Mass spectrometry-based relative quantification of human neutrophil peptides 1, 2, and 3 from biological samples.
Human neutrophil peptides (HNPs) are cysteine-rich antimicrobial peptides stored in neutrophils. The similar structure of HNPs -1, -2, and -3 renders them impossible to study individually in biological samples. For the first time, we describe a method of individually identifying the HNPs -1-3 from exudative neutrophils using matrix-assisted laser desorption ionization/time-of-flight (MALDI-TOF) mass spectrometry, and we demonstrate the ability to quantify the relative changes in the peptides found in biological samples. The study includes tracheal aspirates (TA) from infants with respiratory syncytial virus (RSV) illness at intubation for respiratory failure (acute illness) and at extubation (convalescence). In vitro, convalescent and acute illness TAs are labeled with d0- and d3-acrylamides, respectively, and mixed 1:1. TA proteins are separated by one-dimensional gel electrophoresis and then identified by mass spectrometry-based peptide mass fingerprinting. The ratio of signal intensities for the isotopically normal (d0-labeled) and heavy (d3-labeled) forms of the peptide reveals the relative increase in each peptide with illness.
Thompson
Linda
L
Department of Pediatrics, Critical Care Medicine Section, Baylor College of Medicine, 6621 Fannin Rd WT6-006, Houston, TX 77030, USA. lmthomps@texaschildrenshospital.org
Turko
Illarion
I
Murad
Ferid
F
D000208:Acute Disease; D000595:Amino Acid Sequence; D006801:Humans; D007223:Infant; D008969:Molecular Sequence Data; D009504:Neutrophils / Q000276:immunology; D018357:Respiratory Syncytial Virus Infections / Q000276:immunology; D018113:Respiratory Syncytial Virus, Human; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods*; D014357:Trypsin; D023084:alpha-Defensins / Q000032:analysis* / Q000235:genetics
D023084:alpha-Defensins; C406413:human neutrophil peptide 1; C406416:human neutrophil peptide 2; C406427:human neutrophil peptide 3; D014357:Trypsin