13c615n4 arginine

The SID-SRM-MS assays of selected proteins were developed as described previously (85 (link)). For protein tau, two or three peptides were initially selected, and then the sensitivity and selectivity of these were experimentally evaluated as described previously (85 (link)). The peptide with the best sensitivity and selectivity was selected as the surrogate for that protein. For each peptide, 3–5 SRM transitions were monitored (Table S2). The signature peptides of various ubiquitination linkages were chemically synthesized, incorporating isotopically labeled [¹³C₆¹⁵N₄] arginine or [¹³C₆¹⁵N₂] lysine to a 99% isotopic enrichment (Thermo Scientific). The amount of stable isotope-labeled standard (SIS) peptides was determined by amino acid analysis. After trypsin digestion, the tau peptides were then reconstituted in 30 μl of 5% formic acid-0.01% TFA, and an aliquot of SIS peptides was added to each tryptic digest. These samples were desalted with a ZipTip C18 cartridge and analyzed by LC-SRM-MS as described above.

Seven proteotypic peptides of ERG protein were selected and stable isotope-labeled heavy peptides with C-terminal [¹³C₆¹⁵N₂] lysine or [¹³C₆¹⁵N₄] arginine were synthesized (ThermoFisher Scientific, Waltham, MA) for SRM assay development [33 (link)]. SRM parameters were optimized by direct infusion experiments on a TSQ Quantum Ultra triple quadrupole mass spectrometer (ThermoFisher Scientific), where the 6-8 most intense fragment ions for each peptide were selected as precursor-to-fragment transitions and the collision energy (CE) of each transition was optimized automatically in SRM mode. The peptides were dissolved in a buffer containing 50% acetonitrile and 0.1% formic acid, and the infusion rate was 300 nL/min. The transitions and corresponding optimal CE values from the infusion experiments were further validated for optimal detection of the target peptides in actual LC-SRM analysis. In this step 50 fmol/μL of heavy peptide standards were spiked with 0.5 μg/μL of VCaP-derived tryptic peptides, and 2 μL of the sample was analyzed using a nanoACQUITY UPLC® system (Waters Corporation, Milford, MA) and a TSQ Vantage triple quadrupole mass spectrometer (ThermoFisher Scientific). Transitions with lower intensity or higher level of interference were removed, and the three best transitions were retained for each peptide in developing the final SRM assays.