Stable Isotope Labeling In Cell Culture Research Articles

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.

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.

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.

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.

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.

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.

Nucleolar protein trafficking in response to HIV-1 Tat: rewiring the nucleolus.

The trans-activator Tat protein is a viral regulatory protein essential for HIV-1 replication. Tat trafficks to the nucleoplasm and the nucleolus. The nucleolus, a highly dynamic and structured membrane-less sub-nuclear compartment, is the site of rRNA and ribosome biogenesis and is involved in numerous cellular functions including transcriptional regulation, cell cycle control and viral infection. Importantly, transient nucleolar trafficking of both Tat and HIV-1 viral transcripts are critical in HIV-1 replication, however, the role(s) of the nucleolus in HIV-1 replication remains unclear. To better understand how the interaction of Tat with the nucleolar machinery contributes to HIV-1 pathogenesis, we investigated the quantitative changes in the composition of the nucleolar proteome of Jurkat T-cells stably expressing HIV-1 Tat fused to a TAP tag. Using an organellar proteomic approach based on mass spectrometry, coupled with Stable Isotope Labelling in Cell culture (SILAC), we quantified 520 proteins, including 49 proteins showing significant changes in abundance in Jurkat T-cell nucleolus upon Tat expression. Numerous proteins exhibiting a fold change were well characterised Tat interactors and/or known to be critical for HIV-1 replication. This suggests that the spatial control and subcellular compartimentaliation of these cellular cofactors by Tat provide an additional layer of control for regulating cellular machinery involved in HIV-1 pathogenesis. Pathway analysis and network reconstruction revealed that Tat expression specifically resulted in the nucleolar enrichment of proteins collectively participating in ribosomal biogenesis, protein homeostasis, metabolic pathways including glycolytic, pentose phosphate, nucleotides and amino acids biosynthetic pathways, stress response, T-cell signaling pathways and genome integrity. We present here the first differential profiling of the nucleolar proteome of T-cells expressing HIV-1 Tat. We discuss how these proteins collectively participate in interconnected networks converging to adapt the nucleolus dynamic activities, which favor host biosynthetic activities and may contribute to create a cellular environment supporting robust HIV-1 production.

Quantitative Chemical Proteomics Approach To Identify Post-translational Modification-Mediated Protein–Protein Interactions

Post-translational modifications (PTMs) (e.g., acetylation, methylation, and phosphorylation) play crucial roles in regulating the diverse protein-protein interactions involved in essentially every cellular process. While significant progress has been made to detect PTMs, profiling protein-protein interactions mediated by these PTMs remains a challenge. Here, we report a method that combines a photo-cross-linking strategy with stable isotope labeling in cell culture (SILAC)-based quantitative mass spectrometry to identify PTM-dependent protein-protein interactions. To develop and apply this approach, we focused on trimethylated lysine-4 at the histone H3 N-terminus (H3K4Me(3)), a PTM linked to actively transcribed gene promoters. Our approach identified proteins previously known to recognize this modification and MORC3 as a new protein that binds H3M4Me(3). This study indicates that our cross-linking-assisted and SILAC-based protein identification (CLASPI) approach can be used to profile protein-protein interactions mediated by PTMs, such as lysine methylation.

Mass spectrometric analysis identifies a cortactin–RCC2/TD60 interaction in mitotic cells

Cortactin is an F-actin binding protein that functions as a scaffold to regulate Arp2/3 mediated actin polymerization in lamellipodia and invadopodia formation as well as functioning in cell migration and endocytosis of many different cell types. In light of the fact that regulated actin polymerization is critical for many cellular processes we launched a search for novel cortactin interactions with cellular proteins that might indicate heretofore undescribed biological activities supported by cortactin. Using a modified stable isotope labeling in cell culture (SILAC) approach in HEK293 cells and Flag-tagged cortactin (F-cortactin) as bait, we identified a limited set of cortactin interactions including several proteins which have not previously been identified as cortactin associated proteins. Among these were serine/threonine-protein phosphatase 2A subunit beta (PP2A-beta) and RCC2/TD60, a Rac guanine nucleotide exchange factor (GEF) required for completion of mitosis and cytokinesis. The interaction between cortactin and RCC2/TD60 was verified in cell lysates immunoprecitated with anti-RCC2/TD60 antibody. Furthermore, cortactin was localized by immunofluorescence in the equatorial plane of dividing HeLa cells in the region where RCC2/TD60 has previously been localized thus providing support for a complex containing cortactin and RCC2/TD60 complex that may play a functional role in cells undergoing mitosis.

