85 research outputs found

    Global Analysis of Protein Expression and Phosphorylation Levels in Nicotine-Treated Pancreatic Stellate Cells

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    Smoking is a risk factor in pancreatic disease; however, the biochemical mechanisms correlating smoking with pancreatic dysfunction remain poorly understood. Strategies using multiplexed isobaric tag-based mass spectrometry facilitate the study of drug-induced perturbations on biological systems. Here, we present the first large-scale analysis of the proteomic and phosphoproteomic alterations in pancreatic stellate cells following treatment with two nicotinic acetylcholine receptor (nAChR) ligands: nicotine and α-bungarotoxin. We treated cells with nicotine or α-bungarotoxin for 12 h in triplicate and compared alterations in protein expression and phosphorylation levels to mock-treated cells using a tandem mass tag (TMT9plex)-based approach. Over 8100 proteins were quantified across all nine samples, of which 46 were altered in abundance upon treatment with nicotine. Proteins with increased abundance included those associated with neurons, defense mechanisms, indicators of pancreatic disease, and lysosomal proteins. In addition, we measured differences for ∼16 000 phosphorylation sites across all nine samples using a titanium dioxide-based strategy, of which 132 sites were altered with nicotine and 451 with α-bungarotoxin treatment. Many altered phosphorylation sites were involved in nuclear function and transcriptional events. This study supports the development of future targeted investigations to establish a better understanding for the role of nicotine and associated receptors in pancreatic disease

    Filter-Based Protein Digestion (FPD): A Detergent-Free and Scaffold-Based Strategy for TMT Workflows

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    High-throughput proteome profiling requires thorough optimization to achieve comprehensive analysis. We developed a filter aided sample preparation (FASP)-like, detergent-free method, termed Filter-Based Protein Digestion (FPD). We compared FPD to protein extraction methods commonly used in isobaric tag-based proteome profiling, namely trichloroacetic acid (TCA) and chloroform–methanol (C–M) precipitation. We divided a mammalian whole cell lysate from the SH-SY5Y neuroblastoma cell line for parallel protein processing with TCA (<i>n</i> = 3), C–M (<i>n</i> = 2), and FPD using either 10 kDa (<i>n</i> = 3) or 30 kDa (<i>n</i> = 3) molecular weight cutoff membranes. We labeled each sample with tandem mass tag (TMT) reagents to construct a TMT11-plex experiment. In total, 8654 proteins were quantified across all samples. Pairwise comparisons showed very little deviation for individual protein abundance measurements between the two FPD methods, whereas TCA and FPD showed the most difference. Specifically, membrane proteins were more readily quantified when samples were processed using TCA precipitation than other methods tested. However, globally, only 4% of proteins differed greater than 4-fold in the most divergent pair of protein extraction methods (i.e., FPD10 and TCA). We conclude that the detergent-free FPD strategy, particularly using the faster-flowing 30 kDa filter, is a seamless alteration to high-throughput TMT workflows

    Filter-Based Protein Digestion (FPD): A Detergent-Free and Scaffold-Based Strategy for TMT Workflows

    No full text
    High-throughput proteome profiling requires thorough optimization to achieve comprehensive analysis. We developed a filter aided sample preparation (FASP)-like, detergent-free method, termed Filter-Based Protein Digestion (FPD). We compared FPD to protein extraction methods commonly used in isobaric tag-based proteome profiling, namely trichloroacetic acid (TCA) and chloroform–methanol (C–M) precipitation. We divided a mammalian whole cell lysate from the SH-SY5Y neuroblastoma cell line for parallel protein processing with TCA (<i>n</i> = 3), C–M (<i>n</i> = 2), and FPD using either 10 kDa (<i>n</i> = 3) or 30 kDa (<i>n</i> = 3) molecular weight cutoff membranes. We labeled each sample with tandem mass tag (TMT) reagents to construct a TMT11-plex experiment. In total, 8654 proteins were quantified across all samples. Pairwise comparisons showed very little deviation for individual protein abundance measurements between the two FPD methods, whereas TCA and FPD showed the most difference. Specifically, membrane proteins were more readily quantified when samples were processed using TCA precipitation than other methods tested. However, globally, only 4% of proteins differed greater than 4-fold in the most divergent pair of protein extraction methods (i.e., FPD10 and TCA). We conclude that the detergent-free FPD strategy, particularly using the faster-flowing 30 kDa filter, is a seamless alteration to high-throughput TMT workflows

