Isotope Labeling Of Amino Acids Research Articles

Fibroblasts Mobilize Tumor Cell Glycogen to Promote Proliferation and Metastasis

Successful metastasis requires the co-evolution of stromal and cancer cells. We used stable isotope labeling of amino acids in cell culture coupled with quantitative, label-free phosphoproteomics to study the bidirectional signaling in ovarian cancer cells andhuman-derived, cancer-associated fibroblasts (CAFs) after co-culture. In cancer cells, the interaction with CAFs supported glycogenolysis under normoxic conditions and induced phosphorylation and activation of phosphoglucomutase 1, an enzyme involved in glycogen metabolism. Glycogen was funneled into glycolysis, leading to increased proliferation, invasion, and metastasis of cancer cells co-cultured with human CAFs. Glycogen mobilization in cancer cells was dependent on p38α MAPK activation in CAFs. Invivo, deletion of p38α in CAFs and glycogen phosphorylase inhibition in cancer cells reduced metastasis, suggesting that glycogen is an energy source used by cancer cells to facilitate metastatic tumor growth.

The muscle anabolic effect of protein ingestion during a hyperinsulinaemic euglycaemic clamp in middle-aged women is not caused by leucine alone.

It has been suggested that leucine is primarily responsible for the increase in muscle protein synthesis after protein ingestion because leucine uniquely activates the mTOR-p70S6K signalling cascade. We compared the effects of ingesting protein or an amount of leucine equal to that in the protein during a hyperinsulinaemic-euglycaemic clamp (to eliminate potential confounding as a result of differences in the insulinogenic effect of protein and leucine ingestion) on muscle anabolic signalling and protein turnover in 28 women. We found that protein, but not leucine, ingestion increased muscle p-mTORSer2448 and p-p70S6KThr389 , although only protein, and not leucine, ingestion decreased muscle p-eIF2αSer51 and increased muscle protein synthesis. It has been suggested that leucine is primarily responsible for the increase in muscle protein synthesis (MPS) after protein ingestion because leucine uniquely activates the mTOR-p70S6K signalling cascade. We tested this hypothesis by measuring muscle p-mTORSer2448 , p-p70S6KThr389 and p-eIF2αSer51 , as well as protein turnover (by stable isotope labelled amino acid tracer infusion in conjunction with leg arteriovenous blood and muscle tissue sampling), in 28 women who consumed either 0.45g proteinkg-1 fat-free mass (containing 0.0513g leucinekg-1 fat-free mass) or a control drink (n= 14) or 0.0513g leucinekg-1 fat-free mass or a control drink (n= 14) during a hyperinsulinaemic-euglycaemic clamp procedure (HECP). Compared to basal conditions, the HECP alone (without protein or leucine ingestion) suppressed muscle protein breakdown by ∼20% and increased p-mTORSer2448 and p-p70S6KThr389 by >50% (all P< 0.05) but had no effect on p-eIF2αSer51 and MPS. Both protein and leucine ingestion further increased p-mTORSer2448 and p-p70S6KThr389 , although only protein, and not leucine, ingestion decreased (by ∼35%) p-eIF2αSer51 and increased (by ∼100%) MPS (all P< 0.05). Accordingly, leg net protein balance changed from negative (loss) during basal conditions to equilibrium during the HECP alone and the HECP with concomitant leucine ingestion and to positive (gain) during the HECP with concomitant protein ingestion. These results provide new insights into the regulation of MPS by demonstrating that leucine and mTOR signalling alone are not responsible for the muscle anabolic effect of protein ingestion during physiological hyperinsulinaemia, most probably because they fail to signal to eIF2α to initiate translation and/or additional amino acids are needed to sustain translation.

Dose- and time-dependent effects of triethylene glycol dimethacrylate on the proteome of human THP-1 monocytes.

