adenomas is arrested when Wnt secretion is inhibited, indicating
a dependence of the adenoma stem cells on Wnt produced by
adenoma Paneth cells. In the HEK293T human cancer cell line,
expression of RNF43 blocks Wnt responses and targets surfaceexpressed
frizzled receptors to lysosomes. In the RNF43-mutant
colorectal cancer cell line HCT116, reconstitution of RNF43
expression removes its response to exogenous Wnt. We conclude
that RNF43 and ZNRF3 reduce Wnt signals by selectively ubiquitinating
frizzled receptors, thereby targeting these Wnt receptors
for degradation.

In this manuscript, we present a proof-of-concept study for targeted relative protein quantitation workflow using chemical labeling in the form of dimethylation, coupled with selected reaction monitoring (dimethyl-SRM). We first demonstrate close to complete isotope incorporation for all peptides tested. The accuracy, reproducibility, and linear dynamic range of quantitation are further assessed based on known ratios of nonhuman standard proteins spiked into human cerebrospinal fluid (CSF) as a model complex matrix. Quantitation reproducibility below 20% (CV < 20%) was obtained for analyte concentrations present at a dynamic range of 4 orders of magnitude lower than that of the background proteins. An error of less than 15% was observed when measuring the abundance of 44 out of 45 major human plasma proteins. Dimethyl-SRM was further examined by comparing the relative quantitation of eight proteins in human CSF with the relative quantitation obtained using synthetic heavy peptides coupled to stable isotope dilution-SRM (SID-SRM). Comparison between the two methods reveals that the correlation between dimethyl-SRM and SID-SRM is within 0.3-33% variation, demonstrating the accuracy of relative quantitation using dimethyl-SRM. Dimethyl labeling coupled with SRM provides a fast, convenient, and cost-effective alternative for relative quantitation of a large number of candidate proteins/peptides.

Metal and metal oxide chelating-based phosphopeptide enrichment technologies provide powerful tools for the in-depth profiling of phosphoproteomes. One weakness inherent to current enrichment strategies is poor binding of phosphopeptides containing multiple basic residues. The problem is exacerbated when strong cation exchange (SCX) is used for pre-fractionation, as under low pH SCX conditions phosphorylated peptides with multiple basic residues elute with the bulk of the tryptic digest and therefore require more stringent enrichment. Here, we report a systematic evaluation of the characteristics of a novel phosphopeptide enrichment approach based on a combination of low pH SCX and Ti(4+)-immobilized metal ion affinity chromatography (IMAC) comparing it one-to-one with the well established low pH SCX-TiO(2) enrichment method. We also examined the effect of 1,1,1,3,3,3-hexafluoroisopropanol (HFP), trifluoroacetic acid (TFA), or 2,5-dihydroxybenzoic acid (DHB) in the loading buffer, as it has been hypothesized that high levels of TFA and the perfluorinated solvent HFP improve the enrichment of phosphopeptides containing multiple basic residues. We found that Ti(4+)-IMAC in combination with TFA in the loading buffer, outperformed all other methods tested, enabling the identification of around 5000 unique phosphopeptides containing multiple basic residues from 400 μg of a HeLa cell lysate digest. In comparison, ∼ 2000 unique phosphopeptides could be identified by Ti(4+)-IMAC with HFP and close to 3000 by TiO(2). We confirmed, by motif analysis, the basic phosphopeptides enrich the number of putative basophilic kinases substrates. In addition, we performed an experiment using the SCX/Ti(4+)-IMAC methodology alongside the use of collision-induced dissociation (CID), higher energy collision induced dissociation (HCD) and electron transfer dissociation with supplementary activation (ETD) on considerably more complex sample, consisting of a total of 400 μg of triple dimethyl labeled MCF-7 digest. This analysis led to the identification of over 9,000 unique phosphorylation sites. The use of three peptide activation methods confirmed that ETD is best capable of sequencing multiply charged peptides. Collectively, our data show that the combination of SCX and Ti(4+)-IMAC is particularly advantageous for phosphopeptides with multiple basic residues.

