Isotope Labeling In Cell Culture Research Articles

Glycan/Protein-Stable Isotope Labeling in Cell Culture for Enabling Concurrent Quantitative Glycomics/Proteomics/Glycoproteomics.

The complexity and heterogeneity of protein glycosylation present an analytical challenge to the studies of characterization and quantitation. Various LC-MS-based quantitation strategies have emerged in recent decades. Metabolic stable isotope labeling has been developed to enhance the accurate LC/MS-based quantitation between different cell lines. Stable isotope labeling by amino acids in a cell culture (SILAC) is the most widely used metabolic labeling method in proteomic analysis. However, it can only label the peptide backbone and is thus limited in glycomic studies. Here, we present a metabolic isotope labeling strategy, named GlyProSILC (Glycan Protein Stable Isotope Labeling in Cell Culture), that can label both the glycan motif and peptide backbone from the same batch of cells. It was performed by feeding cells with a heavy medium containing amide-15N-glutamine, 13C6-arginine (Arg6), and 13C6-15N2-lysine (Lys8). No significant change of cell line metabolism after GlyProSILC labeling was observed based on transcriptomic, glycomic, and proteomic data. The labeling conditions, labeling efficiency, and quantitation accuracy were investigated. After quantitation correction, we simultaneously quantified 62 N-glycans, 574 proteins, and 344 glycopeptides using the same batch of mixed 231BR/231 cell lines. So far, GlyProSILC provides an accurate and effective quantitation approach for glycomics, proteomics, and glycoproteomics in a cell culture system.

Abstract 2437: Identification of MDMX/MDM2 metastasis signaling pathways in breast cancer cells with mutant p53

Abstract Human breast cancers often overexpress MDM family proteins MDM2 and MDMX(MDM4). MDM2 is an E3 ubiquitin ligase and MDMX is an E4 that promotes ubiquitination of MDM2 targets (however this is an understudied area). The Cancer Genome Atlas (TCGA) shows high MDMX/MDM2 expression in all breast cancer subtypes. We identified that MDMX depletion reduces circulating tumor cells (CTCs) and metastasis of breast cancers with mtp53. Our ongoing objective is to determine the mechanisms of how MDMX promotes CTCs and metastasis, and the potential MDMX regulated ubiquitinated targets. In TNBC primary tumors, knockdown of mdmx correlates with reduced expression of the G protein coupled receptor, CXCR4. We used mtp53 expressing T47D ER+, and MDA-MB-468, and MDA-MB-231 metastatic TNBC cells and their derived CTC lines, and shRNA-mediated knockdown of MDM2 and MDMX derived lines, to address the potential MDMX targets. We assessed metastatic properties of migration and invasion changed by the knockdown of MDM2/MDMX in parental and CTC cell lines using multi-faceted cell biological and biochemical approaches. Depletion of MDM2 in T47D and MDA-MB-231 cells increased the level of MDMX, demonstrating a critical function of MDM2 for regulating MDMX. MDMX depletion surprisingly did not change MDM2 levels, but did reduce the migration of both MDA-MB-231 cells and their derived CTC lines. Interestingly, the parental MDA-MB-231 cells migrated more rapidly in scratch assays, compared to both their derived CTCs and MDMX-depleted CTCs, suggesting migration to be critical for intravasation, but not for latter cancer cell survival. Experiments are in progress to address if loss of MDMX perturbs CXCL12 ligand communication with the CXCR4 receptor in parental and CTC breast cancer lines. Preliminary data suggests activated MDMX expression correlates with the activation of these metastasis signal transduction pathways. The ubiquitination targets of MDMX/MDM2 in the parental and CTC cell lines are being defined by affinity purification in the presence, and absence, of MDMX/MDM2, and add back of purified MDMX/MDM2 to validate targets. As expected, purified MDMX increases MDM2 E3 ubiquitin ligase activity, and on its own MDMX is inert. Stable isotope labeling in cell culture suggested protein targets involved in DNA replication and repair pathways. This preliminary data informed our interest in studying DNA damage signaling by pharmacologically inhibiting the DNA repair protein PARP. We examined how MDMX/MDM2 pathways respond to DNA damage caused by PARP inhibition. Our preliminary data affirms regulation of the MDMX/MDM2 stress-response circuit by replication stress response pathways, and reveals the requirement of MDMX/MDM2 for their function in the studied cell lines. Disrupting MDMX function has potential to be beneficial for inhibition of breast cancer progression and metastasis. Supported by BCRF-20-011. Citation Format: Rusia Lee, Viola Ellison, Gu Xiao, Dominique Forbes, Pam Leybengrub, Alexandra Kern, Falande Alexandre, George Annor, Jill Bargonetti. Identification of MDMX/MDM2 metastasis signaling pathways in breast cancer cells with mutant p53 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2437.