Triethylene glycol dimethacrylate (TEGDMA) is commonly used in polymer resin‐based dental materials. This study investigated the molecular mechanisms of TEGDMA toxicity by identifying its time‐ and dose‐dependent effects on the proteome of human THP‐1 monocytes. The effects of different concentrations (0.07–5 mM) and exposure times (0–72 h) of TEGDMA on cell viability, proliferation, and morphology were determined using a real‐time viability assay, automated cell counting, and electron microscopy, and laid the fundament for choice of exposure scenarios in the proteomic experiments. Solvents were not used, as TEGDMA is soluble in cell culture medium (determined by photon correlation spectroscopy). Cells were metabolically labeled [using the stable isotope labeled amino acids in cell culture (SILAC) strategy], and exposed to 0, 0.3 or 2.5 mM TEGDMA for 6 or 16 h before liquid chromatography‐tandem mass spectrometry (LC‐MS/MS) analyses. Regulated proteins were analyzed in the STRING database. Cells exposed to 0.3 mM TEGDMA showed increased viability and time‐dependent upregulation of proteins associated with stress/oxidative stress, autophagy, and cytoprotective functions. Cells exposed to 2.5 mM TEGDMA showed diminished viability and a protein expression profile associated with oxidative stress, DNA damage, mitochondrial dysfunction, and cell cycle inhibition. Altered expression of immune genes was observed in both groups. The study provides novel knowledge about TEGDMA toxicity at the proteomic level. Of note, even low doses of TEGDMA induced a substantial cellular response.

Temporal Viral Genome-Protein Interactions Define Distinct Stages of Productive Herpesviral Infection.

ABSTRACTHerpesviruses utilize multiple mechanisms to redirect host proteins for use in viral processes and to avoid recognition and repression by the host. To investigate dynamic interactions between herpes simplex virus type 1 (HSV-1) DNA and viral and host proteins throughout infection, we developed an approach to identify proteins that associate with the infecting viral genome from nuclear entry through packaging. To accomplish this, virus stocks were prepared in the presence of ethynyl-modified nucleotides to enable covalent tagging of viral genomes after infection for analysis of viral genome-protein interactions by imaging or affinity purification. Affinity purification was combined with stable isotope labeling of amino acids in cell culture (SILAC) mass spectrometry to enable the distinction between proteins that were brought into the cell by the virus or expressed within the infected cell before or during infection. We found that input viral DNA progressed within 6 h through four temporal stages where the genomes sequentially (i) interacted with intrinsic antiviral and DNA damage response proteins, (ii) underwent a robust transcriptional switch mediated largely by ICP4, (iii) engaged in replication, repair, and continued transcription, and then (iv) transitioned to a more transcriptionally inert state engaging de novo-synthesized viral structural components while maintaining interactions with replication proteins. Using a combination of genetic, imaging, and proteomic approaches, we provide a new and temporally compressed view of the HSV-1 life cycle based on input genome-proteome dynamics.

Abstract 2499: Identification of functional proteins interacting with NSD2 in multiple myeloma

Abstract Multiple myeloma (MM), characterized by the uncontrolled proliferation of malignant plasma cells, occurs mainly in the elderly population. Recurrent chromosomal translocations are central to the pathogenesis of MM, with t(4;14) being the second most common and associated with poor prognosis. The histone methyltransferase (HMTase) NSD2 is overexpressed in MM due to the t(4;14) translocation and has been suggested to play an important tumorigenic role in t(4;14) MM. However, the detailed molecular mechanism or signaling pathways describing how deregulation of NDS2 contributes to MM cell growth and survival are still uncertain. In this study, stable isotope labeling of amino acid in cell culture (SILAC)-based mass spectrometry analysis was used to determine NSD2-interacting proteins. Mass spectrometry analysis indicated that there were 75 proteins identified in the immunoprecipitation of NSD2 protein complexes. Gene ontology (GO) enrichment analysis showed that these proteins were enriched in processes related to chromatin organization, RNA processing and translation. Five high potential MMSET-interacting proteins from the most enriched functions were selected and verified by Immunoprecipitation and Western blot assays. The results indicated that there were interactions between NSD2 and SMARCA2, topoisomerase IIa, ADAR1, DDX5 and RPS6. In silico analysis with STRING database showed that SMARCA2 interacts with NSD2. SMARCA2 is the ATPase subunit of the SWI/SNF chromatin remodeling complex, which harnesses the energy from ATP to remodel the position of nucleosomes, making the DNA accessible during transcription, replication and DNA repair. Double immunofluorescence showed NSD2 and SMARCA2 co-localized in the cell nucleus. SMARCA2 was knocked down (KD) by shRNAs in t(4;14) MM cells. CellTiter-Glo Luminescent Cell Viability Assay (CTG assay) indicated that SMARCA2 KD by shRNA reduced t(4;14) MM cell growth. SMARCA2 KD also significantly inhibited the capacity to form colonies in t(4;14) MM cells on methylcellulose colony formation assay. Inhibiting SMARCA2 with active DNA-dependent ATPase A domain inhibitor (ADAADi) also reduced t(4;14) MM cell growth. In addition, SMARCA2 KD reduced cell cycle S phase. Western blot analysis showed downregulation of PRL3 and CCND1 protein level upon NSD2 or SMARCA2 KD. This study reveals a protein-protein interaction between NSD2 and SMARCA2. SMARCA2 KD showed SMARCA2 might be a novel therapeutic target for t(4;14) MM. Citation Format: Jing Yuan Chooi, Zhigang Xie, Hannah Lee-Foon Swa, Jayantha Gunaratne, Dennis Kappei, Wee Joo Chng. Identification of functional proteins interacting with NSD2 in multiple myeloma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2499.

