title: abFASP-MS: affinity-based filter-aided sample preparation mass spectrometry for quantitative analysis of chemically labeled protein complexes.
abstract: Affinity purification coupled to 1-D gel-free liquid chromatography mass spectrometry (LC-MS) is a well-established and widespread approach for the analyses of noncovalently interacting protein complexes. In this study, two proteins conjugated to a streptavidin-binding peptide and hemagglutinin double tag were expressed in the respective Flp-In HEK293 cell lines: green fluorescent protein (SH-GFP) and TANK binding kinase 1 (SH-TBK1_MOUSE). Fluorescent anti-HA immunoblots revealed that the expression level of SH-GFP was ∼50% lower than that of SH-TBK1_MOUSE. Subsequently, the input material was normalized to obtain a similar quantity of purified SH-tagged proteins. Optimization of the release of protein complexes from the anti-HA-agarose with different eluting agents was then assessed. With respect to the total number of protein groups identified in the purified complexes, elution with 2% SDS surpassed both 100 mM glycine and 100 mM formic acid. Relative quantitation of the purified protein complexes using TMT 6-plex reagents confirmed the higher efficiency of the 2% SDS elution followed by filter-aided sample preparation (FASP). The data presented in this study provide a new application of FASP to quantitative MS analysis of affinity-purified protein complexes. We have termed the approach abFASP-MS, or affinity-based filter-aided sample preparation mass spectrometry.
authors.affiliation: CeMM Research Center for Molecular Medicine of the Austrian Academy of Sciences , Lazarettgasse 14, 1090 Vienna, Austria.
authors.forename: Marie L
authors.initials: ML
authors.lastname: Huber
authors.forename: Roberto
authors.initials: R
authors.lastname: Sacco
authors.forename: Katja
authors.initials: K
authors.lastname: Parapatics
authors.forename: Anna
authors.initials: A
authors.lastname: Skucha
authors.forename: Kseniya
authors.initials: K
authors.lastname: Khamina
authors.forename: André C
authors.initials: AC
authors.lastname: Müller
authors.forename: Elena L
authors.initials: EL
authors.lastname: Rudashevskaya
authors.forename: Keiryn L
authors.initials: KL
authors.lastname: Bennett
mesh_terms: D015153:Blotting, Western; D002846:Chromatography, Affinity; D056148:Chromatography, Reverse-Phase; D004591:Electrophoresis, Polyacrylamide Gel; D011506:Proteins / Q000032:analysis* / Q000737:chemistry; D053719:Tandem Mass Spectrometry / Q000379:methods*; D014357:Trypsin / Q000737:chemistry
chemical_list: D011506:Proteins; D014357:Trypsin