Project description:LC-MS/MS raw files for Jurkat cell lysate aliquots digested with either Arg-C, Asp-N, Chymotrypsin, Glu-C, Lys-C or Trypsin and fractionated offline to produce 11 fractions (Trypsin only has 10 fractions).
Project description:Proteins present in cellular environments with high levels of reactive oxygen and nitrogen species and/or low levels of antioxidants are highly susceptible to oxidative post-translational modification (PTM). Irreversible oxidative PTMs can generate a complex distribution of modified protein molecules, recently termed as proteoforms. Using ubiquitin as a model system, we mapped oxidative modification sites using trypsin, Lys-C, and Glu-C peptides. Several M+16 Da proteoforms were detected as well as proteoforms that include other previously unidentified oxidative modifications. This work highlights the use of multiple protease digestions to give insights to the complexity of oxidative modifications possible in bottom-up analyses.
Project description:Ancient skeletal proteomes are increasingly utilised for phylogenetic and evolutionary analysis. These proteomes are, however, often small and with low sequence coverage. We expand on previous observations which have shown that parallel digestion of Pleistocene skeletal proteomes increases proteome size and protein sequence coverage. Furthermore, we demonstrate that the consecutive digestion of a skeletal proteome using two proteases, particularly Glu-C or chymotrypsin followed by trypsin digestion, enables the recovery of alternative proteome components not reachable through trypsin digestion alone. The sequential utilisation of several proteases is a promising avenue for the study of highly degraded ancient proteomes for phylogenetic purposes.
Project description:Acid-extracted histones from the 0-hpi_2, 18-hpi_1 and 36-hpi_2 samples previously analyzed for PTMs by MudPIT were digested with endoproteinase Arg-C (RC) or endoproteinase Lys-C followed by Glu-C (KCEC). Digested histone peptides were separated on a 15cm nano-reverse phase (RP) column using a 4-hr RP-HPLC separation. The data was acquired on a LTQ-Velos-Orbitrap in low resolution mode. Based on the available fragmentation data of the modified peptides, we selected between three to seven MS/MS fragment ions for transitions. The criteria for selecting the fragment ions were that the fragments included the amino acid residue(s) modified in the peptide. The Multiple Reaction Monitoring (MRM) method was built by including the parent mass, parent mass selection window, product mass and mass selection windows for these three to seven product ions. Average and monoisotopic m/z values were used for the parent ion mass and the product ions, respectively. The number of simultaneously assayable peptides was limited to 12. SRM and the MS/MS spectra for the same parent ion were collected consecutively within the same run. Each of the 24 precursor ions targeted for SRM was assayed at least twice independently in each of the 3 samples analyzed (Supplementary Files 0-, 18-, 36-hpi_MRM-Assays_Overview.xlsx).
The 24 peptides targeted for SRM were validated manually to confirm the presence of the relevant transitions in the SRM spectra followed by their corresponding MS/MS spectrum. When a peptide was not observed in one of the 3 stages analyzed, the unfiltered PSMs (below cut-off) were also manually assessed to ensure that the modified peptide expression was indeed stage-specific and not missed due to the conservative selection criteria. Annotated MS/MS spectra for all modified peptides are provided in Supplementary Files 0-, 18-, 36-hpi_ MRMs_MS2-Annotated-Spectra.pdf.
Project description:Covalent modifications of proteins with ubiquitin and ubiquitin-like molecules are instrumental to most, if not all biological processes. However, identifying the E3 ligase responsible for these modifications remains a major bottleneck in ubiquitin research. Here, we have developed an E2-thioester-driven identification (E2~dID) method for the targeted identification of substrates of specific E2 and E3 enzyme pairs. E2~dID exploits the central position of E2 conjugating enzymes in the ubiquitination cascade and provides in vitro generated biotinylated E2~ubiquitin thioester conjugates as the sole source for ubiquitination in extracto. This enables purification and identification of modified proteins by mass spectrometry under stringent conditions independently of the biological source of the extract. We demonstrate the sensitivity and specificity of E2-dID by identifying and validating substrates of the APC/C in human cells. Finally, performing E2~dID with SUMO in S. cerevisiae we show that E2-dID can be easily adapted to other ubiquitin-like modifiers and experimental models.