Identification of low abundant secreted proteins and peptides from primary culture supernatants of human T-cells

Finoulst, Inez; Vink, Paul; Rovers, Eric; Pieterse, Mervin; Pinkse, Martijn W. H.; Bos, Ebo; Verhaert, Peter

doi:10.1016/j.jprot.2011.03.034

Cited by 17 publications

(16 citation statements)

References 36 publications

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“…For example, a study that focused on mass spectrometry based detection of interleukin-2 (IL-2) from highly specialized T-cells used a variety of chromatographic, mass spectrometric (Orbitrap), and bioinformatics approaches to weed out IL-2’s signature peptides from the complex sample [174]. That is, in the spectral searches any peptide fragments not related to proteins derived from T-cells were removed from the search.…”

Section: Mass Spectrometrymentioning

confidence: 99%

Bioanalytical chemistry of cytokines – A review

Stenken

Poschenrieder

2015

Analytica Chimica Acta

245

172

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Cytokines are bioactive proteins produced by many different cells of the immune system. Due to their role in different inflammatory disease states and maintaining homeostasis, there is enormous clinical interest in the quantitation of cytokines. The typical standard methods for quantitation of cytokines are immunoassay-based techniques including enzyme-linked immusorbent assays (ELISA) and bead-based immunoassays read by either standard or modified flow cytometers. A review of recent developments in analytical methods for measurements of cytokine proteins is provided. This review briefly covers cytokine biology and the analysis challenges associated with measurement of these biomarker proteins for understanding both health and disease. New techniques applied to immunoassay-based assays are presented along with the uses of aptamers, electrochemistry, mass spectrometry, optical resonator-based methods. Methods used for elucidating the release of cytokines from single cells as well as in vivo collection methods are described.

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Section: Mass Spectrometrymentioning

confidence: 99%

Bioanalytical chemistry of cytokines – A review

Stenken

Poschenrieder

2015

Analytica Chimica Acta

245

172

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“…In one approach, cells are grown in growth medium containing serum. This method usually necessitates extensive protein and/or peptide fractionation to detect low-abundance secreted proteins (in the ng per ml range) against a background of thousands of highly abundant serum proteins (in the mg per ml range) 2,3 . Alternatively, a more common approach is to grow cells using serum-free conditions, thereby reducing analytical interference and increasing the ability to detect secreted proteins 4 .…”

mentioning

confidence: 99%

Selective enrichment of newly synthesized proteins for quantitative secretome analysis

Eichelbaum¹,

Winter²,

et al. 2012

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Secreted proteins constitute a large and biologically important subset of proteins that are involved in cellular communication, adhesion and migration. Yet secretomes are understudied because of technical limitations in the detection of low-abundance proteins against a background of serum-containing media. Here we introduce a method that combines click chemistry and pulsed stable isotope labeling with amino acids in cell culture to selectively enrich and quantify secreted proteins. The combination of these two labeling approaches allows cells to be studied irrespective of the complexity of the background proteins. We provide an in-depth and differential secretome analysis of various cell lines and primary cells, quantifying secreted factors, including cytokines, chemokines and growth factors. In addition, we reveal that serum starvation has a marked effect on secretome composition. We also analyze the kinetics of protein secretion by macrophages in response to lipopolysaccharides.

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“…The different column materials were kept separate from each other by insertion of a piece of glass wool. The LC equipment and solvents used were similar to those used by Finoulst et al (45). Each sample analysis consisted of six fractionations.…”

Section: Methodsmentioning

confidence: 99%

“…Mass spectrometry was performed using a protocol derived from that of Finoulst et al (45). Full-scan MS spectra (from m/z 400 to 1,500; charge states 2 and higher) were acquired at a resolution of 30,000 at m/z 400 after accumulation to a target value of 10 6 ions (automatic gain control).…”

Section: Methodsmentioning

confidence: 99%

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In Vivo Analysis of NH ₄ ⁺ Transport and Central Nitrogen Metabolism in Saccharomyces cerevisiae during Aerobic Nitrogen-Limited Growth