Linker of Activation of T Cells – 2 (LAT2), a Lipid Raft-Associated Adaptor Protein, Is a Novel Target for Therapy in Acute Leukemia

Abstract 737Lipid rafts are highly ordered membrane domains that are enriched in cholesterol and sphingolipids and function in providing a scaffold to signal transduction. Altered raft assembly has been implicated in breast and colorectal cancer progression; rafts are essential to sustained p38K activation by GM-CSF and VEGFa in acute myeloid leukemia (Saulle et al. BJH, 2009). In a previous study, we showed that 10-(Octyloxy) decyl-2-(trimethylammonium) ethyl phosphate (ODPC), an alkylphospholipid, targets raft like domains in model membranes and induces apoptosis in leukemia cells, (ED-50 25mM - NB4), but spares normal hematopoietic and epithelial cells (dos Santos et al. BBACTA 2010). Here, we performed an unbiased quantitative proteomic screen of ODPC targets in lipid rafts. We used stable isotope labeling in cell culture (SILAC) to reveal proteins that are modulated by ODPC before the installation of apoptosis, to gain access to the primary mechanisms of drug action (Fig 1A, B and C). We used successive detergent extraction (Fig 1D) to isolate detergent resistant membranes (DRM) enriched in lipid rafts; blots with specific markers prove the quality of fractionation (Fig 1E). Proteins were considered modulated if: fold change of 2/0.5 or greater plus two or more quantification events and a P value <0.1. A small fraction of proteins was modulated by ODPC (Fig 1F). Of the 1426 proteins identified in the total extract, only one protein (0.07%) was upregulated. Of the 1314 proteins identified in the DRM, 6 proteins (0.45%) were donwregulated. Of these, 4 have predicted or validated palmitoylation sites (Fig 1F). We validated that ODPC downregulates LAT-2 (Fig 2A), an adaptor protein that is target to rafts by palmitoylation and is expressed in B cells and myeloid cells. Interesting, LAT-2 was not expressed in K562 (Fig 2A), a cell resistant to ODPC-induced apoptosis (Fig 1A and B). Moreover, ODPC induced a loss of LAT-2 in the lipid rafts of NB4 cells after 3h of treatment (Fig 2B). Since LAT-2 may function as an adaptor protein in AKT signalization (Roget et al. J.Immunol. 2008) we evaluate the effect of ODPC on the ATK pathway. Fig 2C shows that ODPC induces a downregulation of all components from AKT signaling, compatible with a membrane target drug. However, in functional experiments, ODPC inhibited AKT activation in NB4 cells by myeloid growth factors (MGF), after few minutes of incubation, in a similar way of the specific PI3K inhibitor Wortmannin (Fig 2D). Moreover, in these experiments, MGF induced a rapid and translation-dependent up regulation in LAT-2 expression in control treated cells (Fig 2E). This effect was partially suppressed by ODPC treatment and was suppressed by Wortmannin (Fig 2D). This suggests that LAT-2 translation is highly regulated and dependent on a functional AKT pathway. Indeed, phosphorylation of 4EBP-1 and ribossomal S6 kinase (S6K) with consequent upregulation of translation in the ribosome is downstream of PI3K/AKT/mTOR (Mamane et al. Oncogene, 2006). Moreover, we confirmed a hypophosphorilation state of S6K in the presence of ODPC or Wortmannin (Fig 2D). Interesting, we have identified two potential-palmitoylated ribosomal proteins (RP) to be downregulated in the lipid raft fraction of ODPC treated cells (Fig 1F). This finding confirms previous reports that a raft disruption treatment (methyl-β-cyclodextrin) causes downregulation of several RP (Foster, De Hoog and Mann. PNAS, 2003) and that palmitoylation target ribosome proteins to lipid rafts (Wei et al. MCP, 2010). This indicates that ODPC may present also a direct or indirect inhibitory effect on translational apparatus. Our results strongly indicate that ODPC acts on LAT-2 by interference with its protein-lipid interactions, since experimental-induced disturbances in lipid raft physiology recapitulate or increase ODPC effects on LAT-2 expression (Fig 3A and B). To further clarify the mechanism of LAT-2 downregulation we evaluate if it is degraded by the proteasome after ODPC or 2BrPA treatments. Fig 3C and D reveals that this is the case. This confirms the findings of Orr and McVicar (J.Leuk.Biol. 2011) of ubiquitin-proteasomal regulation of LAT-2 stability. Taken together, our results demonstrate that a lipid raft targeting drug may present several effects on signal transduction, causing a severe toxicity to leukemia cells. Moreover, adaptor proteins like LAT-2 emerge as possible new therapeutic targets in leukemia. [Display omitted] [Display omitted] [Display omitted] Disclosures:No relevant conflicts of interest to declare.

