Positive patient urine by mass spectrometry.
PRM mass spectra showing fragmentation ions (b9, b7, b11, y9) for peptide VERPTGNPDLLK, originating from protein GRA1. A subgroup of 44 urine samples were tested for the presence of 12 T. gondii proteins SAG1, SAG2A, GRA1, GRA3, GRA7, GRA12, ROP4, ROP5, ROP7, RON8, MIC10, and M2AP. Only GRA1, peptide VERPTGNPDLLK was found. VERPTGNPDLLK was identified based on the consistent identification of ion b9+, b11+, b7+, and y9+ consistent retention time across controls and samples. b+ ions extend from the amino end of the peptide, while y+ ions extend from the carboxyl end. The associated number is indicative of how many amino acids were associated with each ion. (A) 1e6 RH strain tachyzoites after incubation with nanoparticles acting as a positive control. (B) Recombinant GRA1 protein after incubation with nanoparticles acting as a 2nd positive control. (C&D) Representative urine samples from patients infected with HIV and possible Toxoplasmic Encephalitis CBBA52 and IQ25. All patients that show a positive result for peptide VERPTGNPDLLK, demonstrate the presence of protein GRA1 in their respective urine samples. Representative sample CBBA52 has retention time (~8.3 min) corresponding to the tachyzoite positive control (A), while representative sample IQ-25 has retention time (~9.8 min) corresponding to the recombinant GRA1 positive control (B). The shift in retention times was due to a necessary change in the LC column. (E) The fragmentation ion key for ions b9, b7, b11, y9; each peak in part (C&D) corresponds to an ion of the documented color in E.
