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

作者：Huber Marie L; Sacco Roberto; Parapatics Katja; Skucha Anna; Khamina Kseniya; Mueller Andre C; Rudashevskaya Elena L^*; Bennett Keiryn L

来源：Journal of Proteome Research, 2014, 13(2): 1147-1155.

DOI：10.1021/pr4009892

• affinity purification
• orbitrap velos
• SDS elution
• normalization
• bait expression
• FASP
• quantitation
• TMT 6-plex

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 I (SH-TBK1_MOUSE). Fluorescent anti-HA immnnoblots revealed that the expression level of SH-GFP was similar to 50% lower than that of SH-TBK1_MOUSE. Subsequently, the input material was normalized to obtain 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.

• 出版日期2014-2
• 单位河北医科大学