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.

Auxin is one of the crucial regulators of plant growth and development. The discovered auxin cytosolic receptor (TIR1) is not involved in the perception of the hormone signal at the plasma membrane. Instead, another receptor, related to the ABP1, auxin binding protein1, is supposed to be responsible for the perception at the plasma membrane. One of the fast and sensitive auxin-induced reactions is an increase of Ca²⁺ cytosolic concentration, which is suggested to be dependent on the activation of Ca²⁺ influx through the plasma membrane. This investigation was carried out with a plasmalemma enriched vesicle fraction, obtained from etiolated maize coleoptiles. The magnitude of Ca²⁺ efflux through the membrane vesicles was estimated according to the shift of potential dependent fluorescent dye diS-C₃-(5). The obtained results showed that during coleoptiles ageing (3rd, 4th and 5th days of seedling etiolated growth) the magnitude of Ca²⁺ efflux from inside-out vesicles was decreased. Addition of ABP1 led to a recovery of Ca²⁺ efflux to the level of the youngest and most sensitive cells. Moreover, the efflux was more sensitive, responding from 10⁻⁸ to 10⁻⁶ M 1-NAA, in vesicles containing ABP1, whereas native vesicles showed the highest efflux at 10⁻⁶ M 1-NAA. We suggest that auxin increases plasma membrane permeability to Ca²⁺ and that ABP1 is involved in modulation of this reaction.