All other reagents were from Sigma (Saint Quentin Fallavier,. France). Apo(a) quantifications were performed by LC-MS/MS, as follows. Standard solutions of the.
ONLINE APPENDIX SUPPLEMENTAL METHODS LC-MS/MS
quantification
of
apo(a)
-
Synthetic apo(a) peptides
LFLEPTQADIALLK,
GTYSTTVTGR, LFLEPTQADIALL[K(13C615N2)], and GTYSTTVTG[R(13C615N4)] were purchased from Thermo Fisher Scientific (Darmstadt, Germany); acetonitrile, water, and 99% formic acid from Biosolve (Valkenswaard, Netherlands). All other reagents were from Sigma (Saint Quentin Fallavier, France). Apo(a) quantifications were performed by LC-MS/MS, as follows. Standard solutions of the LFLEPTQADIALLK and GTYSTTVTGR peptides were prepared by serial dilutions in 0.5% sodium deoxycholate. Cell media, cell extracts, and standard solutions (150 µL) were mixed with 150 µL of a 50 mM ammonium bicarbonate buffer (pH 8) containing 2.5 nM of the LFLEPTQADIALL[K( 13C615N2)] or GTYSTTVTG[R(13C615N4)] labeled peptide as internal standards, respectively, 15 µL of 10% sodium deoxycholate, and 4 µL of 500 mM dithiothreitol. Samples were reduced at 60°C for 30 minutes and alkylated with 4 µL of fresh iodoacetamide solution (1 M in 1 M NaOH) for 60 minutes at room temperature in the dark. Samples were digested overnight with 15 µL of trypsin (0.1 mg/mL in 1 mM HCl); 15 µL of 20% formic acid was added to stop the reaction allowing sodium deoxycholate to precipitate. Samples were spun at 4°C for 15 minutes at 20,000 g. Supernatants (300 µL) were dried under nitrogen stream at room temperature, resuspended in 30 µL of an acetonitrile/water (5/95) mixture containing 0.1% formic acid, and transferred into glass vials. Measurements were performed on an LCMS/MS system consisting of a Xevo Triple-Quadrupole mass spectrometer equipped with Acquity HClass UPLC device (Waters Corporation, Milford, MA, USA). Data acquisition and analyses were performed using MassLynx and TargetLynx software. Peptides were separated on an Acquity BEH C18 column (2.1 × 100 mm, 1.7 µm, Waters) at 60°C with a linear gradient of mobile phase B (acetonitrile containing 0.1% formic acid) in mobile phase A (5% acetonitrile in water containing 0.1% formic acid) and at a flow rate of 600 µL/min. Mobile phase B was linearly increased from 1% to 50% for 5 minutes, kept constant for 1 min, returned to the initial condition in 1 min, and kept constant for 1 minute before the next injection. Ten microliters of each sample were injected into the LC column. Peptides were then detected by the mass spectrometer equipped with an electrospray interface operating in the positive ion mode at a capillary voltage of 4 kV. The multiple reaction-monitoring mode was applied for MS/MS detection with cone and collision voltages set at set at 55 V and 27 V (LFLEPTQADIALLK) or 35 V and 22 V (GTYSTTVTGR), respectively. Selected MRM transitions were (i) m/z 786.9 > 1069.7 (LFLEPTQADIALLK > PTQADIALLK) and 790.8 > 1077.7 (LFLEPTQADIALL[K(13C615N2)] > PTQADIALL[K(13C615N2)]) and (ii) m/z 522>721.6 (GTYSTTVTGR > STTVTGR) and 527.2 > 731.4 (GTYSTTVTG[R(13C615N4)] > STTVTG[R(13C615N4)]), for apo(a) proteotypic peptides and internal standards, respectively. Chromatographic peak area ratios between targeted peptides and internal standards constituted the detector responses. Standard solutions were used to plot calibration curves for quantification.
1
Supplemental Table 1: Dermal Fibroblasts Mutations Status Fibroblast Genotype Wild type NHDF HeHF MD136 GM03040 HoFH GM01915 defective SA260 YV134 NO4339 EW6421 AO5063 HoFH GM00488 negative GM00701 GM02000 TD176
LDLR mutation wild-type/wild type T315_G396del/wild type G197del/G197del C176F/D283N D154N/E80K D154N/V408M D206E/D154N D206E/V408M D206E/D206E C646Y/C646Y C660X/C660X E387K/E387K T315_G396del/T315_G396del
2
Supplemental Figure 1: Labeling of Lp(a) with bodipy does not alter its electrophoretic mobility. Human plasma and purified lipoprotein fractions [LDL and Lp(a)] were separated on agarose gels and stained for lipids with Sudan Black.
Supplemental Figure 2: PCSK9 does not modulate the uptake of Lp(a) in primary hepatocytes across a range of Lp(a) concentrations. Cells were treated with or without recombinant PCSK9 and incubated with increasing concentrations of Lp(a)-bodipy. Fluorescent Lp(a) uptake was measured. Also refer to legend of Figure 3.
3
Supplemental Figure 3: PCSK9 reduces LDL-bodipy but not Lp(a)-bodipy internalization. Primary human hepatocytes treated with or without recombinant PCSK9 and incubated with LDL-bodipy or Lp(a)-bodipy, were analyzed for lipoprotein uptake after three standard washes with PBS or with highly stringent buffers [three times with PBS-0.8% BSA, twice with PBS-0.8% BSA supplemented with 0.2 M epsilon aminocaproic acid, and twice with 0.2 M acetic acid/0.5 M NaCl solution (pH 2.5)]. * p