Extensive Identification and In-depth Validation of Importin 13 Cargoes

Importin 13 is a member of the importin β family of transport receptors. Unlike most family members, importin 13 mediates both, nuclear protein import and export. To search for novel importin 13 cargoes, we used stable isotope labeling of amino acids in cell culture (SILAC) and mass spectrometry. Using stringent criteria, we identified 255 importin 13 substrates, including the known cargoes Ubc9, Mago and eIF1A, and validate many of them as transport cargoes by extensive biochemical and cell biological characterization. Several novel cargoes can also be transported by the export receptor CRM1, demonstrating a clear redundancy in receptor choice. Using importin 13 mutants, we show that many of the novel substrates contact regions on the transport receptor that are not used by Ubc9, Mago or eIF1A. Together, this study significantly expands the repertoire of importin 13 cargoes and sets the basis for a more detailed characterization of this extremely versatile transport receptor.

Abstract 972: Defining the molecular context of MYC and WDR5 at chromatin

Abstract MYC is an oncoprotein transcription factor that is overexpressed in the majority of malignancies and contributes to the deaths of ~100,000 Americans each year. The tumorigenic functions of MYC stem from its influence on the transcriptional program of a cell, promoting the expression of thousands of genes involved in cell cycle progression, growth, and metabolism. In order to understand how MYC promotes cancer, we must understand the mechanisms by which MYC selects its target genes. Epigenetic signatures, DNA sequence motifs, and protein-protein interactions have all been implicated to play a role in MYC target gene selection, but a deep molecular understanding of the determinants of target gene recognition by MYC is missing. Our laboratory discovered that the chromatin-associated scaffolding protein WDR5 directly interacts with MYC and co-localizes on chromatin at a majority of loci (Thomas et al., Mol. Cell, 58: 440-452, 2015). The MYC-WDR5 interaction is disrupted by point mutation of three charged residues in MYC. While this MYC mutant retains the ability to bind to naked DNA, the mutant does not bind target loci in the context of chromatin and is unable to form tumors in mice. From these findings we propose that WDR5 is a crucial factor for enabling MYC activity in cancer. Both MYC and WDR5 are known to be highly networked proteins with multiple simultaneous interactions. Our aim is to tease apart this complicated network and define the molecular context specifically for the MYC-WDR5 interaction. To do this, we are employing our WDR5-binding deficient MYC mutant as a tool to genetically distinguish MYC interactions that are dependent upon WDR5. Our approach is to use Stable Isotope Labeling of Amino acids in Cell Culture (SILAC) because it enables us to quantitatively compare wild-type and mutant MYC and reduces background noise common to non-quantitative MYC-centered proteomics. Once identified by SILAC, interacting proteins are validated with biochemical methods and by proximity ligation assay in cells. This dual validation confirms the interaction in two ways, and provides information on the sub-cellular localization of the interaction. Thus far we have identified a preliminary set of ~25 interacting proteins that are currently undergoing validation. Characterization of these new factors by genetic, genomic, and biochemical approaches will expand our understanding of MYC biology, and potentially identify new factors that can be therapeutically targeted in cancer. Citation Format: Alissa D. Guarnaccia, William P. Tansey. Defining the molecular context of MYC and WDR5 at chromatin [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 972.

