title: iFASP: combining isobaric mass tagging with filter-aided sample preparation.
abstract: Careful, clean and controlled preparation of samples for mass spectrometry proteomics is crucial to obtain reproducible and reliable data. This is especially important when carrying out quantitative proteomics by chemical isobaric labeling (aka tandem mass tagging), since the differentially labeled samples are combined quite late during the sample processing. Addressing this need for robust and reliable sample processing for quantitative proteomics, we describe here iFASP, a simple protocol for combining isobaric mass tagging with the recently introduced filter-aided sample preparation (FASP) method. iFASP provides a quick, simple and effective method for obtaining clean samples, ensuring efficient digestion and providing excellent labeling yields for quantitative proteomics experiments. We have carried out our iFASP protocol using several highly complex Xenopus laevis egg and embryo lysates and compared the labeling yields and number of high-confidence peptide identifications to a standard in-solution digestion and labeling protocol. Although the labeling efficiency with both techniques is in the 99+% range, the number of peptides identified with a 1% false discovery rate and the corresponding number of quantified peptide spectral matches are as much as doubled with iFASP compared to the corresponding non-FASP-based method.
authors.affiliation: Proteomics Center and Department of Pathology, Boston Children's Hospital, Boston, Massachusetts 02115, USA.
authors.forename: Gary S
authors.initials: GS
authors.lastname: McDowell
authors.forename: Aleksandr
authors.initials: A
authors.lastname: Gaun
authors.forename: Hanno
authors.initials: H
authors.lastname: Steen
mesh_terms: D029845:Amphibian Proteins / Q000737:chemistry* / Q000302:isolation & purification; D000818:Animals; D002853:Chromatography, Liquid; D004625:Embryo, Nonmammalian / Q000737:chemistry*; D005260:Female; D005374:Filtration; D008970:Molecular Weight; D040901:Proteomics / Q000379:methods*; D013194:Staining and Labeling / Q000379:methods*; D053719:Tandem Mass Spectrometry; D014982:Xenopus laevis / Q000378:metabolism; D015053:Zygote / Q000737:chemistry*
chemical_list: D029845:Amphibian Proteins