Project description:Digested Escherichia coli protein extract were mixed with 89 different isotopically-labelled reference heavy peptides (5 pmol, 15N2,13C6-Lys, 15N4,13C6-Arg), and with different amounts of the corresponding light peptides (10 pmol, 5 pmol, 2.5 pmol), as 1, 2 and 4, respectively. Light peptides were dived in three different subsets and five different mixtures were prepared in triplicates. SRM measurements for the light-heavy synthetic peptides were performed on a hybrid triple quadrupole/ion trap mass spectrometer (4000 Q-Trap, AB Sciex) equipped with a nano-LC electrospray ionization source. Details are available in description.pdf
Project description:U2OS cells were split into two aliquots and cultured in one of
the following media (DMEM + 10 % dialysed FCS):
i) light medium: contains Arg-0 and Lys-0
ii) heavy medium: contains Arg-10 and Lys-8
Cells were passaged at least three times in the respective medium to ensure complete
labelling of proteins. SILAC experiments were performed in a forward and a reverse
reaction: for the forward experiment, heavy-labelled cells were irradiated with 20 Gy and
harvested 45 minutes after irradiation, while light-labelled cells were left untreated. In the
reverse experiment, labels were swapped and light-labelled cells were irradiated, while
heavy labelled cells were left untreated.
Project description:A novel stable isotope labelling strategy was developed to quantify methionine oxidation in an unstressed human proteome. Cell extracts were oxidized with 18O labelled hydrogen peroxide following cell lysis in order to convert all unoxidized methionine residues to an oxidized version with a heavy label. The heavy labelled peptides were used to generate a custom search and quantification of the relative ratios between heavy and light labelled methionine sulfoxide containing peptides. Where the light labelled peptides were in vivo oxidized
Project description:A novel stable isotope labelling stategy was developed to quantify methionine oxidation in an unstressed human proteome. Cell extracts were oxidized with 18O labelled hydrogen peroxide following cell lysis in order to convert all unoxidized methionine residues to an oxidized version with a heavy label. The heavy labelled peptides were used to generate a custom search and quantification of the relative ratios between heavy and light labelled methionine sulfoxide containing peptides. Where the light labelled peptides were in vivo oxidized
Project description:Today, the most widely used bottom-up proteomics studies necessitate a proteolysis step prior to MS analysis. Trypsin is often the best protease in choice due to its high specificity and MS-favored proteolytic products. Trypsin has been challenged for its low cleavage efficiency at Lys-X bonds, and tandem Lys-C/trypsin digestion approach has been developed to alleviate the problem. However, the identification capacity of the two digestions often disagrees among different research groups. Herein, we report a new tandem digestion using Lys-C and Arg-C (Lys-C/Arg-C), which we have proven to be more efficient and specific than trypsin and Lys-C/trypsin approaches. Analysis of our previous data (Anal. Chem. 2018, 90, 1554-1559) revealed that both Lys-C and Arg-C perform better in trypsin-mode digestion. In particular for Arg-C, the identification capacity is increased to 2.6 times and even comparable with trypsin. The good complementarity, high digestion efficiency and high specificity of Lys-C and Arg-C prompt a tandem Lys-C/Arg-C digestion. We systematically evaluated the Lys-C/Arg-C digestion using qualitative and quantitative proteomics approaches and confirmed its superior performance in digestion efficiency, specificity and identification capacity to the currently widely used trypsin and Lys-C/trypsin digestions. As a result, we concluded that Lys-C/Arg-C digestion approach would be the choice of next-generation digestion approach for better results in both qualitative and quantitative proteomics studies.
Project description:Ubiquitination is a post-translational modification that signals multiple processes, including protein degradation, trafficking and DNA repair. Polyubiquitin accumulates globally during the oxidative stress response, and this has been mainly attributed to increased ubiquitin conjugation and perturbations in protein degradation. Here we show that the unconventional Lys 63 (K63)-linked polyubiquitin accumulates in the yeast Saccharomyces cerevisiae in a highly sensitive and regulated manner as a result of exposure to peroxides. We demonstrate that hydrogen peroxide inhibits the deubiquitinating enzyme Ubp2, leading to accumulation of K63 conjugates assembled by the Rad6 ubiquitin conjugase and the Bre1 ubiquitin ligase. Using linkage-specific isolation methods and stable isotope labeling by amino acids in cell culture (SILAC)–based quantitative proteomics, we identified >100 new K63-polyubiquitinated targets, which were substantially enriched in ribosomal proteins. Finally, we demonstrate that impairment of K63 ubiquitination during oxidative stress affects polysome stability and protein expression, rendering cells more sensitive to stress, and thereby reveal a new redox-regulatory role for this modification.
Project description:TtT/GF is a mouse pituitary cell line derived from radiothyroidectomy-induced thyrotropic pituitary tumor and has been recognized as a model of folliculostellate cells. Our previous studies suggested that TtT/GF cells have cellular plasticity, rather than representing a model only for folliculostellate cells. We found that transforming growth factor beta (TGFβ) reinforces pericyte properties (Tsukada et al. Cell and Tissue Research, 371: 339-350, 2018). In order to extensively identify TGFβ-induced proteins in TtT/GF cells, the present study performed mass spectrometry analysis combined with the stable isotope labeling of amino acids in cell culture (SILAC) system. TtT/GF cells were cultured in either light medium (containing normal Arg/Lys) or heavy medium (containing 13C6-labelled Arg/Lys) in the presence of vehicle (light), TGFβ (heavy), or selective TGFβ receptor I inhibitor (SB431542, heavy). Collected proteins were then analyzed by mass spectrometry. We successfully found 51 up-regulated and 112 down-regulated proteins, which are related to actin cytoskeleton, cell adhesion, extracellular matrix, and DNA replication. The result also showed up-regulation of many pericyte markers/pointers, supporting our previous hypothesis. Intriguingly, we found down-regulation of several pituitary adenoma markers. Mass spectrometry data using SILAC will provide valuable information about pathological processes in pituitary adenomas, as well as pericyte differentiation.
Project description:Methionine COFRADIC combined with Arg and Lys SILAC labeling was used to quantify proteins upon induction of miR-17-92 in SHEP neuroblastoma cells. Differently labeled cells treated with tetracycline for 72 h or left untreated, were harvested and mixed lysates were analyzed by methionine COFRADIC to isolate methionyl peptides followed by identification of these peptides by LC-MS/MS and subsequent quantification.
Project description:Mass spectrometry-based quantitative proteome profiling is most commonly performed by label-free quantification (LFQ), stable isotopic labeling with amino acids in cell culture (SILAC), and reporter-ion based isobaric labeling methods (TMT and iTRAQ). Isobaric peptide termini labeling (IPTL) was described as an alternative to these methods and is based on crosswise labeling of both peptide termini and MS2 quantification. High quantification accuracy was assumed for IPTL because multiple quantification points are obtained per identified MS2 spectrum. A direct comparison of IPTL with other quantification methods has not been performed yet because IPTL commonly requires digestion with endoproteinase Lys-C. To enable tryptic digestion of IPTL samples, a novel labeling for IPTL was developed which combines metabolic labeling (Arg-0/Lys-0 and Arg-d4/Lys-d4, respectively) with crosswise N-terminal dimethylation (d4 and d0, respectively). The comparison of IPTL with LFQ revealed significantly more protein identifications for LFQ above homology ion scores but not above identity ion score. However, the quantification accuracy was superior for LFQ despite the many quantification points obtained with IPTL. A reason for this outcome is probably because of the significantly higher signal intensities in MS1 compared to MS2.