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Supplemental Methods
Kinetic Study Infusion Protocol
Apolipoprotein kinetics were measured under constantly-fed conditions as previously
described (3). Briefly, subjects were fed a standardized meal representing 1/20 th of their
daily caloric intake hourly for 20 hours. After the initial 5 hours of feeding, participants
were given a bolus of [5,5,5-D3]-leucine (D3-leucine) (10 µmol/kg) immediately followed
by a constant infusion of D3-leucine (10 µmol/kg per hour) for the remaining 15 hours.
Blood samples were collected at selected time points during the infusion period.
Plasma Lipoprotein Fraction Preparation by Ultracentrifugation
Lipoprotein fractions were isolated by sequential ultracentrifugation. Briefly, plasma (5
mL) was transferred to Quick-Seal tubes (BeckmanCoulter) and centrifuged at 32,000
rpm in a Ti50.4 rotor. The upper fraction containing a mixture of the triglyceride-rich
lipoproteins (TRL) chylomicrons and VLDL with density < 1.006 g/mL was isolated by
tube slicing. The infranatant was adjusted to density = 1.019 g/mL with KBr, transferred
to Quick-Seal tubes (BeckmanCoulter) and centrifuged at 32,000 rpm in a Ti50.4 rotor
(110,000 RCF) to isolate the IDL faction (d =1.006-1.019 g/mL). Similarly, the LDL
(density = 1.019-1.063 g/mL) and HDL (density = 1.063 -1.21 g/mL) fractions by using
18 hr and 48 hour centrifugation times, respectively.
Tracer Enrichment Measurement by GC-MS
VLDL apoB100 was isolated from the TRL fraction and apoA-I was isolated from the
HDL fraction (density = 1.063-1.21 g/mL) using SDS-PAGE followed by staining with
Coomassie Blue R250, and analysis of the free amino acids by GC-MS as previously
2
described (8). Briefly, proteins were identified by comparison to standards of known
molecular weight. Bands of interest were cut from gels using a scalpel and transferred
to 4 mL glass vials with Teflon-lined caps. The protein bands were hydrolyzed with 6 N
HCl at 100° C for 24 hr.
Free amino acids were isolated from dried apoA-I and apoB100 protein hydrolysates by
cation exchange chromatography (AG-50W-X8, Bio-Rad Laboratories, Richmond, CA).
Recovered amino acids were derivatized to the N-heptafluorobutyryl n-propyl esters,
dissolved in ethyl acetate, and analyzed by a GC-MS (Agilent 5973 mass spectrometer
coupled to an Agilent 6890 gas chromatograph) in the methane-negative chemical
ionization mode with selective ion monitoring at m/z 349 and 352 (M+0, M+3 isotopomer
ions, respectively). Tracer enrichments ratio was determined by regression analysis of
the instrument measured isotopomer ratio against standards of known enrichments ratio
(4).
Automated In-gel Digestion
Protein gel bands were excised into 96-well plates and processed using an automated
in-gel digestion protocol developed on a TECAN Freedom EVO automated liquid
handler with EVOware standard software.
Briefly, gel plugs were destained in 1:1
acetonitrile:100 mM AMBIC (pH 8), dehydrated in 100% acetonitrile, reduced in 10 mM
DTT for 30 minutes at 40°C, and alkylated in 55 mM iodoacetamide for 30 minutes at
room temperature. The samples were then washed in 1:1 acetonitrile:100 mM AMBIC,
dehydrated with 100% acetonitrile and incubated in a 5 ng/µL solution of sequencing
3
grade trypsin (Promega) at 37°C overnight. Digested peptides were then extracted two
times with a 5% formic acid, 50% acetonitrile solution followed by one extraction with
100% acetonitrile. Extracted peptides were dried down and reconstituted in 10 µL 0.1%
formic acid prior to MS analysis.
Standard dilution of synthetic peptide DYVSQFEGSALGK in complex background.
Digested bovine serum albumin was obtained from Michrom Bioresources (Auburn, CA)
and reconstituted as per manufacturer’s instructions to 100 fmol/µL. Synthetic peptide,
DYVSQFEGSALGK 10 nmoles (lyophilized) obtained from Ana Spec (Freemont, CA)
was reconstituted in 50 µL glacial Acetic acid + 950 µL solvent "A" (0.1.% Formic acid)
to a final concentration of 10 pmol/µL. A 2000 fmol/µL stock dilution solution was made
by adding 80 µL of 100 fmol digested BSA, and 20 µL from 10 pmol/µL synthetic
peptide. Dilution series is as follows:
Dilution#
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
Final Synthetic
Peptide
Concentration
(fmol/µL)
2000.00
1000.00
500.00
250.00
125.00
62.50
31.25
15.62
7.81
3.90
1.95
0.97
0.48
0.24
0.12
0.06
0.03
100
fmol/µL
BSA
(µL)
80
50
50
50
50
50
50
50
50
50
50
50
50
50
50
50
50
Synthetic peptide added
20 µL 10 pmol/µL
50 µL from 2000 fmol/µL
50 µL from 1000 fmol/µL
50 µL from 500 fmol/µL
50 µL from 250 fmol/µL
50 µL from 125 fmol/µL
50 µL from 62.50 fmol/µL
50 µL from 31.25 fmol/µL
50 µL from 15.62 fmol/µL
50 µL from 7.81 fmol/µL
50 µL from 3.90 fmol/µL
50 µL from 1.95 fmol/µL
50 µL from 0.97 fmol/µL
50 µL from 0.48 fmol/µL
50 µL from 0.24 fmol/µL
50 µL from 0.12 fmol/µL
50 µL from 0.06 fmol/µL
Final
Volume
(µL)
50
50
50
50
50
50
50
50
50
50
50
50
50
50
50
50
100
4
Analysis by Liquid Chromatography and Mass Spectrometry.
Recovered mixtures of native and metabolically labeled tryptic peptides were analyzed
by LC-MS/MS using a Quantum™ triple quadruple mass spectrometer (Thermo-Fisher)
fitted with a microanalytical HPLC (Agilent) and conventional electrospray ionization
source. The recovered peptides for ApoA-I, ApoB, ApoE, ApoC1, ApoC2, ApoC3, and
PCSK9 were resolved by reverse-phase chromatography on a 1 x 50 mm Acquity UPLC
BEH column packed with 1.7 µm C18 particles (Waters) that was maintained at 50ºC.
Sample injection volumes varied from 2-8 µL. Peptides were eluted at a flow rate of 40
µL/min, with linear gradients of 0-65% acetonitrile and 0.1% formic acid, during 10 min,
followed by 5 min at 95% of acetonitrile and 0.1% formic acid. Peptides were ionized
using a 4000V needle potential and 300º C capillary temperature.
Tandem mass
spectrometry data was acquired using Argon as the collision gas at an uncalibrated cell
pressure of 1.5 mTorr.
Selected reaction monitoring was used to acquire ion
chromatograms for the labeled and unlabeled peptides using the precursor and product
ion transitions shown Supplemental Table 1.
The resolution of the first and third
quadrupole mass filter was set to 0.7 FWHM and an electron multiplier gain of 2x106
was used for all analytes. Chromatographic peak data were integrated using LC-Quan
(Thermo-Fisher). The recovered peptides for CETP were analyzed separately using a
modified chromatographic method that employed a 1 x 150 mm MC-15-C18WSS320EU column (MicroTech), an 8 µL/min flow rate and a linear gradient from 0-80% of
acetonitrile and 0.1% formic acid in 13 minutes. A sample injection volume of 5 µL and
275º C capillary temperature was used for the analysis of CETP peptides.