Abstract 352: DNA replication licensing factor MCM5 is a target for SETD8 mediated methylation

Abstract SETD8 methyltransferase activity is implicated in several fundamental cellular processes such as transcriptional regulation and heterochromatin formation as well as processes that ensures genomic stability including DNA replication and the DNA damage response. It has been proposed that SETD8 affects DNA replication as a positive regulator of origin licensing through H4K20 methylation and by supporting Okazaki fragment processing through PCNA methylation. However, there is no evidence whether other key element in the replication machinery is directly modified by SETD8. To address this question, we used a combination of SILAC-based BPPM (Bioorthogonal Profiling of Protein Methylation) to profile new substrates for SETD8. We genetically engineered SETD8 and identified mutants amenable to accommodate non-native SAM analogues containing a terminal alkyne moiety for click chemistry. The engineered SETD8 can transfer this unique chemical moiety into target proteins for subsequent identification of the modified substrates. Stable cell lines expressing catalytically active or catalytically dead SETD8 mutants were systematically cultured with distinct stable isotope labeled amino acids allowing quantitative proteomic analysis of the identified substrates. With this method we could validate previously known substrates of SETD8 and identify novel targets, including MCM5, a subunit of the hexameric minichromosome maintenance (MCM) DNA helicase complex. MCM5 directly interacts with MCM2 and 3 to form the MCM2-7 hexamer which associates with the origins of DNA replication to form part of the pre-replicative complex (preRC), playing a major role during replication initiation and elongation. We found that SETD8 mediated methylation of MCM5 directly affects its binding affinity to MCM2 and 3. Amino acid substitutions at the methylated lysine further evidenced a stronger binding to its interacting partners contributing to the formation of the MCM hexamer. In addition, CRISPR based modifications on MCM5 methylated lysine affected the replication process resulting in defective cell cycle progression. Taken together our results indicate that MCM5 methylation contributes to the assembly of the MCM complex revealing a novel role for SETD8 in replication initiation. Our findings brings new perspectives on the biological importance of SETD8 during DNA replication. Citation Format: Fabio Pittella, Gil Blum, Yongxia Zhu, Chamara Senevirathne, Minkui Luo. DNA replication licensing factor MCM5 is a target for SETD8 mediated methylation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 352.

Rapid Cue-Specific Remodeling of the Nascent Axonal Proteome

SummaryAxonal protein synthesis and degradation are rapidly regulated by extrinsic signals during neural wiring, but the full landscape of proteomic changes remains unknown due to limitations in axon sampling and sensitivity. By combining pulsed stable isotope labeling of amino acids in cell culture with single-pot solid-phase-enhanced sample preparation, we characterized the nascent proteome of isolated retinal axons on an unparalleled rapid timescale (5 min). Our analysis detects 350 basally translated axonal proteins on average, including several linked to neurological disease. Axons stimulated by different cues (Netrin-1, BDNF, Sema3A) show distinct signatures with more than 100 different nascent protein species up- or downregulated within the first 5 min followed by further dynamic remodeling. Switching repulsion to attraction triggers opposite regulation of a subset of common nascent proteins. Our findings thus reveal the rapid remodeling of the axonal proteomic landscape by extrinsic cues and uncover a logic underlying attraction versus repulsion.

2342 Protein production as an early pharmacodynamics biomarker for RNA-targeting therapies

OBJECTIVES/SPECIFIC AIMS: We aimed to develop an assay to measure new protein synthesis after Antisense Oligonucleotide treatment, which we hypothesized to be the earliest biochemical identification of RNA-targeting therapy efficacy. METHODS/STUDY POPULATION: We treated 2 transgenic animal models expressing proteins implicated in neurodegenerative disease: human tau protein (hTau) and human superoxide dismutase 1 (hSOD1), with ASO against these mRNA transcripts. Animals received isotope-labeled 13C6-Leucine via drinking water to label newly synthesized proteins. We assayed target protein synthesis and concentration after ASO treatment to determine the earliest identification of ASO target engagement. RESULTS/ANTICIPATED RESULTS: hTau ASO treatment in transgenic mice lowered hTau protein concentration 23 days post-treatment in cortex (95% CI: 0.05%–64.0% reduction). In the same tissue, we observed lowering of hTau protein synthesis as early as 13 days (95% CI: 29.4%–123%). In hSOD1 transgenic rats, we observed lowering of 13C6-leucine-labeled hSOD1 in the cerebrospinal fluid 30 days after ASO treatment compared with inactive ASO control (95% CI: 12.0%–48.4%). DISCUSSION/SIGNIFICANCE OF IMPACT: In progressive neurodegenerative diseases, it is crucial to develop measurements that identify treatment efficacy early to improve patient outcomes. These data support the use of stable isotope labeling of amino acids to measure new protein synthesis as an early pharmacodynamics measurement for therapies that target RNA and inhibit the translation of proteins.

