Efficient Strategy for Screening Chemical Modifications on Human Serum Albumin: Use of LC/MS/MS and Differential Analysis Software

Chemical modifications on proteins can provide significant information about chemical stresses during some physiological events. In this study, we have demonstrated a comprehensive LC/MS/MS-based strategy for screening various chemical modifications of human serum albumin (HSA), the most abundant blood plasma protein. A complementary use of different proteases (trypsin and Glu-C), LC columns (C18 and HILIC), and ion detection modes (positive and negative) improved the number of detected peptides, sequence coverage, and identification of the target modifications. A database search using Mascot or Proteome Discoverer revealed 16 and 13 modifications from HSAs treated by in vitro oxidation and nitration, respectively. A commercially available albumin depletion kit was used to clean up HSA from pooled plasma. The database analysis enabled it to find 7 types of 36 modification sites, including Met⁸⁷, Trp²¹⁴, and Cys³⁴ oxidation, and the glycation of Lys⁵²⁵. The differential analysis between the plasma sample and HSA standard using XCMS online revealed that 13 peptides had more than 3.5-fold changes with p < 0.05.