Quantitative Translation Proteomics Using mePROD.

Multiplexed enhanced protein dynamic mass spectrometry (mePROD MS) enables robust quantification of translation in cell culture. Tandem mass tags (TMT) are combined with pulsed stable isotope labeling in cell culture (pSILAC) to monitor newly synthesized proteins on a proteome wide scale. While approaches combining pSILAC and TMT typically require long labeling times to reach sufficient intensity of the newly synthesized peptides in the mass spectrometer, mePROD uses a carrier signal that boosts the survey scan intensity and strongly increases identification rates. Hence, this protocol provides an easy and cost-efficient method to profile proteome-wide translatome changes at a temporal resolution of minutes.

Advances in stable isotope labeling: dynamic labeling for spatial and temporal proteomic analysis.

The field of proteomics is continually improving, requiring the development of new quantitative methods. Stable isotope labeling in cell culture (SILAC) is a metabolic labeling technique originating in the early 2000s. By incorporating isotopically labeled amino acids into the media used for cell culture, unlabeled versus labeled cells can be differentiated by the mass spectrometer. Traditional SILAC labeling has been expanded to pulsed applications allowing for a new quantitative dimension of proteomics - temporal analysis. The complete introduction of Heavy SILAC labeling chased with surplus unlabeled medium mimics traditional pulse-chase experiments and allows for the loss of heavy signal to track proteomic changes over time. In a similar fashion, pulsed SILAC (pSILAC) monitors the initial incorporation of a heavy label across a period of time, which allows for the rate of protein label integration to be assessed. These innovative techniques have aided in inspiring numerous SILAC-based temporal and spatial labeling applications, including super SILAC, spike-in SILAC, spatial SILAC, and a revival in label multiplexing. This review reflects upon the evolution of SILAC and the pulsed SILAC application, introduces advances in SILAC labeling, and proposes future perspectives for this novel and exciting field.

Quantification of Modified Nucleosides in the Context of NAIL-MS.

Recent progress in epitranscriptome research shows an interplay of enzymes modifying RNAs and enzymes dedicated for RNA modification removal. One of the main techniques to study RNA modifications is liquid chromatography-coupled tandem mass spectrometry (LC-MS/MS) as it allows sensitive detection of modified nucleosides. Although RNA modifications have been found to be highly dynamic, state-of-the-art LC-MS/MS analysis only gives a static view on modifications and does not allow the investigation of temporal modification placement. Here, we present the principles of nucleic acid isotope labeling coupled with mass spectrometry, termed NAIL-MS, which overcomes these limitations by stable isotope labeling in human cell culture and gives detailed instructions on how to label cells and process samples in order to get reliable results. For absolute quantification in the context of NAIL-MS, we explain the production of internal standards in detail. Furthermore, we outline the requirements for stable isotope labeling in cell culture and all subsequent steps to receive nucleoside mixtures of native RNA for NAIL-MS analysis. In the final section of this chapter, we describe the distinctive features of NAIL-MS data analysis with a special focus toward absolute quantification of modified nucleosides.

Instrument Logic Increases Identifications during Mutliplexed Translatome Measurements.

