FIGURE 2.

MALDI-TOF analysis of purified fractions. Fractions corresponding to the elution peak of solubilized SERCA1a (see Fig. 1) were collected, and an aliquot of each was submitted to MS analysis. Spectra were acquired either in a large range of molecular masses (m/z from 3000 to 140,000 Da) or on a smaller range, from 2000–6000 Da (zoom in the 3600–4300 Da region in the left insets). Conditions for sample preparations, before (A) or after (B, C) hydroxylamine treatment are indicated on each spectrum. The precision on the mass determination is quite high considering the large range studied and the resulting difficulty to achieve an accurate calibration in these particular acquisition conditions: the experimental mass for SERCA1a is about 109,493.9 ± 93.1 Da for an expected mass of 109,532 Da, and the experimental mass for SLN is about 3772.0 ± 1.1 Da for an expected mass of 3774 Da (NB: in this figure and in all the other figures we report the masses of the protonated forms (M + H)⁺). Right inset of panel A: SR vesicles (lane 1), DOC-extracted SR vesicles (lane 2), SEC purified SERCA1a/SLN complex (lane 3), and the mixed micelles peak (lane 4) (V_e = 6.5–7.8 ml and V_e = 8.6–9.6 ml, respectively) were loaded on a TGX Stain-Free Precast 4–20% SDS-PAGE for in-gel fluorescence analysis (Bio-Rad). A band corresponding to SLN was visualized at an apparent molecular weight of 8–10 kDa as expected (see “Results”). In this experiment about 20–25 μg of protein has been applied in each lane. Under these conditions the Ca²⁺-ATPase band is oversaturated but SLN can easily be spotted. For quantitative comparison, dilutions of the same samples were loaded too (not shown). A profile analysis was done to estimate the intensity of fluorescence of SERCA1a and SLN, which respectively contain 13 and only one tryptophan residue. Treatment by hydroxylamine had no effect on the SERCA1a/SLN ratio (not shown).