Project description:High specificity and ease of use make trypsin the most used enzyme in proteomics. Proteases with complementary cleavage specificity to trypsin have been applied to obtain additional data. However, use of proteases with broad specificity proved especially challenging. In this work, we analyzed the characteristics of five protease alternatives to trypsin for protein identification and sequence coverage when applied to S. pombe whole cell lysates. The specificity of the protease heavily impacted on the number of proteins identified. Proteases with higher specificity let to the identification of more proteins than proteases with lower specificity. However, AspN, GluC, chymotrypsin and proteinase K largely benefited from being paired with trypsin in sequential digestion, as had been shown by us for elastase before. In the most extreme case, the addition of trypsin to a proteinase K digest increased the number of identified proteins by 524 %. Also, AspN (82 %) and GluC (74 %) protein identifications largely improved following the additional digestion with trypsin. In general, protein identifications improved most over the use of the single protease when the enzymes followed on an initial digestion with trypsin. In the most extreme case, the sequential digest with trypsin and AspN yielded even higher number of protein identifications than digesting with trypsin alone.
Project description:Gliadin triggers T-cell mediated immunity in celiac disease, and has cytotoxic effects on enterocytes mediated through obscure mechanisms. In addition, gliadin transport mechanisms, potential cell surface receptors and gliadin-activated downstream signaling pathways are not completely understood. In order to screen for novel downstream gliadin target genes we performed a systematic whole genome expression study on intestinal epithelial cells. Undifferentiated Caco-2 cells were exposed to pepsin- and trypsin- digested gliadin (PT-G), a blank pepsin-trypsin control (PT) and to a synthetic peptide corresponding to gliadin p31-43 peptide for six hours. RNA from four different experiments was used for hybridization on Agilent one color human whole genome DNA microarray chips. The microarray data were analyzed using the Bioconductor package LIMMA. Genes with nominal p < 0.01 were considered statistically significant. Compared to the untreated cells 1705, 1755 and 211 probes were affected by PT-G, PT and p31-43 respectively. 46 probes were significantly different between PT and PT-G treated cells. Among the p31-43 peptide affected probes, 10 and 21 probes were affected by PT-G and PT respectively. Only PT-G affected genes could be validated by quantitative real-time polymerase chain reaction. All the genes were, nonetheless, also affected to a comparable level by PT treated negative controls. In conclusion, we could not replicate previously reported direct effects of gliadin peptides on enterocytes. The PT-G affected genes in the microarray analysis were validated by qRT-PCR, however these genes were also affected by PT treated negative controls suggesting that certain epitopes derived from pepsin and trypsin may also affect epithelial cell gene transcription. Our study demonstrates novel non-enzymatic effects of pepsin and trypsin on cells and calls for proper controls in pepsin and trypsin digested gliadin experiments. It is conceivable that gliadin effects on enterocytes are secondary mediated through oxidative stress, NFkB activation and IL-15 up-regulation.
Project description:Gliadin triggers T-cell mediated immunity in celiac disease, and has cytotoxic effects on enterocytes mediated through obscure mechanisms. In addition, gliadin transport mechanisms, potential cell surface receptors and gliadin-activated downstream signaling pathways are not completely understood. In order to screen for novel downstream gliadin target genes we performed a systematic whole genome expression study on intestinal epithelial cells. Undifferentiated Caco-2 cells were exposed to pepsin- and trypsin- digested gliadin (PT-G), a blank pepsin-trypsin control (PT) and to a synthetic peptide corresponding to gliadin p31-43 peptide for six hours. RNA from four different experiments was used for hybridization on Agilent one color human whole genome DNA microarray chips. The microarray data were analyzed using the Bioconductor package LIMMA. Genes with nominal p < 0.01 were considered statistically significant. Compared to the untreated cells 1705, 1755 and 211 probes were affected by PT-G, PT and p31-43 respectively. 46 probes were significantly different between PT and PT-G treated cells. Among the p31-43 peptide affected probes, 10 and 21 probes were affected by PT-G and PT respectively. Only PT-G affected genes could be validated by quantitative real-time polymerase chain reaction. All the genes were, nonetheless, also affected to a comparable level by PT treated negative controls. In conclusion, we could not replicate previously reported direct effects of gliadin peptides on enterocytes. The PT-G affected genes in the microarray analysis were validated by qRT-PCR, however these genes were also affected by PT treated negative controls suggesting that certain epitopes derived from pepsin and trypsin may also affect epithelial cell gene transcription. Our study demonstrates novel non-enzymatic effects of pepsin and trypsin on cells and calls for proper controls in pepsin and trypsin digested gliadin experiments. It is conceivable that gliadin effects on enterocytes are secondary mediated through oxidative stress, NFkB activation and IL-15 up-regulation. In total, 16 samples were analyzed of which 4 were control (MED) samples, 4 samples of p31-43 treatment, 4 samples of PT treatmetn and 4 samples of PT-G treatment
Project description:We have comprehensively profiled the time-series changes in gene expression dependent on enzymatic treatment using trypsin, and attempted to elucidate the global changes suppressed by treatment with cold active protease in a low-temperature environment.
Project description:Whole-cell lysates were digested with trypsin, fractionated by reverse phase chromatography into 24 fractions and analyzed by liquid chromatography - tandem mass spectrometry
Project description:To investigate whether samples digested with trypsin, GluC, or AspN benefit from PSM rescoring, we rescored a timsTOF Pro dataset that was digested using different proteases. For detailed information on data acquisition, please refer to the original publication by Fossati et al. (PXD025671). In brief, for spectral library generation 500 ng for each fraction were acquired using DDA PASEF on the timsTOF Pro (Bruker Daltonik, Germany).
Individual spectrum peak files were searched against a combined human-Mtb database encompassing the *Mycobacterium Tuberculosis* proteome (4,081 entries, downloaded from Uniprot on 12/02/2021) and Homo Sapiens proteome (20,397 entries, downloaded on 07/01/2021). The number of missed cleavages was fixed to 2, using cysteine carbamidomethylation as fixed modification, and N-terminal acetylation and methionine oxidation as variable modifications. The search results were rescored by integrating Prosit's fragment ion intensity predictions, using Oktoberfest version 0.5.3 (https://github.com/wilhelm-lab/oktoberfest).
PSM rescoring of the samples cleaved with AspN, GluC, and trypsin resulted in 1.5-fold, 1.7-fold, and 1.4-fold increases in unique identified peptides, respectively.
Project description:The level of trypsin-2 has been shown to correlate with the malignancy and metastatic potential of many cancer. We used microarrays to detail how tryspin-2 overexpression changes the gene profile of human tongue squamous cell carcinoma cell line (HSC-3). We compared trypsin-2 overexpressing HSC-3 line to HSC-3 which was transfected with control vector and because of that did not expressed trypsin-2 at all.