Pulsed Stable Isotope Labeling in Cell culture (SILAC) approaches allow measurement of protein dynamics, including protein translation and degradation. However, its use for quantifying acute changes has been limited due to low labeled peptide stoichiometry. Here, we describe the use of instrument logic to select peaks of interest via targeted mass differences (TMD) for overcoming this limitation. Comparing peptides artificially mixed at low heavy-to-light stoichiometry measured using standard data dependent acquisition with or without TMD revealed 2–3-fold increases in identification without significant loss in quantification precision for both MS2 and MS3 methods. Our benchmarked method approach increased throughput by reducing the necessary machine time. We anticipate that all pulsed SILAC measurements, combined with tandem mass tagging (TMT) or not, would greatly benefit from instrument logic based approaches.

Isotopic Labeling and Quantitative Proteomics of Acetylation on Histones and Beyond.

Lysine acetylation is an important posttranslational modification (PTM) that regulates the function of proteins by affecting their localization, stability, binding, and enzymatic activity. Aberrant acetylation patterns have been observed in numerous diseases, most notably cancer, which has spurred the development of potential therapeutics that target acetylation pathways. Mass spectrometry (MS) has become the most adopted tool not only for the qualitative identification of acetylation sites but also for their large-scale quantification. By using heavy isotope labeling in cell culture combined with MS, it is now possible to accurately quantify newly synthesized acetyl groups and other PTMs, allowing differentiation between dynamically regulated and steady-state modifications. Here, we describe MS-based protocols to identify acetylation sites and quantify acetylation rates on both proteins in general and in the special case of histones. In the experimental approach for the former, 13C-glucose and D3-acetate are used to metabolically label protein acetylation in cells with stable isotopes, thus allowing isotope incorporation to be tracked over time. After protein extraction and digestion, acetylated peptides are enriched via immunoprecipitation and then analyzed by MS. For histones, a similar metabolic labeling approach is performed, followed by acid extraction, derivatization with propionic anhydride, and trypsin digestion prior to MS analysis. The procedures presented may be adapted to investigate acetylation dynamics in a broad range of experimental contexts, including different cell types and stimulation conditions.

Silac-based quantitative proteomic analysis of Lactobacillus reuteri CRL 1101 response to the presence of selenite and selenium nanoparticles

Stable isotope labeling in cell culture (SILAC) was applied for the first time on a lactic acid bacterium strain (L. reuteri CRL1101) for analyzing differential protein expression associated to selenite(Na2SeO3) and selenium nanoparticles (SeNPs) exposure. 57 and 47 proteins were found de-regulated by >1,5 fold in presence of selenite and SeNPs, respectively. Only 16 out of 104 proteins differentially expressed were commonly altered by selenite and SeNPs. The use of a clustered heat map allows us to visualize relations between the de-regulated proteins and exposure conditions. We identified a number of proteins involved in diverse functions and biological processes such as metabolism of carbohydrates, selenium and lipids; folding, sorting and degradation; environmental information and processing. In presence of both, selenite and SeNPs, proteins related to selenium metabolism such as cystathione beta-lyase and oxidoreductases (thioredoxine reductase and NAD/FAD oxidoreductase) were over expressed. Interestingly, the over expression of thioredoxin reductase could protect the host from oxidizing compounds. An over expression of phage proteins and chaperones with selenite was observed; this result and the fact that a lower cell count was detected when selenite was added could indicate that this latter Se species has a more deleterious effect than the nanoparticles.

Site-Specific Quantification of Protein Palmitoylation by Cysteine-Stable Isotope Metabolic Labeling.