Location, location, location: differential profiling of the nucleolar proteome of T-cells expressing HIV-1 Tat reveals the coordinated spatial control of distinct cellular networks

Background The trans-activator Tat protein is a viral regulatory protein essential for HIV-1 replication. Tat localises in both the nucleoplasm and the nucleolus. The nucleolus, a highly structured and dynamic membrane-less subnuclear compartment, is the site of rRNA and ribosome biogenesis and is involved in numerous cellular functions including transcriptional regulation, cell cycle control, RNA trafficking and viral infection. Importantly, transient nucleolar trafficking of both Tat and HIV-1 viral transcripts are critical in HIV-1 replication, however, the role(s) of the nucleolus in HIV-1 replication remains to be elucidated. To better understand how the interaction of Tat with the nucleolar machinery contributes to HIV-1 pathogenesis, we investigated the quantitative changes in the composition of the nucleolar proteome of T-cells stably expressing HIV-1 Tat. Materials and methods We employed an organellar proteomic approach using mass spectrometry coupled with Stable Isotope Labelling in Cell culture (SILAC). Identification, quantification and analysis of the purified nucleolar proteins was performed using MaxQuant, combined with Perseus, and cytoscape for pathway analysis and network reconstruction. Results

Adhesion and Degranulation Promoting Adapter Protein (ADAP) Is a Central Hub for Phosphotyrosine-Mediated Interactions in T Cells

TCR stimulation leads to an increase in cellular adhesion among other outcomes. The adhesion and degranulation promoting adapter protein (ADAP) is known to be rapidly phosphorylated after T cell stimulation and relays the TCR signal to adhesion molecules of the integrin family. While three tyrosine phosphorylation sites have been characterized biochemically, the binding capabilities and associated functions of several other potential phosphotyrosine motifs remain unclear. Here, we utilize in vitro phosphorylation and mass spectrometry to map novel phosphotyrosine sites in the C-terminal part of human ADAP (486–783). Individual tyrosines were then mutated to phenylalanine and their relevance for cellular adhesion and migration was tested experimentally. Functionally important tyrosine residues include two sites within the folded hSH3 domains of ADAP and two at the C-terminus. Furthermore, using a peptide pulldown approach in combination with stable isotope labeling in cell culture (SILAC) we identified SLP-76, PLCγ, PIK3R1, Nck, CRK, Gads, and RasGAP as phospho-dependent binding partners of a central YDDV motif of ADAP. The phosphorylation-dependent interaction between ADAP and Nck was confirmed by yeast two-hybrid analysis, immunoprecipitation and binary pulldown experiments, indicating that ADAP directly links integrins to modulators of the cytoskeleton independent of SLP-76.

