An immunoaffinity liquid chromatography-tandem mass spectrometry assay for the quantitation of matrix metalloproteinase 9 in mouse serum.
An immunoaffinity liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed for the quantitation of the zinc endopeptidase matrix metalloproteinase 9 (MMP-9) from mouse serum. Sample preparation for the assay included magnetic bead-based enrichment using an MMP-9 antibody and was performed in a 96-well plate format using a liquid-handling robotic platform. The surrogate peptide GSPLQGPFLTAR derived from MMP-9 by trypsin digestion was monitored using an on-line capillary flow trap-release chromatography setup incorporating a series of trap columns (C18, strong cation exchange, and another C18) prior to nanoflow chromatography and nanospray ionization with selected reaction monitoring (SRM) detection. The assay was fit-for-purpose validated and found to be accurate (<15% interbatch relative error) and precise (<15% interbatch coefficient of variation) across a range from 0.03 to 7.3nM mouse MMP-9. Finally, the method was employed to measure MMP-9 concentrations in 30 naïve mouse serum samples, and results were compared with those obtained by an immunoassay.
Ocaña
Mireia Fernández
MF
Experimental Biological Sciences, Pfizer, Kent CT13 9NJ, UK. mireia.fernandezocana@pfizer.com
Neubert
Hendrik
H
D000595:Amino Acid Sequence; D000818:Animals; D002853:Chromatography, Liquid; D018189:Immunomagnetic Separation / Q000379:methods*; D020780:Matrix Metalloproteinase 9 / Q000097:blood* / Q000302:isolation & purification; D051379:Mice; D010455:Peptides / Q000032:analysis; D053719:Tandem Mass Spectrometry / Q000379:methods*; D014357:Trypsin / Q000378:metabolism
D010455:Peptides; D014357:Trypsin; D020780:Matrix Metalloproteinase 9