Palmitoylation is one of the most important protein translational modifications and plays vital roles in many key biological processes. Aberrant palmitoylation has been associated with a variety of human diseases. So it is of great significance to profile the palmitoylated proteomes qualitatively and quantitatively. Here, we described a novel method based on the cysteine-stable isotope labeling in cell culture (cysteine-SILAC) to facilitate the quantitation of palmitoylated proteins by mass spectrometry (MS), in which "light" or "heavy" samples could be pooled and subjected to the subsequent analysis procedures simultaneously, minimizing systematic errors caused by parallel operations and improving quantitative accuracy and precision. The mass tags lay on the cysteine residues, which were the potential palmitoylated sites, indicating that all the putative modified sites/peptides could be quantified, including the C-terminal peptide of one protein. Due to the isotopically labeled cysteine, the nonspecifically adsorbed peptide without cysteine was singlet in MS spectra, whereas pair peaks should be the signals of putative palmitoylated peptides, which could reduce spectral complexity and achieve double verification for the putative palmitoylated peptides. Finally, the palmitoylome in hepatocellular carcinoma (HCC) cells with different metastasis potentials (MHCC-97L and HCC-LM3 cells) were analyzed for the first time. Totally, 151 proteins were found to be differentially palmitoylated with high confidence, including many important proteins involved in a variety of biological processes, such as protein palmitoylation, cell proliferation, signal transduction, regulation of cell migration, and so on.

Peptide Level Turnover Measurements Enable the Study of Proteoform Dynamics

The coordination of protein synthesis and degradation regulating protein abundance is a fundamental process in cellular homeostasis. Today, mass spectrometry-based technologies allow determination of endogenous protein turnover on a proteome-wide scale. However, standard dynamic SILAC (Stable Isotope Labeling in Cell Culture) approaches can suffer from missing data across pulse time-points limiting the accuracy of such analysis. This issue is of particular relevance when studying protein stability at the level of proteoforms because often only single peptides distinguish between different protein products of the same gene. To address this shortcoming, we evaluated the merits of combining dynamic SILAC and tandem mass tag (TMT)-labeling of ten pulse time-points in a single experiment. Although the comparison to the standard dynamic SILAC method showed a high concordance of protein turnover rates, the pulsed SILAC-TMT approach yielded more comprehensive data (6000 proteins on average) without missing values. Replicate analysis further established that the same reproducibility of turnover rate determination can be obtained for peptides and proteins facilitating proteoform resolved investigation of protein stability. We provide several examples of differentially turned over splice variants and show that post-translational modifications can affect cellular protein half-lives. For example, N-terminally processed peptides exhibited both faster and slower turnover behavior compared with other peptides of the same protein. In addition, the suspected proteolytic processing of the fusion protein FAU was substantiated by measuring vastly different stabilities of the cleavage products. Furthermore, differential peptide turnover suggested a previously unknown mechanism of activity regulation by post-translational destabilization of cathepsin D as well as the DNA helicase BLM. Finally, our comprehensive data set facilitated a detailed evaluation of the impact of protein properties and functions on protein stability in steady-state cells and uncovered that the high turnover of respiratory chain complex I proteins might be explained by oxidative stress.

Labeling Carboxyl Groups of Surface-Exposed Proteins Provides an Orthogonal Approach for Cell Surface Isolation.

Quantitative profiling of cell surface proteins is critically important for the understanding of cell-cell communication, signaling, tissue development, and homeostasis. Traditional proteomics methods are challenging for cell surface proteins due to their hydrophobic nature and low abundance, necessitating alternative methods to efficiently identify and quantify this protein group. Here we established carboxyl-reactive biotinylation for selective and efficient biotinylation and isolation of surface-exposed proteins of living cells. We assessed the efficiency of carboxyl-reactive biotinylation for plasma membrane proteins by comparing it with a well-established protocol, amine-reactive biotinylation, using SILAC (stable isotope labeling in cell culture). Our results show that carboxyl-reactive biotinylation of cell surface proteins is both more selective and more efficient than amine-reactive biotinylation. We conclude that it is a useful approach, which is partially orthogonal to amine-reactive biotinylation, allowing us to cast a wider net for a comprehensive profiling of cell surface proteins.

