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 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.