    Filter-Based Protein Digestion (FPD): A Detergent-Free and Scaffold-Based Strategy for TMT Workflows

    No full text
    High-throughput proteome profiling requires thorough optimization to achieve comprehensive analysis. We developed a filter aided sample preparation (FASP)-like, detergent-free method, termed Filter-Based Protein Digestion (FPD). We compared FPD to protein extraction methods commonly used in isobaric tag-based proteome profiling, namely trichloroacetic acid (TCA) and chloroform–methanol (C–M) precipitation. We divided a mammalian whole cell lysate from the SH-SY5Y neuroblastoma cell line for parallel protein processing with TCA (<i>n</i> = 3), C–M (<i>n</i> = 2), and FPD using either 10 kDa (<i>n</i> = 3) or 30 kDa (<i>n</i> = 3) molecular weight cutoff membranes. We labeled each sample with tandem mass tag (TMT) reagents to construct a TMT11-plex experiment. In total, 8654 proteins were quantified across all samples. Pairwise comparisons showed very little deviation for individual protein abundance measurements between the two FPD methods, whereas TCA and FPD showed the most difference. Specifically, membrane proteins were more readily quantified when samples were processed using TCA precipitation than other methods tested. However, globally, only 4% of proteins differed greater than 4-fold in the most divergent pair of protein extraction methods (i.e., FPD10 and TCA). We conclude that the detergent-free FPD strategy, particularly using the faster-flowing 30 kDa filter, is a seamless alteration to high-throughput TMT workflows

    Streamlined Tandem Mass Tag (SL-TMT) Protocol: An Efficient Strategy for Quantitative (Phospho)proteome Profiling Using Tandem Mass Tag-Synchronous Precursor Selection-MS3

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    Mass spectrometry (MS) coupled toisobaric labeling has developed rapidly into a powerful strategy for high-throughput protein quantification. Sample multiplexing and exceptional sensitivity allow for the quantification of tens of thousands of peptides and, by inference, thousands of proteins from multiple samples in a single MS experiment. Accurate quantification demands a consistent and robust sample-preparation strategy. Here, we present a detailed workflow for SPS-MS3-based quantitative abundance profiling of tandem mass tag (TMT)-labeled proteins and phosphopeptides that we have named the streamlined (SL)-TMT protocol. We describe a universally applicable strategy that requires minimal individual sample processing and permits the seamless addition of a phosphopeptide enrichment step (“mini-phos”) with little deviation from the deep proteome analysis. To showcase our workflow, we profile the proteome of wild-type <i>Saccharomyces cerevisiae</i> yeast grown with either glucose or pyruvate as the carbon source. Here, we have established a streamlined TMT protocol that enables deep proteome and medium-scale phosphoproteome analysis

    Improved Method for Determining Absolute Phosphorylation Stoichiometry Using Bayesian Statistics and Isobaric Labeling

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    Phosphorylation stoichiometry, or occupancy, is one element of phosphoproteomics that can add useful biological context (Gerber et al. <i>Proc. Natl. Acad. Sci. U. S. A</i>. 2003, <i>100</i>, 6940–5). We previously developed a method to assess phosphorylation stoichiometry on a proteome-wide scale (Wu et al. <i>Nat. Methods</i> 2011, <i>8</i>, 677–83). The stoichiometry calculation relies on identifying and measuring the levels of each nonphosphorylated counterpart peptide with and without phosphatase treatment. The method, however, is problematic in that low stoichiometry phosphopeptides can return negative stoichiometry values if measurement error is larger than the percent stoichiometry. Here, we have improved the stoichiometry method through the use of isobaric labeling with 10-plex TMT reagents. In this way, five phosphatase treated and five untreated samples are compared simultaneously so that each stoichiometry is represented by five ratio measurements with no missing values. We applied the method to determine basal stoichiometries of HCT116 cells growing in culture. With this method, we analyzed five biological replicates simultaneously with no need for phosphopeptide enrichment. Additionally, we developed a Bayesian model to estimate phosphorylation stoichiometry as a parameter confined to an interval between 0 and 1 implemented as an R/Stan script. Consequently, both point and interval estimates are consistent with the plausible range of values for stoichiometry. Finally, we report absolute stoichiometry measurements with credible intervals for 6772 phosphopeptides containing at least a single phosphorylation site

    Combining Amine Metabolomics and Quantitative Proteomics of Cancer Cells Using Derivatization with Isobaric Tags

