abFASP-MS: Affinity-Based Filter-Aided Sample Preparation Mass Spectrometry for Quantitative Analysis of Chemically Labeled Protein Complexes

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 doub...

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Published in:Journal of proteome research 2014-02, Vol.13 (2), p.1147-1155
Main Authors: Huber, Marie L, Sacco, Roberto, Parapatics, Katja, Skucha, Anna, Khamina, Kseniya, Müller, André C, Rudashevskaya, Elena L, Bennett, Keiryn L
Format: Article
Language:English
Subjects:
Blotting, Western
Chromatography, Affinity
Chromatography, Reverse-Phase
Electrophoresis, Polyacrylamide Gel
Proteins - analysis
Proteins - chemistry
Tandem Mass Spectrometry - methods
Technical Note
Trypsin - chemistry
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Summary: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.
ISSN:1535-3893
1535-3907
DOI:10.1021/pr4009892