MALDI-TOF mass spectrometry technology for detecting biomarkers of antimicrobial resistance: current achievements and future perspectives.

The laboratory diagnosis of infections is based on pathogen identification and antimicrobial susceptibility determination. The gold standard of cultivation, isolation and susceptibility testing is a time-consuming procedure and in some cases this can be threatening for patients' outcome. In the current review the applications of mass spectrometry in pathogen identification and especially in detecting biomarkers of antimicrobial resistance are analyzed. MALDI-TOF (matrix-assisted laser desorption/ionization time-of-flight) mass spectrometry is a new technology that has revolutionized pathogen identification and has also proven to accelerate detection of antimicrobial resistance compared to the traditional antibiotic susceptibility tests (AST) as well as DNA amplification methodologies. The technology has incorporated up to know four different methodologies: (I) the detection of differences of mass spectra of susceptible and resistant isolates of a given microorganism using the classical strain typing methodology; (II) the analysis of bacterial induced hydrolysis of β-lactam antibiotics; (III) the detection of stable (non-radioactive) isotope-labeled amino acids; and (IV) the analysis of bacterial growth in the presence and absence of antibiotics using an internal standard. The implementation of MALDI-TOF methodologies has improved detection of resistance in aerobic, Gram-positive and Gram-negative bacteria, mycobacteria, anaerobic bacteria, fungi and viruses. The MALDI-TOF is an easy to use, rapid, reliable, economical, and environmentally friendly methodology. However, this technology needs further development of research protocols that will be validated for routine application.

SILAC\u2013based quantitative MS approach for real-time recording protein-mediated cell-cell interactions

In tumor microenvironment, interactions among multiple cell types are critical for cancer progression. To understand the molecular mechanisms of these complex interplays, the secreted protein analysis between malignant cancer cells and the surrounding nonmalignant stroma is a good viewpoint to investigate cell-cell interactions. Here, we developed two stable isotope labeling of amino acids in cell culture (SILAC)-based mass spectrometry (MS)/MS approaches termed spike-in SILAC and triple-SILAC to quantify changes of protein secretion level in a cell co-cultured system. Within the co-culture system of CT26 and Ana-1 cells, the spike-in SILAC and triple-SILAC MS approaches are sensitive to quantitatively measure protein secretion changes. Three representative quantified proteins (Galectin-1, Cathepsin L1 and Thrombospondin-1) by two SILAC-based MS methods were further validated by Western blotting, and the coming result matched well with SILACs’. We further applied these two SILACs to human cell lines, NCM460 and HT29 co-culture system, for evaluating the feasibility, which confirmed the spike-in and triple SILAC were capable of monitoring the changed secreted proteins of human cell lines. Considering these two strategies in time consuming, sample complexity and proteome coverage, the triple-SILAC way shows more efficiency and economy for real-time recording secreted protein levels in tumor microenvironment.

Metabolic Patterns in Spirodela polyrhiza Revealed by 15N Stable Isotope Labeling of Amino Acids in Photoautotrophic, Heterotrophic, and Mixotrophic Growth Conditions

In this study we describe a [15N] stable isotopic labeling study of amino acids in Spirodela polyrhiza (common duckweed) grown under three different light and carbon input conditions which represent unique potential metabolic modes. Plants were grown with a light cycle, either with supplemental sucrose (mixotrophic) or without supplemental sucrose (photoautotrophic) and in the dark with supplemental sucrose (heterotrophic). Labeling patterns, pool sizes (both metabolically active and inactive), and kinetics/turnover rates were estimated for 17 of the proteinogenic amino acids. Estimation of these parameters followed several overall trends. First, most amino acids showed plateaus in labeling patterns of <100% [15N]-labeling, indicating the possibility of a large proportion of amino acids residing in metabolically inactive metabolite pools. Second, total pool sizes appear largest in the dark (heterotrophic) condition, whereas active pool sizes appeared to be largest in the light with sucrose (mixotrophic) growth condition. In contrast turnover measurements based on pool size were highest overall in the light with sucrose experiment, with the exception of leucine/isoleucine, lysine, and arginine, which all showed higher turnover in the dark. K-means clustering analysis also revealed more rapid turnover in the light treatments with many amino acids clustering in lower-turnover groups. Emerging insights from other research were also supported, such as the prevalence of alternate pathways for serine metabolism in non-photosynthetic cells. These data provide extensive novel information on amino acid pool size and kinetics in S. polyrhiza and can serve as groundwork for future metabolic studies.