Nedd4 Deficiency in Vascular Smooth Muscle Promotes Vascular Calcification by Stabilizing pSmad1

The nonosseous calcification process such as atherosclerosis is one of the major complications in several types of metabolic diseases. In a previous study, we uncovered that aberrant activity of transforming growth factor β (TGF-β) signaling pathway could contribute to the vascular smooth muscle cells' (VSMCs) calcification process. Also, we identified NEDD4 E3 ligase as a key suppressor of bone morphogenetic protein (BMP)/Smad pathway via a polyubiquitination-dependent selective degradation of C-terminal phosphorylated Smad1 (pSmad1) activated by TGF-β. Here, we further validated and confirmed the role of Nedd4 in in vivo vascular calcification progression. First, Nedd4 deletion in SM22α-positive mouse tissues (Nedd4fl/fl ;SM22α-Cre) showed deformed aortic structures with disarranged elastin fibers at 24 weeks after birth. Second, vitamin D-induced aorta vascular calcification rate in Nedd4fl/fl ;SM22α-Cre mice was significantly higher than their wild-type littermates. Nedd4fl/fl ;SM22α-Cre mice showed a development of vascular calcification even at very low-level injection of vitamin D, but this was not exhibited in wild-type littermates. Third, we confirmed that TGF-β1-induced pSmad1 levels were elevated in Nedd4-deficient primary VSMCs isolated from Nedd4fl/fl ;SM22α-Cre mice. Fourth, we further found that Nedd4fl/fl ;SM22α-Cre mVSMCs gained mesenchymal cell properties toward osteoblast-like differentiation by a stable isotope labeling in cell culture (SILAC)-based proteomics analysis. Finally, epigenetic analysis revealed that methylation levels of human NEDD4 gene promoter were significantly increased in atherosclerosis patients. Collectively, abnormal expression or dysfunction of Nedd4 E3 ligase could be involved in vascular calcification of VSMCs by activating bone-forming signals during atherosclerosis progression. © 2016 American Society for Bone and Mineral Research.

Posttranscriptional Regulation in Adenovirus Infected Cells.

A deeper understanding of how viruses reprogram their hosts for production of progeny is needed to combat infections. Most knowledge on the regulation of cellular gene expression during adenovirus infection is derived from mRNA studies. Here, we investigated the changes in protein expression during the late phase of adenovirus type 2 (Ad2) infection of the IMR-90 cell line by stable isotope labeling in cell culture with subsequent liquid chromatography-high resolution tandem mass spectrometric analysis. Two biological replicates of samples collected at 24 and 36 h post-infection (hpi) were investigated using swapped labeling. In total, 2648 and 2394 proteins were quantified at 24 and 36 hpi, respectively. Among them, 659 and 645 were deregulated >1.6-fold at the two time points. The protein expression was compared with RNA expression using cDNA sequencing data. The correlation was surprisingly low (r = 0.3), and several examples of posttranscriptional regulation were observed; e.g., proteins related to carbohydrate metabolism were up-regulated at the protein level but unchanged at the RNA level, whereas histone proteins were down-regulated at the protein level but up-regulated at the RNA level. The deregulation of cellular gene expression by adenovirus is mediated at multiple levels and more complex than hitherto believed.

Time-resolved Analysis of Proteome Dynamics by Tandem Mass Tags and Stable Isotope Labeling in Cell Culture (TMT-SILAC) Hyperplexing

Recent advances in mass spectrometry have enabled system-wide analyses of protein turnover. By globally quantifying the kinetics of protein clearance and synthesis, these methodologies can provide important insights into the regulation of the proteome under varying cellular and environmental conditions. To facilitate such analyses, we have employed a methodology that combines metabolic isotopic labeling (Stable Isotope Labeling in Cell Culture - SILAC) with isobaric tagging (Tandem Mass Tags - TMT) for analysis of multiplexed samples. The fractional labeling of multiple time-points can be measured in a single mass spectrometry run, providing temporally resolved measurements of protein turnover kinetics. To demonstrate the feasibility of the approach, we simultaneously measured the kinetics of protein clearance and accumulation for more than 3000 proteins in dividing and quiescent human fibroblasts and verified the accuracy of the measurements by comparison to established non-multiplexed approaches. The results indicate that upon reaching quiescence, fibroblasts compensate for lack of cellular growth by globally downregulating protein synthesis and upregulating protein degradation. The described methodology significantly reduces the cost and complexity of temporally-resolved dynamic proteomic experiments and improves the precision of proteome-wide turnover data.

