Dataset Information

ChaFRADIC: A novel SCX-based approach for N-terminal Enrichment

ABSTRACT: Step 1: Cell lysis of yeast sphaeroblast (Saccharomyces cerevisiae) and carbamidomethylation. Step 2: Isotopic labeling with dimethylation (light + medium). Step 3: Proteolytic digestion with trypsin (ArgC specificity because of blocked lysines by dimethylation). Step 4: Enrichment via ChaFRADIC method Step 5: Nano-LC-MS/MS measurements Step 6: Data interpretation. Specifications: --ESI spray ionisation o Q Exactive mass spec. --Quadrupole selection, HCD fragmentation and orbitrap detection o Identification + Quantification: PD version 1.3.0039. Search engine: Mascot 2.4. SGD database (Sep 2011), 6717 target sequences. Special nodes: precursor ions quantifier (within a 1 min retention time window), percolator (default settings). Protease: SemiArgC with max. 2 missed cleavage sites. Used modifications: Yeast_N-term_Enriched_Search_Acetylation Variable -- N-terminal Acetylation (42.0105 Da) -- Dimethylation light (28.0313 Da) at Lys -- Dimethylation medium (34.0689 Da) at Lys fixed -- Carbamidomethylation at Cys (57.0214 Da) Quantification -- Peptides with labeled lysine Yeast_N-term_Enriched_Search_Dimethylation Variable -- N-terminal Dimethylation light (28.0313 Da) -- N-terminal Dimethylation medium (34.0689 Da) -- Dimethylation light (28.0313 Da) at Lys -- Dimethylation medium (34.0689 Da) at Lys fixed -- Carbamidomethylation at Cys (57.0214 Da) Quantification -- Peptides with labeled N-terminus -- Peptides with labeled N-terminus and labeled lysine. Mass tolerance: 10 ppm for MS, 0.02 Da for MS/MS. Applied filters: high confidence (FDR <1%), search engine rank 1

INSTRUMENT(S): Q Exactive

ORGANISM(S): Saccharomyces Cerevisiae (baker's Yeast)

SUBMITTER: Saskia Venne

PROVIDER: PXD000292 | Pride | 2013-08-09

REPOSITORIES: Pride

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Publications

Novel highly sensitive, specific, and straightforward strategy for comprehensive N-terminal proteomics reveals unknown substrates of the mitochondrial peptidase Icp55.

Venne A Saskia AS Vögtle F-Nora FN Meisinger Chris C Sickmann Albert A Zahedi René P RP

Journal of proteome research 20130821 9

We present a novel straightforward method for enrichment of N-terminal peptides, utilizing charge-based fractional diagonal chromatography (ChaFRADIC). Our method is robust, easy to operate, fast, specific, and more sensitive than existing methods, enabling the differential quantitation of 1459 nonredundant N-terminal peptides between two S. cerevisiae samples within 10 h of LC-MS, starting from only 50 μg of protein per condition and analyzing only 40% of the obtained fractions. Using ChaFRADIC ...[more]

PMID: 23964590

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Project description:Metastasizing tumor cells exit the vascular system through dynamic interactions with endothelial cells that line the internal surface of vessels. While extravasation is a key event within the metastatic cascade, the signals regulating tumor cell adhesion to the endothelium and their subsequent transendothelial migration are poorly understood. Here, we combined Stable Isotope Labeling by Amino acids in Cell culture (SILAC) and phosphoproteomic analysis to identify cell-specific signaling pathways regulated between interacting breast cancer cells and endothelial cells (see PRIDE repository PXD001558). Further co-culture experiments were performed alongside the phosphoproteomic analysis in order to control for the protein quantity. These are presented here. Using SILAC, cell-specific labels were introduced into MDA-MB-231-LM2 cells (Human Breast cancer) and HUVECs (Human endothelial cells) to ensure each cell type had a distinct and traceable phosphoproteome when tumor and endothelial cells were co-cultured. To probe regulatory signaling events triggered in cancer cells following contact with endothelial cells, we labeled LM2 cells with medium or heavy isotopomers of arginine and lysine (Arg+6 Da, Lys+4 Da and Arg+10 Da, Lys+8 Da respectively). Heavy-labeled LM2 cells were collected by enzyme-free cell dissociation buffer, thereby preserving membrane proteins and adhesion receptors, and seeded onto a monolayer of light-labeled (Arg+0 Da, Lys+0 Da) HUVECs. Following 15 min of co-culture, non-adherent LM2 cells were gently removed and cancer cells that had attached to the endothelial layer were lysed together with the HUVECs. In parallel, medium-labeled LM2 cells were collected under the same conditions and maintained as suspension cells in monoculture to represent circulating tumor cells prior to any contact with the endothelium. These were then added to the harvested LM2-HUVEC co-culture in a 1:1 ratio of heavy:medium-labeled cells to provide a point of reference. Based on the SILAC labeling of the different cell populations, light-labeled peptides were assigned to HUVECs, medium-labeled peptides to monocultured LM2 cells in suspension, and heavy-labeled peptides to LM2 cells that had made contact with HUVECs. As such, the heavy/medium ratio for each peptide was used to quantify phosphorylation-dependent signaling changes occurring specifically in the LM2 cells upon contact with HUVECs. Conversely, to elucidate signaling events in HUVECs that were initiated by contacting cancer cells, light-labeled LM2 cells were seeded on top of a confluent monolayer of heavy-labeled HUVECs, while medium-labeled HUVECs were maintained in monoculture. Following 15 min of co-culture, the unattached LM2 cells were removed. Cells were lysed, mixed in a 1:1 ratio of heavy:medium HUVECs, followed by membrane fractionation and phosphoproteomic analysis as described above. A variation in phospho-peptide quantity could be due to the experimental difficulties in mixing heavy and medium labels in a 1:1 ratio or a quick regulation of the protein quantity through modification of its expression/degradation balance. Thus, we performed relative quantification of non-phosphorylated peptides in parallel with the phosphoproteomic analysis to account for changes in total protein abundance (data presented here) or correct for experimental bias. Cytoplasmic fractions were analyzed by LC-MS/MS before TiO2 enrichment and their median log2(H/M) were used for normalization of the phosphoproteomic dataset. In order to increase the number of proteins quantified in the LM2 cells, we performed a supplementary co-culture experiment and fractionated the peptides by SDS-PAGE. In order to get a confident relative quantification of Ephrin type-A receptor 2 (EPHA2), we performed 2 independent experiments followed by EPHA2 IP and LC-MSMS.

Dataset Information

ChaFRADIC: A novel SCX-based approach for N-terminal Enrichment

Publications

Novel highly sensitive, specific, and straightforward strategy for comprehensive N-terminal proteomics reveals unknown substrates of the mitochondrial peptidase Icp55.

Similar Datasets

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