Adaptation of a commonly used, chemically defined medium for human embryonic stem cells to stable isotope labeling with amino acids in cell culture (SILAC)

ABSTRACT: Metabolic labeling with stable isotopes is a prominent technique for comparative quantitative proteomics and stable isotope labeling with amino acids in cell culture (SILAC) is the most commonly used approach. SILAC is, however, traditionally limited to simple tissue culture regiments and only rarely employed in the context of complex culturing conditions as those required for human embryonic stem cells (hESCs). Classic hESC culture is based on the use of mouse embryonic fibroblasts (MEFs) as a feeder layer and as a result possible xenogeneic contamination, contribution of unlabeled amino acids by the feeders, inter-laboratory variability of MEF preparation and the overall complexity of the culture system are all of concern in conjunction with SILAC. We demonstrate a feeder-free SILAC culture system based on a customised version of a commonly used chemically defined hESC medium developed by Ludwig et al. (2006) and commercially available as mTeSR1 . This medium, together with adjustments to the culturing protocol, facilitates reproducible labeling which is easily scalable to the protein amounts required by proteomic work flows. It greatly enhances the usability of quantitative proteomics as a tool for the study of mechanisms underlying hESCs differentiation and self-renewal. Mass spectrometric data was analysed using the MaxQuant suite of algorithms [version 1.3.0.5]. The data was searched against the Homo sapiens UniProtKB protein sequence database (downloaded on 10/24/2012, 68,108 entries including canonical and isoform sequence data). Enzyme specificity was set to trypsin, allowing for cleavage N-terminal to proline and between aspartic acid and proline. Carbamidomethylcysteine was set as a fixed modification, and oxidized methionine and N-acetylation were set as variable modifications. Labeled Arg and Lys were used as peptide modifications for quantitation. MaxQuant’s requantitation option was not used where incorporation efficiency and arginine to proline conversion were analyzed. The maximum mass deviation allowed was set at 20 ppm for the initial search, and 6 ppm for the main search for monoisotopic precursor ions; the mass error tolerance was set at 20 ppm for MS/MS peaks. A maximum of two missed cleavages and three labeled amino acids (arginine and lysine) were allowed. The required false discovery rates were set to 1% at the peptide and protein level (the protein level filter was disabled for incorporation efficiency and arginine to proline conversion testing), and the minimum required peptide length was set to 6 amino acids. Statistical analysis and plotting were performed using the R Statistical Programming Environment. The analyses performed are documented in an R package provided in the proteomeXchange repository and based on a publicly available collection of tools (RCFPD, URL: http://sourceforge.net/projects/rcfpd/).

INSTRUMENT(S): Q Exactive

ORGANISM(S): Homo Sapiens (human)

SUBMITTER: Johannes Graumann  

LAB HEAD: Johannes Graumann

PROVIDER: PXD000151 | Pride | 2013-06-17

REPOSITORIES: Pride