Cueto-Rojas

Seifar

Pierick

et al. 2016

Appl Environ Microbiol

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Ammonium is the most common N source for yeast fermentations. Although its transport and assimilation mechanisms are well documented, there have been only a few attempts to measure the in vivo intracellular concentration of ammonium and assess its impact on gene expression. Using an isotope dilution mass spectrometry (IDMS)-based method, we were able to measure the intracellular ammonium concentration in N-limited aerobic chemostat cultivations using three different N sources (ammonium, urea, and glutamate) at the same growth rate (0.05 h ؊1 ). The experimental results suggest that, at this growth rate, a similar concentration of intracellular (IC) ammonium, about 3.6 mmol NH 4 ؉ /liter IC , is required to supply the reactions in the central N metabolism, independent of the N source. Based on the experimental results and different assumptions, the vacuolar and cytosolic ammonium concentrations were estimated. Furthermore, we identified a futile cycle caused by NH 3 leakage into the extracellular space, which can cost up to 30% of the ATP production of the cell under N-limited conditions, and a futile redox cycle between Gdh1 and Gdh2 reactions. Finally, using shotgun proteomics with protein expression determined relative to a labeled reference, differences between the various environmental conditions were identified and correlated with previously identified N compound-sensing mechanisms. IMPORTANCEIn our work, we studied central N metabolism using quantitative approaches. First, intracellular ammonium was measured under different N sources. The results suggest that Saccharomyces cerevisiae cells maintain a constant NH 4 ؉ concentration (around 3 mmol NH 4 ؉ /liter IC ), independent of the applied nitrogen source. We hypothesize that this amount of intracellular ammonium is required to obtain sufficient thermodynamic driving force. Furthermore, our calculations based on thermodynamic analysis of the transport mechanisms of ammonium suggest that ammonium is not equally distributed, indicating a high degree of compartmentalization in the vacuole. Additionally, metabolomic analysis results were used to calculate the thermodynamic driving forces in the central N metabolism reactions, revealing that the main reactions in the central N metabolism are far from equilibrium. Using proteomics approaches, we were able to identify major changes, not only in N metabolism, but also in C metabolism and regulation. Saccharomyces cerevisiae is a versatile organism that can grow on a large variety of N sources, namely, ammonium (NH 4 ϩ ), urea, citrulline, ornithine, gamma aminobutyric acid (GABA), allatoin, allantoate, and all proteinogenic L-amino acids, with the exceptions of L-lysine, L-histidine, and L-cysteine (1, 2). Therefore, S. cerevisiae cells require complex machinery to achieve metabolic regulation for efficient uptake and anabolic and catabolic processing of N compounds.In particular, S. cerevisiae can differentiate between preferred and nonpreferred N sources using the nitrogen catabolite repression (NCR) pa...

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Identification of low abundant secreted proteins and peptides from primary culture supernatants of human T-cells

Cited by 17 publications

References 36 publications

Bioanalytical chemistry of cytokines – A review

Bioanalytical chemistry of cytokines – A review

Selective enrichment of newly synthesized proteins for quantitative secretome analysis

In Vivo Analysis of NH ₄ ⁺ Transport and Central Nitrogen Metabolism in Saccharomyces cerevisiae during Aerobic Nitrogen-Limited Growth

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Identification of low abundant secreted proteins and peptides from primary culture supernatants of human T-cells

Cited by 17 publications

References 36 publications

Bioanalytical chemistry of cytokines – A review

Bioanalytical chemistry of cytokines – A review

Selective enrichment of newly synthesized proteins for quantitative secretome analysis

In Vivo Analysis of NH 4 + Transport and Central Nitrogen Metabolism in Saccharomyces cerevisiae during Aerobic Nitrogen-Limited Growth

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Product

Resources

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In Vivo Analysis of NH ₄ ⁺ Transport and Central Nitrogen Metabolism in Saccharomyces cerevisiae during Aerobic Nitrogen-Limited Growth