Quantification of matrix metalloprotease-9 in bronchoalveolar lavage fluid by selected reaction monitoring with microfluidics nano-liquid-chromatography–mass spectrometry

Quantitative protein analysis by liquid chromatography–tandem mass spectrometry (LC–MS/MS) in the selected reaction monitoring (SRM) mode was used to quantify matrix metalloprotease-9 (MMP-9; ∼90 kDa) in bronchoalveolar lavage fluid (BALF) from patients having undergone lung transplantation. We developed an SRM assay for microfluidics-based nanoLC–MS/MS on a triple quadrupole mass spectrometer based on two signature peptides. Samples were prepared by chloroform–methanol precipitation followed by trypsin digestion in the presence of stable-isotope-labeled internal peptide standards. The method allows accurate quantification of MMP-9 in BALF with an LLOQ of 2.9 ng/mL and an LLOD of 0.25 ng/mL without the use of extensive fractionation or antibodies.

► Highly sensitive and specific LC–MS/MS method for MMP-9 in BALF. ► First application of LC–MS/MS in the SRM mode for protein quantification in BALF. ► Accurate quantification at the amol level by microfluidics-nanoLC–MS/MS. ► Application to real-life samples from lung transplantation patients.