With the completion of the human genome project, analysis of enriched phosphotyrosyl proteins from epidermal growth factor (EGF)-induced phosphotyrosine proteome permits the identification of novel downstream substrates of the EGF receptor (EGFR). Using cICAT-based LC-MS/MS method, we identified and relatively quantified the tyrosine phosphorylation levels of 21 proteins between control and EGF-treated A431 human cervical cancer cells. Of these, Endofin, DCBLD2, and KIAA0582 were validated to be novel tyrosine-phosphorylation targets of EGF signaling and Iressa, a highly selective inhibitor of EGFR. In addition, EGFR activity was shown to be necessary for EGF-induced localization of Endofin, an FYVE domain-containing protein regulated by phosphoinositol lipid and engaged in endosome-mediated receptor modulation. Although several groups have conducted phosphoproteomics of EGF signaling in recent years, our study is the first to identify and validate Endofin, DCBLD2, and KIAA0582 as part of a complex EGF phosphotyrosine signaling network. These novel data will provide new insights into the complex EGF signaling and may have implications on target-directed cancer therapeutics.

Embryonic stem (ES) cells are pluripotent cells with self-renewing property. Nanog is a homeobox transcription factor required to maintain ES cells in a non-differentiated state. Using affinity purification coupled to liquid chromatography-tandem mass spectrometry analysis, we identified Sall4 as a Nanog co-purified protein. Co-immunoprecipitation and glutathione S-transferase pulldown experiments confirmed the interaction between Nanog and Sall4. We showed that Nanog and Sall4 co-occupied Nanog and Sall4 enhancer regions in living ES cells. Knockdown of Nanog or Sall4 by RNA interference led to a reduction in Nanog and Sall4 enhancer activities, providing evidence that these factors are positively regulating these enhancers. Importantly, co-transfection of Sall4 with these ES cell-specific enhancers led to transactivation in heterologous somatic cells. Chromatin immunoprecipitation experiments also showed that Sall4 co-occupied many Nanog binding sites in ES cells. Our data implicate Sall4 as an important component of the transcription regulatory networks in ES cells by cooperating with Nanog. We suggest that Sall4 and Nanog form a regulatory circuit similar to that of Oct4 and Sox2. This study highlights the extensive regulatory loops connecting genes, which encode for key transcription factors in ES cells.

We have developed a rapid microwave-assisted protein digestion technique based on classic acid hydrolysis reaction with 2% formic acid solution. In this mild chemical environment, proteins are hydrolyzed to peptides, which can be directly analyzed by MALDI-MS or ESI-MS without prior sample purification. Dilute formic acid cleaves proteins specifically at the C-terminal of aspartyl (Asp) residues within 10 min of exposure to microwave irradiation. By adjusting the irradiation time, we found that the extent of protein fragmentation can be controlled, as shown by the single fragmentation of myoglobin at the C-terminal of any of the Asp residues. The efficacy and simplicity of this technique for protein identification are demonstrated by the peptide mass maps of in-gel digested myoglobin and BSA, as well as proteins isolated from Escherichia coli K12 cells.

Thioacetamide (TAA) administration is an established technique for generating rat models of liver fibrosis and cirrhosis. Oxidative stress is believed to be involved as TAA-induced liver fibrosis is initiated by thioacetamide S-oxide, which is derived from the biotransformation of TAA by the microsomal flavine-adenine dinucleotide (FAD)-containing monooxygense (FMO) and cytochrome P450 systems. A two-dimensional gel electrophoresis-mass spectrometry approach was applied to analyze the protein profiles of livers of rats administered with sublethal doses of TAA for 3, 6 and 10 weeks respectively. With this approach, 59 protein spots whose expression levels changed significantly upon TAA administration were identified, including three novel proteins. These proteins were then sorted according to their common biochemical properties and functions, so that pathways involved in the pathogenesis of rat liver fibrosis due to TAA-induced toxicity could be elucidated. As a result, it was found that TAA-administration down-regulated the enzymes of the primary metabolic pathways such as fatty acid β-oxidation, branched chain amino acids and methionine breakdown. This phenomenon is suggestive of the depletion of succinyl-CoA which affects heme and iron metabolism. Up-regulated proteins, on the other hand, are related to oxidative stress and lipid peroxidation. Finally, these proteomics data and the data obtained from the scientific literature were integrated into an “overview model” for TAA-induced liver cirrhosis. This model could now serve as a useful resource for researchers working in the same area.