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    Quantitative metabolomics and proteomics technologies are powerful approaches to explore cellular metabolic regulation. Unfortunately, combining the two technologies typically requires different LC-MS setups for sensitive measurement of metabolites and peptides. One approach to enhance the analysis of certain classes of metabolites is by derivatization with various types of tags to increase ionization and chromatographic efficiency. We demonstrate here that derivatization of amine metabolites with tandem mass tags (TMT), typically used in multiplexed peptide quantitation, facilitates amino acid analysis by standard nanoflow reversed-phase LC-MS setups used for proteomics. We demonstrate that this approach offers the potential to perform experiments at the MS1-level using duplex tags or at the MS2-level using novel 10-plex reporter ion-containing isobaric tags for multiplexed amine metabolite analysis. We also demonstrate absolute quantitative measurements of amino acids conducted in parallel with multiplexed quantitative proteomics, using similar LC-MS setups to explore cellular amino acid regulation. We further show that the approach can also be used to determine intracellular metabolic labeling of amino acids from glucose carbons

    Proteome-Wide Evaluation of Two Common Protein Quantification Methods

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    Proteomics experiments commonly aim to estimate and detect differential abundance across all expressed proteins. Within this experimental design, some of the most challenging measurements are small fold changes for lower abundance proteins. While bottom-up proteomics methods are approaching comprehensive coverage of even complex eukaryotic proteomes, failing to reliably quantify lower abundance proteins can limit the precision and reach of experiments to much less than the identifiedlet alone totalproteome. Here we test the ability of two common methods, a tandem mass tagging (TMT) method and a label-free quantitation method (LFQ), to achieve comprehensive quantitative coverage by benchmarking their capacity to measure 3 different levels of change (3-, 2-, and 1.5-fold) across an entire data set. Both methods achieved comparably accurate estimates for all 3-fold-changes. However, the TMT method detected changes that reached statistical significance three times more often due to higher precision and fewer missing values. These findings highlight the importance of refining proteome quantitation methods to bring the number of usefully quantified proteins into closer agreement with the number of total quantified proteins

    Streamlined Tandem Mass Tag (SL-TMT) Protocol: An Efficient Strategy for Quantitative (Phospho)proteome Profiling Using Tandem Mass Tag-Synchronous Precursor Selection-MS3

    No full text
    Mass spectrometry (MS) coupled toisobaric labeling has developed rapidly into a powerful strategy for high-throughput protein quantification. Sample multiplexing and exceptional sensitivity allow for the quantification of tens of thousands of peptides and, by inference, thousands of proteins from multiple samples in a single MS experiment. Accurate quantification demands a consistent and robust sample-preparation strategy. Here, we present a detailed workflow for SPS-MS3-based quantitative abundance profiling of tandem mass tag (TMT)-labeled proteins and phosphopeptides that we have named the streamlined (SL)-TMT protocol. We describe a universally applicable strategy that requires minimal individual sample processing and permits the seamless addition of a phosphopeptide enrichment step (“mini-phos”) with little deviation from the deep proteome analysis. To showcase our workflow, we profile the proteome of wild-type <i>Saccharomyces cerevisiae</i> yeast grown with either glucose or pyruvate as the carbon source. Here, we have established a streamlined TMT protocol that enables deep proteome and medium-scale phosphoproteome analysis

    A High-Throughput, Multiplexed Kinase Assay Using a Benchtop Orbitrap Mass Spectrometer To Investigate the Effect of Kinase Inhibitors on Kinase Signaling Pathways

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    Protein phosphorylation is an important and ubiquitous post-translational modification in eukaryotic biological systems. The KAYAK (<u>K</u>inase <u>A</u>ctivit<u>Y</u> <u>A</u>ssay for <u>K</u>inome profiling) assay measures the phosphorylation rates of dozens of peptide substrates simultaneously, directly from cell lysates. Here, we simplified the assay by removing the phosphopeptide enrichment step, increasing throughput while maintaining similar data quality. We term this new method, direct-KAYAK, because kinase activities were measured directly from reaction mixtures after desalting. In addition, new peptides were included to profile additional kinase pathways and redundant substrate peptides were removed. Finally, the method is now performed in 96-well plate format using a benchtop orbitrap mass spectrometer and the Pinpoint software package for improved data analysis. We applied the new high-throughput method to measure IC<sub>50</sub> values for kinases involved in monocyte-to-macrophage differentiation, a process important for inflammation and the immune response
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