TLC analysis of the hydrolysis of different OPs.

<p>A, Boc-Val-Leu-Lys-MCA (shOP1), Pro-Phe-Arg-MCA (shOP2), and Boc-Ile-Glu-Gly-Arg-MCA (shOP3) (5 mM) were incubated for 24 h without Abs (lanesC) and in the presence of 0.05 mg/ml MS IgG_mix or SLE IgG_mix preparations (lanes shown on the panel) demonstrating comparable relative activities in the hydrolysis of intact MBP. B, Nonspecific in-OP1 and in-OP2 were incubated for 24 h without Abs (lanes 1) and in the presence of 0.05 mg/ml SLE IgG_mix (lanes 2); lanes C correspond to in-OP1 and in-OP2 before incubation. C, Specific X-OP21 (C) and X-OP25 oligopeptides were incubated for 7 h with hd-IgG_mix (lane C) and in the presence of 0.02 mg/ml sle-IgG_mix. These OPs were used in different concentrations (mM): 0.05 (lanes 1), 0.1 (lanes 2), 0.2 (lanes 3), 0.3 (lanes 4), 0.4 (lanes 5), and 0.5 (lanes 6).</p

Complete sequence of human MBP (on the top) and all sites of cleavage of X-OP21 (C) and X-OP25 (F) determined using a combination of RPhC, TLC, and massspectrometry of detectable major and minor products of these OPs hydrolysis by sle-IgG_mix.

<p>The positions of OP21 and OP25 sequences in the human MBP sequence are shown in bold. Panels <b>A</b> and <b>D</b> show all possible sites of these OPs cleavage by trypsin, while panels <b>B</b> and <b>E</b> demonstrate the major cleavage sites of MBP, which were found previously in the case of hydrolysis of globular intact MBP by MS IgGs <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0051600#pone.0051600-Ponomarenko1" target="_blank">[31]</a>. All sites corresponding to major and moderate products of the cleavage are shown by long and short arrows respectively, while to minor ones by diamonds (panels <b>C</b> and <b>F</b>).</p

The data of RPhC, TLC, and MALDI analysis of molecular masses of fluorescent ligopeptides forming after incubation of X-OP25 with sle-IgG_mix.

*<p>Free fluorescent compound; all analyzed OPs contained fluorescent X-component.</p>**<p>The same products of the hydrolysis separated by RPhC (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0051600#pone-0051600-g005" target="_blank">Fig. 5A</a>) were revealed in several peaks by MALDI spectrometry; the main peaks are marked in bold, while additional peaks are shown in parentheses. C1 reflects the presence of signal corresponding to the analyzed product in the spectrum of total reaction mixture.</p>***<p>The same several products of the hydrolysis corresponding to each peak after RPhC (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0051600#pone-0051600-g005" target="_blank">Fig. 5A</a>) were revealed not only by MALDI spectrometry, but also by TLC (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0051600#pone-0051600-g005" target="_blank">Fig. 5B</a>)</p

The data of RPhC, TLC, and MALDI analysis of molecular masses of fluorescent oligopeptides forming after incubation of X-OP21 with sle-IgG_mix.

*<p>Free fluorescent compound; all analyzed OPs contained fluorescent X-component.</p>**<p>The same products of the hydrolysis separated by RPhC (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0051600#pone-0051600-g003" target="_blank">Fig. 3A</a>) were revealed in several peaks by MALDI spectrometry; the main peaks are marked in bold, while additional peaks containing low amount of the same OPs are shown in parentheses. C1 reflects the presence of signal corresponding to the analyzed product in spectrum of total reaction mixture.</p>***<p>The same several products of the hydrolysis corresponding to each peak after RPhC (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0051600#pone-0051600-g003" target="_blank">Fig. 3A</a>) were revealed not only by MALDI spectrometry, but also by TLC (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0051600#pone-0051600-g003" target="_blank">Fig. 3B</a>).</p>§<p>The relative content of different products after incomplete hydrolysis of X-OP21 was performed taking into account the data of TLC analysis (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0051600#pone-0051600-g003" target="_blank">Fig. 3B</a>); a range of the values from three repeats is given.</p

Determination of the <i>K_m</i> and <i>V</i>_max values for OP21 (A) and OP25 (B) in the reaction catalyzed by sle-IgG_mix (0.1 µM) using a Lineweaver–Burk plot.