Development of online microdialysis-mass spectrometry for continuous minimally invasive measurement of soil solution dynamics

The rate that amino acids are removed from the soil solution is poorly known but vitally important. It is possible to determine the time course of soil solution concentrations by extracting soils at different time points after adding labelled compounds, but this approach either lacks sufficient temporal resolution or generates large number of samples that require subsequent offline analysis. The aim of this study was to develop online microdialysis-mass spectrometry to enable the minimally invasive measurement of the time-course of isotope labelled amino acid added to soil. The method was subsequently tested by examining the fate of isotope labelled L- and d-alanine added to sterile and non-sterile soils. One concern with application of microdialysis to soil is if calibrations are affected by inorganic ion composition of the perfusate and the external (soil) solution. Tests showed that the presence/absence of inorganic ions in perfusate and external solution did not affect dialysate concentrations, suggesting that perfusing with an artificial soil solution matching the inorganic ion composition of the external solution does not convey any benefits. Hence water was used as perfusate for development of online microdialysis-mass spectrometry. The online system took around one minute to equilibrate to step-changes in concentration and had detection limits around 0.5 μmol L−1 for alanine. Addition of isotope labelled alanine to soils led to an almost instantaneous increase and subsequent decrease in dialysate alanine concentration. With sterile soils there was a slow abiotic decrease in dialysate concentrations, presumably due to development of a depletion shell around the microdialysis probe and adsorption of alanine to the soil. For non-sterile soils there was an additional more rapid biotic decrease in dialysate concentrations that presumably reflected microbial uptake. For l-alanine added to non-sterile soil much of the compound was taken up before it reached the probe surface and concentrations decreased to below detection limits within 5–20 min. Thus microdialysis afforded a graphic illustration of the ephemeral nature of intact l-alanine in non-sterile soil, while parallel measurements showed that added d-alanine was removed from soil solution several times more slowly.

Assessing the Feasibility of Neutralizing Osteopontin with Various Therapeutic Antibody Modalities

Osteopontin is a secreted glycophosphoprotein that is highly implicated in many physiological and pathological processes such as biomineralization, cell-mediated immunity, inflammation, fibrosis, cell survival, tumorigenesis and metastasis. Antibodies against osteopontin have been actively pursued as potential therapeutics for various diseases by pharmaceutical companies and academic laboratories. Many studies have demonstrated the efficacy of osteopontin inhibition in a variety of preclinical models of diseases such as rheumatoid arthritis, cancer, nonalcoholic steatohepatitis, but clinical utility has not yet been demonstrated. To evaluate the feasibility of osteopontin neutralization with antibodies in a clinical setting, we measured its physiological turnover rate in humans, a sensitive parameter required for mechanistic pharmacokinetic and pharmacodynamic (PK/PD) modeling of biotherapeutics. Results from a stable isotope-labelled amino acid pulse-chase study in healthy human subjects followed by mass spectrometry showed that osteopontin undergoes very rapid turnover. PK/PD modeling and simulation of different theoretical scenarios reveal that achieving sufficient target coverage using antibodies can be very challenging mostly due to osteopontin’s fast turnover, as well as its relatively high plasma concentrations in human. Therapeutic antibodies against osteopontin would need to be engineered to have much extended PK than conventional antibodies, and be administered at high doses and with short dosing intervals.

Quantifying extracellular matrix turnover in human lung scaffold cultures

Remodelling of the extracellular matrix is accomplished by altering the balance between matrix macromolecule production and degradation. However, it is not well understood how cells balance production of new matrix molecules and degradation of existing ones during tissue remodelling and regeneration. In this study, we used decellularized lung scaffolds repopulated with allogenic lung fibroblasts cultured with stable isotope labelled amino acids to quantify the balance between matrix production and degradation at a proteome-wide scale. Specific temporal dynamics of different matrisome proteins were found to correspond to the proliferative activity of the repopulating cells and the degree of extracellular deposition. The remodeling of the scaffold was characterized by an initial phase with cell proliferation and high production of cell adhesion proteins such as emilin-1 and fibronectin. Extended culture time resulted in increased levels of core matrisome proteins. In a comparison with monolayer cultures on plastic, culture in lung scaffolds lead to a pronounced accumulation of proteoglycans, such as versican and decorin, resulting in regeneration of an extracellular matrix with greater resemblance to native lung tissue compared to standard monolayer cultures. Collectively, the study presents a promising technique for increasing the understanding of cell- extracellular matrix interactions under healthy and diseased conditions.

