Quantitative mass spectrometric analysis of C-terminal 36 amino acid peptides of alpha-1 antitrypsin in plasma using survey spectra

C-terminal peptides of alpha-1 antitrypsin (AAT) may serve as biomarkers for diseases such as sepsis, chronic obstructive pulmonary disease, liver disease, and autoimmune disorders. In this study, we present a robust and straightforward MS (mass spectrometry)-based method for quantifying AAT peptides 388–418 (C36) and its polymorphic variant (E400D, C36D) in plasma samples. Absolute quantification was accomplished using MALDI-MS reflectron spectra and ESI-MS MS1 scans, implemented in two independent laboratories. Two plasma preparation methods, methanol precipitation and ultrafiltration, were evaluated, with methanol precipitation yielding significantly higher recovery rates. The impact of freeze–thaw cycles on C36 levels was also assessed, revealing a significant increase in C36 levels after each cycle. Comparisons between MALDI-MS and ESI-MS showed strong concordance in C36 and C36D measurements. Furthermore, C36 and C36D levels correlated strongly with post-precipitation protein content across both MS methods. Normalizing C36 levels to protein content effectively mitigated variability. This method should be straightforward to implement in other laboratories, facilitating clinical studies to evaluate the diagnostic and prognostic significance of C36 peptides across various diseases.