<p>The reactions were performed as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0051600#s4" target="_blank">Materials and Methods</a>. The average error in the initial rate determination from two experiments for each substrate concentration did not exceed 7–10%.</p

Profile of RPhC of SLE IgG_mix-dependent products of X-OP21 relatively deep hydrolysis (A) and analysis of X-OP products of the hydrolysis corresponding to different peaks after RPhC by TLC (B) or massspectrometry (C–E): (–), relative fluorescence (A).

<p>Numbers of lines in panel B coincide with numbers of peaks on panel A; lanes C1, C2, and C3 correspond to the products of reaction mixture before their separation by RPhC, X-OP25 incubated in the absence of IgGs, and a free fluorescent compound X, respectively. The arrows (and numerals on panel B) indicate the positions of OPs of different length and compound X. Panels C, D, and E demonstrate the MALDI spectrum signals corresponding to the products eluted under RPhC in peaks 2, 5 and 6, respectively. See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0051600#s4" target="_blank">Materials and Methods</a> for other details.</p

Profile of RPhC of sle-IgG_mix-dependent products of a complete hydrolysis of X-OP21 (A) and analysis of the products corresponding to different peaks after RPhC by TLC (B) or massspectrometry (C–E): (–), relative fluorescence (A).

<p>Numbers of lines in panel B coincide with numbers of peaks on panel A; lane C1 corresponds to the products of reaction mixture before their separation by RPhC. The arrows (and numerals on panel B) indicate the positions of OPs of different length and compound X. Panels C, D, and E demonstrate the MALDI spectra corresponding to the products of the hydrolysis eluted under RPhC in peaks 2, 3, and 4, respectively (Fig. 4A). See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0051600#s4" target="_blank">Materials and Methods</a> for other details.</p

Gel filtration of the SPC proteins on a Sepharose 4B column (A) or Superdex 200 (B) corresponding to one placenta donor after the SPC treatment with buffer D with or without DTT (A and B): (—), absorbance at 280 nm (A₂₈₀).

<p>Fig. <b>C</b> shows gel filtration of the SPC on Superdex 200 after its previous proteins separation using Centricon membranes retaining molecules with MMs ≥100 kDa (bold line), with MMs 50–100 kDa (most bold line; the protein fraction passing-through membrane ≥100 kDa and then retained on membrane ≥50 kDa), and with MMs >10–30 kDa (thin line; the protein fraction passing-through membrane ≥30 and then proteins <10 kDa was partially removed from this fraction using membrane passing-through proteins with MMs <10 kDa). For details, see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0111234#s4" target="_blank">Materials and methods</a>.</p

SDS-PAGE analysis of the SPC proteins (30–40 µg) corresponding the extract of placenta of one donor using 3–16% gradient gel before (A) and after the SPC treatment with 50 mM DTT (B).

<p>The central fraction of SPC peak (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0111234#pone-0111234-g001" target="_blank">Fig. 1A</a>) was used. The arrows (lane C) indicate the positions of molecular mass markers. MALDI mass spectrometry spectra of all the SPC proteins corresponding to central part of its peak after gel filtration of untreated stable complex (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0111234#pone-0111234-g001" target="_blank">Fig. 1A</a>). See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0111234#s4" target="_blank">Materials and Methods</a> for other details.</p

MALDI mass spectrometry spectra of several SPC small proteins; fragments of the complete spectra corresponding to 20–25 ml fractions after 50–100 kDa SPC protein (treated with buffer D) fractionation on Superdex 200 (two individual fractions, 1 ml, were used; Fig. 3C).

<p>Panel A and B show possible modifications of several small proteins. See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0111234#s4" target="_blank">Materials and Methods</a> for other details.</p