Shotgun EM of mycobacterial protein complexes during stationary phase stress.
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ABSTRACT:
SUBMITTER: Kirykowicz AM
PROVIDER: S-EPMC8244302 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:The aim of this study was to determine the fate of ribosomes and r-proteins. In this respect, SILAC (Stable Isotope Labeled Amino acids in cell Culture) based experimental approach was used. E.coli cells were grown in MOPS medium supplemented with “heavy” labeled arginine (Arg10) and lysine (Lys8). At the mid-log phase, the culture was further supplemented with a 20-fold molar excess of “light” unlabeled arginine (Arg0) and lysine (Lys0), divided into 8 aliquots, and grown for 14 days. Cell samples were collected at day one (24h), day two (48h), and subsequently in 48h intervals over the following 12 days. The quantities of r-proteins in the proteome were determined using SILAC based LC-MS/MS and normalized to the corresponding values of day one.
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Project description:Large-scale genomics has enabled proteomics by creating sequence infrastructures that can be used with mass spectrometry data to identify proteins. Although protein sequences can be deduced from nucleotide sequences, posttranslational modifications to proteins, in general, cannot. We describe a process for the analysis of posttranslational modifications that is simple, robust, general, and can be applied to complicated protein mixtures. A protein or protein mixture is digested by using three different enzymes: one that cleaves in a site-specific manner and two others that cleave nonspecifically. The mixture of peptides is separated by multidimensional liquid chromatography and analyzed by a tandem mass spectrometer. This approach has been applied to modification analyses of proteins in a simple protein mixture, Cdc2p protein complexes isolated through the use of an affinity tag, and lens tissue from a patient with congenital cataracts. Phosphorylation sites have been detected with known stoichiometry of as low as 10%. Eighteen sites of four different types of modification have been detected on three of the five proteins in a simple mixture, three of which were previously unreported. Three proteins from Cdc2p isolated complexes yielded eight sites containing three different types of modifications. In the lens tissue, 270 proteins were identified, and 11 different crystallins were found to contain a total of 73 sites of modification. Modifications identified in the crystallin proteins included Ser, Thr, and Tyr phosphorylation, Arg and Lys methylation, Lys acetylation, and Met, Tyr, and Trp oxidations. The method presented will be useful in discovering co- and posttranslational modifications of proteins.
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