title: Development of a Protein Standard Absolute Quantification (PSAQ™) assay for the quantification of Staphylococcus aureus enterotoxin A in serum.
abstract: Enterotoxin A (SEA) is a staphylococcal virulence factor which is suspected to worsen septic shock prognosis. However, the presence of SEA in the blood of sepsis patients has never been demonstrated. We have developed a mass spectrometry-based assay for the targeted and absolute quantification of SEA in serum. To enhance sensitivity and specificity, we combined an immunoaffinity-based sample preparation with mass spectrometry analysis in the selected reaction monitoring (SRM) mode. Absolute quantification of SEA was performed using the PSAQ™ method (Protein Standard Absolute Quantification), which uses a full-length isotope-labeled SEA as internal standard. The lower limit of detection (LLOD) and lower limit of quantification (LLOQ) were estimated at 352pg/mL and 1057pg/mL, respectively. SEA recovery after immunocapture was determined to be 7.8±1.4%. Therefore, we assumed that less than 1femtomole of each SEA proteotypic peptide was injected on the liquid chromatography column before SRM analysis. From a 6-point titration experiment, quantification accuracy was determined to be 77% and precision at LLOQ was lower than 5%. With this sensitive PSAQ-SRM assay, we expect to contribute to decipher the pathophysiological role of SEA in severe sepsis. This article is part of a Special Issue entitled: Proteomics: The clinical link.
authors.affiliation: CEA, IRTSV, Biologie à Grande Echelle, F-38054 Grenoble, France.
authors.forename: Annie
authors.initials: A
authors.lastname: Adrait
authors.forename: Dorothée
authors.initials: D
authors.lastname: Lebert
authors.forename: Mathieu
authors.initials: M
authors.lastname: Trauchessec
authors.forename: Alain
authors.initials: A
authors.lastname: Dupuis
authors.forename: Mathilde
authors.initials: M
authors.lastname: Louwagie
authors.forename: Christophe
authors.initials: C
authors.lastname: Masselon
authors.forename: Michel
authors.initials: M
authors.lastname: Jaquinod
authors.forename: Benoît
authors.initials: B
authors.lastname: Chevalier
authors.forename: François
authors.initials: F
authors.lastname: Vandenesch
authors.forename: Jérôme
authors.initials: J
authors.lastname: Garin
authors.forename: Christophe
authors.initials: C
authors.lastname: Bruley
authors.forename: Virginie
authors.initials: V
authors.lastname: Brun
mesh_terms: D000595:Amino Acid Sequence; D001774:Blood Chemical Analysis / Q000379:methods* / Q000592:standards*; D004768:Enterotoxins / Q000032:analysis* / Q000097:blood* / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D006801:Humans; D007120:Immunochemistry / Q000379:methods / Q000592:standards; D013058:Mass Spectrometry / Q000379:methods / Q000592:standards; D008954:Models, Biological; D010446:Peptide Fragments / Q000032:analysis / Q000737:chemistry; D011506:Proteins / Q000032:analysis / Q000737:chemistry; D040901:Proteomics / Q000379:methods / Q000592:standards; D012015:Reference Standards; D012680:Sensitivity and Specificity; D013203:Staphylococcal Infections / Q000097:blood* / Q000175:diagnosis / Q000378:metabolism / Q000382:microbiology; D013211:Staphylococcus aureus / Q000302:isolation & purification / Q000378:metabolism*
chemical_list: D004768:Enterotoxins; D010446:Peptide Fragments; D011506:Proteins; C026066:enterotoxin A, Staphylococcal