Pierce peptide desalting spin columns

HDL total protein concentration was determined by using the BradfordUltra test, following the manufacturer's instructions. Bovine serum albumin was used as standard. Briefly, 3 µg of isolated HDL were diluted in 300 μL of digestion buffer (50 mM ammonium bicarbonate) and then reduced with 20 mM DTT for 20 min at 60 °C before alkylation with 40 mM iodoacetamide (30 min at room temperature in the dark). Samples were subjected to proteolytic digestion with 3 µg of trypsin-tosylphenylalanyl chloromethyl ketone (Sigma) overnight at 37 °C. Digested peptides were acidified with 20% trifluoroacetic acid. After 15 min at 4 °C, peptides were collected by centrifugation at 10,000×g for 15 min at 10 °C and desalted using Pierce peptide desalting spin columns (Thermo Fisher Scientific, part no. 2162704). Eluted peptides were dried in a Speed Vacuum before being concentrated and purified again using C18 Tips (Thermo Fischer Scientific, part no. 87782). Peptides were eluted with 20 µL of 40% acetonitrile in 0.1% TFA and dried under vacuum prior to LC-MS/MS analysis.

Samples were fully resuspended in 50 µL of 10-mM DTT in 8-M urea and incubated in a thermomixer for 1 h at 37 °C. A total 50 µL of alkylation reagent mixture (97.5% acetonitile (ACN), 0.5% triethylphosphine, 2% iodoethanol) was added to each sample, and again incubated for 1 h in a thermomixer at 37 °C. After alkylation, samples were dried in a vacuum centrifuge and resuspended in 200 µL of 0.05 µg/uL Lys-C/Trypsin (Promega) dissolved in 25-mM ABC. Samples were transferred to a barocycler (50 °C, 60 cycles; 50 s at 20 kPSI and 10 s at 1 ATM), in which proteolysis was carried out. Peptides were desalted with the Pierce Peptide Desalting Spin Columns (Thermo Fisher Scientific, USA). A total of 20 μg of peptides from each sample were saved for global analysis. The remainder was used for phosphopeptide enrichment, performed with PolyMac spin tips (Tymora Analytical, West Lafayette, IN, USA), following manufacturer’s recommendations.

Phosphopeptides were enriched automatically using magnetic immobilized metal affinity chromatography (IMAC) beads (ReSyn Biosciences, South Africa) using a Qiagen BioSprint 96 (Qiagen, Germany) magnetic particle handler. For each sample, four aliquots of peptides from 500 μg of digested protein were processed in parallel—two with Ti-IMAC HP beads and two with Zr-IMAC HP beads—and the resulting phosphopeptides were combined. Briefly, the manufacturer’s protocol was followed: desalted and dried peptides from 500 μg of digested protein were resuspended in load buffer (1.0 M glycolic acid–80% ACN–5% trifluoroacetic acid [TFA; Optima LC/MS grade, Fisher Chemical, USA] for Ti-IMAC HP beads, 0.1 M glycolic acid–80% ACN–5% TFA for Zr-IMAC HP beads) and incubated with beads. Beads were washed in load buffer, then 80% ACN–1% TFA, and then 10% ACN–0.2% TFA, and then eluted with 2% (vol/vol) ammonium hydroxide. The eluted peptide solution was acidified with TFA, desalted on Pierce peptide desalting spin columns (Thermo Fisher Scientific no. 89851), and dried in a vacuum concentrator.

The sample was reduced with DL-dithiothreitol (DTT, 100 mM) at 60°C for 30 min and digested with trypsin using a modified filter-aided sample preparation (FASP) method [74 (link)]. In short, the reduced sample was transferred to a Microcon-30 kDa centrifugal filter (Merck), then washed repeatedly with 8 M Urea, 50 mM triethylammonium bicarbonate (TEAB) and once with digestion buffer (0.5% sodium deoxycholate (SDC), 50 mM TEAB). The reduced cysteine side chains were alkylated with 10 mM methyl methanethiosulfonate (MMTS) in the digestion buffer for 30 min at room temperature. The sample was repeatedly washed with a digestion buffer and digested with trypsin (0.2 μg, Pierce MS grade Trypsin, Thermo Fisher Scientific) at 37°C overnight. An additional portion of trypsin (0.2 μg) was added and incubated for another 3 h the next day. The peptides were collected by centrifugation, and SDC was removed by acidification with 10% trifluoroacetic acid. The sample was purified using High Protein and Peptide Recovery Detergent Removal Spin Column (Thermo Fisher Scientific) and Pierce peptide desalting spin columns (Thermo Fisher Scientific) according to the manufacturer’s instructions. The purified peptide sample was dried on a vacuum centrifuge and reconstituted in 3% acetonitrile and 0.2% formic acid for the LC-MS/MS analysis.

Sample volumes corresponding to 25 µg of total protein of each sample were digested using single-pot, solid-phase enhanced sample preparation (SP3). Briefly, the reduced (10 mM DTT for 1 h at 56 °C) and alkylated (55 mM iodoacetamide (IAA) for 30 min at RT) proteins were bound to SP3 beads (10:1 bead:protein ratio, GE Healthcare, Chicago, IL, USA), washed with 80% ethanol and acetonitrile, and subjected to on-bead digestion with trypsin/LysC Mix (1:25 protease:protein ratio, Promega, Madison, WI, USA) overnight at 37 °C in 50 mM ammonium bicarbonate, pH 8.5. After elution, peptides were desalted using Pierce Peptide Desalting Spin Columns (Thermo Fisher Scientific, Waltham, MA, USA) according to the manufacturer’s instructions. The eluates were dried in a vacuum concentrator and reconstituted in 0.1% trifluoroacetic acid.

Dried samples were fully resuspended in 60 µL 8 M urea, 10 mM DTT and incubated at 37 °C for 1 h. 60 µL of alkylation reagent mixture (97.5% Acetonitile (ACN), 0.5% Triethylphosphine, 2% Iodoethanol) was added to samples, which were again incubated at 37 °C for 1 h. Samples were dried in a vacuum centrifuge and resuspended in 200 µL of 0.05 µg/uL Lys-C/Trypsin (Promega, WI, USA) in 25 mM Ammonium Bicarbonate. Proteolysis was carried out using a barocycler (50 °C, 60 cycles: 50 s at 20 kPSI and 10 s at 1 ATM). Digested samples were desalted with Pierce Peptide Desalting Spin Columns (Thermo Fisher Scientific, IL, USA). Phosphopeptide enrichment was subsequently performed using PolyMac spin tips (Tymora Analytical, IN, USA), following manufacturer’s recommendations.