Sep pak c18 plus light cartridge

Parts of the gills (see SI appendix Supplementary Table S7) of three “B.” childressi specimen were used to prepare tryptic digests following the filter-aided sample preparation (FASP) protocol of Wisniewski et al. [102 (link)] with minor modifications [61 (link)]. Prior to FASP, cells were disrupted by beat-beating samples in SDT lysis buffer (4% (w/v) SDS, 100 mM Tris-HCl [pH 7.6], 0.1 M DTT) using lysing matrix D tubes (MP Biomedicals) before heating to 95 °C for 10 min.
To allow binding of peptides to the SCX column for 2D-LC methods, peptides were desalted using Sep-Pak C18 Plus Light Cartridges (Waters) according to the manufacturer’s instructions. A centrifugal vacuum concentrator was used to exchange acetonitrile after peptide elution with 0.2% (v/v) formic acid. The Pierce Micro BCA assay (Thermo Scientific) was used to determine peptide concentrations, following the manufacturer’s instructions.

Cells in mock community samples were disrupted by bead-beating (6.0 m/s, 45 s) in lysing matrix B tubes (MP Biomedicals) in SDT lysis buffer [4% (w/v) SDS, 100 mM Tris-HCl pH 7.6, 0.1 M DTT] followed by heating to 95°C for 10 min. Tryptic digests of protein extracts were prepared according to the filter-aided sample preparation (FASP) protocol described by Wiśniewski et al. (2009) (link). Peptides were desalted with Sep-Pak C18 Plus Light Cartridges (Waters). Acetonitrile from the peptide elution step was exchanged for 0.1% formic acid (v/v) using a centrifugal vacuum concentrator. The desalting step was necessary to enable binding of peptides to the SCX column during sample loading for the 2D-LC methods. Peptide concentrations were determined using the Pierce Micro BCA assay (Thermo Scientific Pierce) according to manufacturer’s instructions. The peptide mixture was aliquoted and frozen at −80°C. For mass spectrometric analyses, fresh aliquots were regularly thawed and formic acid concentration increased to 0.2% (v/v). All aliquots used here were prepared at the same time from the same peptide mixture to eliminate between-sample variation.

Reagents [(NH₄)₂CO₃; NH₄HCO₃; CH₃COONH₄; CH₃COOH; NH₃ solution in H₂O; D₂O; TCTU; DIEA; TIS] and solvents (MeCN, DMF; DCM; MeOH) were purchased from Sigma–Aldrich (St. Louis, MO, USA) and used without further purification. SepPak C18 Plus Light cartridges (Waters Corp., Milford, MA, USA) were used for desalting (parameters: 130 mg sorbent per cartridge, 55–105 μm particle size). Formic acid (99 %) was purchased from Merck (Darmstadt, Germany).