Identification of the targets of biologically active small molecules using quantitative proteomics.

Currently, cell-based screenings yield a multitude of small molecule modulators of diverse biological processes. The most demanding step in the course of elucidation of the mode of action of biologically active compounds is the identification of the target proteins. Although there is no generic approach available, affinity-based chemical proteomics is the most widely applied methodology. Particularly, quantitative chemical proteomics has proven very powerful in the identification of the putative targets of small molecules. Here we describe the procedure for identification of target proteins for small molecules employing affinity chromatography and the stable isotope labeling in cell culture (SILAC) for quantitative proteomics.

Analysis of T4SS-induced signaling by H. pylori using quantitative phosphoproteomics.

Helicobacter pylori is a Gram-negative bacterial pathogen colonizing the human stomach. Infection with H. pylori causes chronic inflammation of the gastric mucosa and may lead to peptic ulceration and/or gastric cancer. A major virulence determinant of H. pylori is the type IV secretion system (T4SS), which is used to inject the virulence factor CagA into the host cell, triggering a wide range of cellular signaling events. Here, we used a phosphoproteomic approach to investigate tyrosine signaling in response to host-pathogen interaction, using stable isotope labeling in cell culture (SILAC) of AGS cells to obtain a differential picture between multiple infection conditions. Cells were infected with wild type H. pylori P12, a P12Δ CagA deletion mutant, and a P12Δ PAI deletion mutant to compare signaling changes over time and in the absence of CagA or the T4SS. Tryptic peptides were enriched for tyrosine (Tyr) phosphopeptides and analyzed by nano-LC-Orbitrap MS. In total, 85 different phosphosites were found to be regulated following infection. The majority of phosphosites identified were kinases of the MAPK family. CagA and the T4SS were found to be key regulators of Tyr phosphosites. Our findings indicate that CagA primarily induces activation of ERK1 and integrin-linked factors, whereas the T4SS primarily modulates JNK and p38 activation.

High-Throughput Quantitative Proteomic Analysis of Dengue Virus Type 2 Infected A549 Cells

Disease caused by dengue virus is a global health concern with up to 390 million individuals infected annually worldwide. There are no vaccines or antiviral compounds available to either prevent or treat dengue disease which may be fatal. To increase our understanding of the interaction of dengue virus with the host cell, we analyzed changes in the proteome of human A549 cells in response to dengue virus type 2 infection using stable isotope labelling in cell culture (SILAC) in combination with high-throughput mass spectrometry (MS). Mock and infected A549 cells were fractionated into nuclear and cytoplasmic extracts before analysis to identify proteins that redistribute between cellular compartments during infection and reduce the complexity of the analysis. We identified and quantified 3098 and 2115 proteins in the cytoplasmic and nuclear fractions respectively. Proteins that showed a significant alteration in amount during infection were examined using gene enrichment, pathway and network analysis tools. The analyses revealed that dengue virus infection modulated the amounts of proteins involved in the interferon and unfolded protein responses, lipid metabolism and the cell cycle. The SILAC-MS results were validated for a select number of proteins over a time course of infection by Western blotting and immunofluorescence microscopy. Our study demonstrates for the first time the power of SILAC-MS for identifying and quantifying novel changes in cellular protein amounts in response to dengue virus infection.

