title: Identification of free phosphopeptides in different biological fluids by a mass spectrometry approach.
abstract: Human body fluids have been rediscovered in the post-genomic era as a great source of biological markers and perhaps as source of potential biomarkers of disease. Recently, it has been found that not only proteins but also peptides and their modifications can be indicators of early pathogenic processes. This paper reports the identification of free phosphopeptides in human fluids using an improved IMAC strategy coupled to iterative mass spectrometry-based scanning techniques (neutral loss, precursor ion, multiple reaction monitoring). Many peptides were detected in the enriched extract samples when submitted to the MS-integrated strategy, whereas they were not detected in the initial extract samples. The combination of the IMAC-modified protocol with selective "precursor ion" and constant "neutral loss" triple quadrupole scan modes confers a high sensitivity on the analysis, allowing rapid phosphopeptide identification and characterization, even at low concentrations. To the best of our knowledge this work represents the first report exclusively focused on the detection of free phosphorylated peptides in biological fluids.
authors.affiliation: Department of Organic Chemistry and Biochemistry, Federico II University of Naples, 80126, Naples, Italy.
authors.forename: Claudia
authors.initials: C
authors.lastname: Cirulli
authors.forename: Giovanni
authors.initials: G
authors.lastname: Chiappetta
authors.forename: Gennaro
authors.initials: G
authors.lastname: Marino
authors.forename: Pierluigi
authors.initials: P
authors.lastname: Mauri
authors.forename: Angela
authors.initials: A
authors.lastname: Amoresano
mesh_terms: D000595:Amino Acid Sequence; D002364:Caseins / Q000032:analysis / Q000737:chemistry; D011232:Chemical Precipitation; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D029968:Escherichia coli Proteins / Q000032:analysis / Q000737:chemistry; D005340:Fibrinogen / Q000032:analysis / Q000737:chemistry; D006801:Humans; D036103:Nanotechnology / Q000379:methods; D010446:Peptide Fragments / Q000032:analysis / Q000737:chemistry; D010748:Phosphopeptides / Q000032:analysis* / Q000097:blood / Q000378:metabolism / Q000652:urine; D012463:Saliva / Q000737:chemistry* / Q000378:metabolism; D020539:Sequence Analysis, Protein / Q000379:methods; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods; D053719:Tandem Mass Spectrometry / Q000379:methods*
chemical_list: D002364:Caseins; D029968:Escherichia coli Proteins; D010446:Peptide Fragments; D010748:Phosphopeptides; C114674:alpha-casein (142-149); C464794:fibrinogen Aalpha; D005340:Fibrinogen