Figure 1.
The proteolytic 18O labeling procedure uses a single tube.
*The photoreceptor OS membrane protein was prepared by precipitation with acetone to remove the lipids from the plasma and disc membranes, but depending on the nature of sample, this step may not be required. If acetone precipitation is not required, the excess of the TEP/IETH can be removed by speed-vac at 45°C. ** After every reconstitution step the sample was sonicated in a water bath for 10 sec. Formic acid can also be used instead of TFA. ***100 mM ammonium formate buffer (pH 6) can also be used. TEP (triethylphosphene), IETH (iodoethanol), ABC (ammonium bicarbonate), PFOA (perfluorooctanoic acid), ETAC (ethylacetate), EtOH (ethanol), TFA (trifluoroacetic acid), and NEM-AA (n-ethyl morpholine-acetic acid).
Table 1.
Solubilization of membrane proteins by different reagents.
Figure 2.
Different solubilizing agents affected the activity of trypsin.
The amidase activity of trypsin was measured by monitoring the initial rate of the hydrolysis of Ac-Lys-p-nitroanilide. Each line in the graph represents the concentration dependent decrease in tryptic activity in the presence of various concentrations of PFOA (◊), SDS (Δ), urea (○), and Gdn-HCl (x).
Figure 3.
Total ion current chromatograms of the tryptic digest of OS membrane proteins.
(A) Residual PFOA in the tryptic digest of OS membrane proteins quantified by flow injection analysis. Different concentrations of the PFOA standard solution (50–350 pg) and samples in 1 µL of 0.1% formic acid and 50% acetonitrile were injected into a flowing carrier stream consisting of 0.1% formic acid and 50% acetonitrile at 40 µL/min that was directly connected to a mass spectrometer (QStar, Applied Biosystems, Foster City, CA) equipped with an electrospray ion source. The (M–H)− ion (m/z 413) of PFOA was monitored. An aliquot of the digest was analyzed by flow injection analysis after three cycles of evaporation in ethanol∶ethylacetate∶water∶TFA (0.33∶0.33∶0.33∶0.01, v/v/v/v) at 25°C in a speed-vac concentrator under low pressure of <10 mTorr (Sample 2). The remaining digest was subjected to another three cycles of evaporation in ethanol∶ethylacetate∶water∶TFA (0.33∶0.33∶0.33∶0.01, v/v/v/v) at 60°C in a speed-vac concentrator under atmospheric pressure of 760 Torr (Sample 1). (B) 1 µg of the digest from sample 2 analyzed by LC-MS/MS. (C) 1 µg of the digest from sample 1 analyzed by LC-MS/MS.
Figure 4.
Plots compare the intensities of 16O- and 18O-labeled peptides.
Linear regression analysis was performed on a total of 377 peptides. The equations and R2 values for the regression line are shown.