Multiplexed proteome analysis with neutron-encoded stable isotope labeling in cells and mice.

We describe a protocol for multiplexed proteomic analysis using neutron-encoded (NeuCode) stable isotope labeling of amino acids in cells (SILAC) or mice (SILAM). This method currently enables simultaneous comparison of up to nine treatment and control proteomes. Another important advantage over traditional SILAC/SILAM is that shorter labeling times are required. Exploiting the small mass differences that correspond to subtle differences in the neutron-binding energies of different isotopes, the amino acids used in NeuCode SILAC/SILAM differ in mass by just a few milliDaltons. Isotopologs of lysine are introduced into cells or mammals, via the culture medium or diet, respectively, to metabolically label the proteome. Labeling time is ∼2 weeks for cultured cells and 3-4 weeks for mammals. The proteins are then extracted, relevant samples are combined, and these are enzymatically digested with lysyl endopeptidase (Lys-C). The resultant peptides are chromatographically separated and then mass analyzed. During mass spectrometry (MS) data acquisition, high-resolution MS1 spectra (≥240,000 resolving power at m/z = 400) reveal the embedded isotopic signatures, enabling relative quantification, while tandem mass spectra, collected at lower resolutions, provide peptide identities. Both types of spectra are processed using NeuCode-enabled MaxQuant software. In total, the approximate completion time for the protocol is 3-5 weeks.

Sulfur-34S and 36S Stable Isotope Labeling of Amino Acids for Quantification (SULAQ34/36) of Proteome Analyses.

Quantitative proteome profiling of microorganisms by isotopic labeling of amino acids is still a challenge, because only microorganisms with auxotrophic character are able to embed amino acids into their biomass in a quantitatively correct manner. Here, we describe an isotopic labeling technique (sulfur stable isotope labeling of amino acids for quantification, SULAQ) for the sulfur-containing amino acids cysteine and methionine in a broad range of organisms. The metabolic labeling approach is suitable for gel-based and gel-free protein analysis.

Revival of 2DE-LC/MS in Proteomics and Its Potential for Large-Scale Study of Human Proteoforms

Two-dimensional gel electrophoresis coupled with liquid chromatography/mass spectrometry (2DE-LC/MS) is a classic and conventional approach to separate and identify proteins in a proteome. However, this approach is conventionally looked upon as a seemingly “low-throughput” technique because only one to two proteins per 2D spot are often achieved; thus, it has gradually dimmed in the field of proteomics compared to seemingly “high-throughput” bottom-up non-gel approaches such as isobaric tags for relative and absolute quantification (iTRAQ), peptide tandem mass tag (TMT), stable isotope labeling of amino acids in cell culture (SILAC), and label-free. With the rapid development and application of high-sensitivity mass spectrometry in proteomics, an average of over 50 or even hundreds of proteins can be identified in every 2D gel spot in an analysis of a complex human proteome, mostly low-abundance proteins, and 2DE-LC/MS can detect the protein species, to break through the conventional concept of 2DE-LC/MS, and assist its revival in the field of proteomics. Splicing and post-translational modifications are fundamental to the proteome, and are the main factors to clarify proteoforms, which enrich the concept of the proteome. The top-down and high-throughput nature of stable isotope-labeled 2DE-LC/MS for the detection, identification, and quantification of proteoforms provides a solid methodological support for the large-scale study of human proteoforms and disease-related proteoforms to clarify mechanisms of a disease and to discover reliable biomarkers for the prediction, diagnosis, and prognostic assessment of a disease.

The RiboPuromycylation Method (RPM): an Immunofluorescence Technique to Map Translation Sites at the Sub-cellular Level.

While isotopic labeling of amino acids remains the reference method in the field for quantifying translation rate, it does not provide any information on spatial localization of translation sites. The rationale behind developing the ribopuromycylation method (RPM) was primarily to map translation sites at the sub-cellular level while avoiding detection of newly synthesized proteins released from ribosomes. RPM visualizes actively translating ribosomes in cells via standard immunofluorescence microscopy in fixed and permeabilized cells using a puromycin-specific monoclonal antibody to detect puromycylated nascent chains trapped on ribosomes treated with a chain elongation inhibitor.