Cover Picture: Proteomics 13'09

AbstractHuman Growth Hormone: Variants vary in sugar coatingIn the last issue we commented on the age and variants of the heat shock proteins, in this issue we take a brief look at another old family with connections, the growth hormones. Human growth hormone (hGH) is produced in at least eight different flavors and two different tissues with apparent molecular weights ranging from 5 to 45 kDa, from anterior pituitary and placenta. Functional roles include growth promotion and insulin potentiation. Bustamante et al. characterized the structure of the O‐linked oligosaccharide they had found earlier attached to hGH‐24. The study required ESI‐ and MALDI TOF‐MS/MS, and high‐performance anion exchange chromatography with pulsed amperometric detection to determine the point of attachment and carbohydrate branching structure. A theoretical 3‐D model placed the carbohydrate in the hGH‐24 high affinity binding site 1.Bustamante, J. et al., Proteomics 2009, 9, 3474–3488.Phospho‐tyrosine targets: T‐ and B‐cell receptor activationPhosphorylation at serine, threonine or tyrosine is one of the most common post‐translational modifications of eukaryotic proteins. The response of genetically similar proteins to phosphorylation is distinct and separate. Matsumoto et al. report here a catalog of the upstream and downstream effects of the phosphorylation of tyrosine in T‐cell (Jurkat cell line, TCR) and B‐cell (Namalwa cell line, BCR) receptors. Proteins were identified by SDS‐PAGE followed by in‐gel digestion, LC and MS/MS. Tyrosine phosphorylated proteins were selectively enriched using an immunoaffinity approach while quantitative aspects were determined by SILAC (stable isotope labeling in cell culture). Status of the receptor endocytosis machinery was checked. A total of 1243 proteins were identified. Alternative criteria yielded 725 proteins and 128 of those were found under all conditions. Many known proteins were found and an interesting new one – PLCγ1 was phosphorylated in Jurkat but not Namalwa cells, PLCγ2 was the reverse.Matsumoto, M. et al., Proteomics 2009, 9, 3549–3563.Tipsy PIPs lose grip on proteasomesProteasomes are the principal “protein garbage eaters” of the eukaryotic cell. The core is a 20S, 28 subunit complex, which is assembled with other subunits for various purposes, including immune clean‐up in response to interferon release, destruction of ubiquitin‐tagged proteins and short‐lived or misfolded proteins. It also has regulatory functions, controlling cell cycles, hypoxic response and transcription. Chronic alcohol ingestion causes severe damage to rat and human liver. Bousquet‐Dubouch et al. report on facets of the proteasome affected by chronic ethanol administration (at ≥2×human drunk driving limit in many US states) for a month. After careful nondenaturing isolation of proteasomes from drunk and sober rats, they identified proteasome interacting proteins (PIPs) by nanoLC‐ESI‐QTOF‐MS/MS and quantitated by Western blots and ICAT techniques. A number of ubiquitin‐associated proteins were downregulated, one was missing, as was a stabilizer of 20S‐activator complexes.Bousquet‐Dubouch, M.‐P. et al., Proteomics 2009, 9, 3609–3622.

In this issue

AbstractHuman Growth Hormone: Variants vary in sugar coatingpp. 3474–3488In the last issue we commented on the age and variants of the heat shock proteins, in this issue we take a brief look at another old family with connections, the growth hormones. Human growth hormone (hGH) is produced in at least eight different flavors and two different tissues with apparent molecular weights ranging from 5 to 45 kDa, from anterior pituitary and placenta. Functional roles include growth promotion and insulin potentiation. Bustamante et al. characterized the structure of the O‐linked oligosaccharide they had found earlier attached to hGH‐24. The study required ESI‐ and MALDI TOF‐MS/MS, and high‐performance anion exchange chromatography with pulsed amperometric detection to determine the point of attachment and carbohydrate branching structure. A theoretical 3‐D model placed the carbohydrate in the hGH‐24 high affinity binding site 1.magnified imagePhospho‐tyrosine targets: T‐ and B‐cell receptor activationpp. 3549–3563Phosphorylation at serine, threonine or tyrosine is one of the most common post‐translational modifications of eukaryotic proteins. The response of genetically similar proteins to phosphorylation is distinct and separate. Matsumoto et al. report here a catalog of the upstream and downstream effects of the phosphorylation of tyrosine in T‐cell (Jurkat cell line, TCR) and B‐cell (Namalwa cell line, BCR) receptors. Proteins were identified by SDS‐PAGE followed by in‐gel digestion, LC and MS/MS. Tyrosine phosphorylated proteins were selectively enriched using an immunoaffinity approach while quantitative aspects were determined by SILAC (stable isotope labeling in cell culture). Status of the receptor endocytosis machinery was checked. A total of 1243 proteins were identified. Alternative criteria yielded 725 proteins and 128 of those were found under all conditions. Many known proteins were found and an interesting new one – PLCγ1 was phosphorylated in Jurkat but not Namalwa cells, PLCγ2 was the reverse.magnified imageTipsy PIPs lose grip on proteasomespp. 3609–3622Proteasomes are the principal “protein garbage eaters” of the eukaryotic cell. The core is a 20S, 28 subunit complex, which is assembled with other subunits for various purposes, including immune clean‐up in response to interferon release, destruction of ubiquitin‐tagged proteins and short‐lived or misfolded proteins. It also has regulatory functions, controlling cell cycles, hypoxic response and transcription. Chronic alcohol ingestion causes severe damage to rat and human liver. Bousquet‐Dubouch et al. report on facets of the proteasome affected by chronic ethanol administration (at ≥2×human drunk driving limit in many US states) for a month. After careful nondenaturing isolation of proteasomes from drunk and sober rats, they identified proteasome interacting proteins (PIPs) by nanoLC‐ESI‐QTOF‐MS/MS and quantitated by Western blots and ICAT techniques. A number of ubiquitin‐associated proteins were downregulated, one was missing, as was a stabilizer of 20S‐activator complexes.magnified image