Proteome-Wide Measurement of Protein Half-Lives and Translation Rates in Vasopressin-Sensitive Collecting Duct Cells

Vasopressin regulates water excretion, in part, by controlling the abundances of the water channel aquaporin-2 (AQP2) protein and regulatory proteins in the renal collecting duct. To determine whether vasopressin-induced alterations in protein abundance result from modulation of protein production, protein degradation, or both, we used protein mass spectrometry with dynamic stable isotope labeling in cell culture to achieve a proteome-wide determination of protein half-lives and relative translation rates in mpkCCD cells. Measurements were made at steady state in the absence or presence of the vasopressin analog, desmopressin (dDAVP). Desmopressin altered the translation rate rather than the stability of most responding proteins, but it significantly increased both the translation rate and the half-life of AQP2. In addition, proteins associated with vasopressin action, including Mal2, Akap12, gelsolin, myosin light chain kinase, annexin-2, and Hsp70, manifested altered translation rates. Interestingly, desmopressin increased the translation of seven glutathione S-transferase proteins and enhanced protein S-glutathionylation, uncovering a previously unexplored vasopressin-induced post-translational modification. Additional bioinformatic analysis of the mpkCCD proteome indicated a correlation between protein function and protein half-life. In particular, processes that are rapidly regulated, such as transcription, endocytosis, cell cycle regulation, and ubiquitylation are associated with proteins with especially short half-lives. These data extend our understanding of the mechanisms underlying vasopressin signaling and provide a broad resource for additional investigation of collecting duct function (http://helixweb.nih.gov/ESBL/Database/ProteinHalfLives/index.html).

A Novel Peptide-Based SILAC Method to Identify the Posttranslational Modifications Provides Evidence for Unconventional Ubiquitination in the ER-Associated Degradation Pathway

The endoplasmic reticulum-associated degradation (ERAD) pathway is responsible for disposing misfolded proteins from the endoplasmic reticulum by inducing their ubiquitination and degradation. Ubiquitination is conventionally observed on lysine residues and has been demonstrated on cysteine residues and protein N-termini. Ubiquitination is fundamental to the ERAD process; however, a mutant T-cell receptor α (TCRα) lacking lysine residues is targeted for the degradation by the ERAD pathway. We have shown that ubiquitination of lysine-less TCRα occurs on internal, non-lysine residues and that the same E3 ligase conjugates ubiquitin to TCRα in the presence or absence of lysine residues. Mass-spectrometry indicates that WT-TCRα is ubiquitinated on multiple lysine residues. Recent publications have provided indirect evidence that serine and threonine residues may be modified by ubiquitin. Using a novel peptide-based stable isotope labeling in cell culture (SILAC) approach, we show that specific lysine-less TCRα peptides become modified. In this study, we demonstrate that it is possible to detect both ester and thioester based ubiquitination events, although the exact linkage on lysine-less TCRα remains elusive. These findings demonstrate that SILAC can be used as a tool to identify modified peptides, even those with novel modifications that may not be detected using conventional proteomic work flows or informatics algorithms.

Antibody crossreactivity between the tumour suppressor PHLPP1 and the proto‐oncogene β‐catenin

During recent years, the PH domain leucine‐rich repeat protein phosphatase 1 (PHLPP1) has received increasing attention as a tumour suppressor that functions by dephosphorylating and antagonizing the survival‐promoting protein kinases AKT, PKC and S6K1 [1]. Previous studies reported that PHLPP1 co‐localizes with the tumour suppressor Scribble at cell–cell contacts [2], its expression is lost in colorectal cancer tumours [3] and it correlates with expression of PTEN, a tumour suppressor phosphatase that functions as a negative regulator upstream from AKT [2,4]. We warn that the PHLPP1 antibodies used in these studies (Bethyl IHC‐00382 and A300‐660A) crossreact strongly with β‐catenin, a proto‐oncogene product known to localize to adherens junctions. When probing Caco‐2 whole cell lysates (WCLs) for PHLPP1 with commonly used commercial PHLPP1 antibodies (Bethyl IHC‐00382 and A300‐660A—different types of the same antibody), we observed an additional prominent immunoreactive band around 90 kDa (Fig 1C), which was still apparent on PHLPP1 immunoprecipitation using the same antibodies (Fig 1A). In contrast with full length PHLPP1, the 90 kDa band was not responsive to siRNA‐mediated knockdown of PHLPP1 (Fig 1A,C), arguing against a PHLPP1 variant and suggesting it reflected antibody crossreactivity. To characterize further this putative crossreactivity, we used stable isotope labelling in cell culture and …