Ziptip c18 tip

Protein content was measured by the Bradford method (Bio-Rad, Hercules, CA, USA). Firstly, 1 µL of plasma was diluted in 20 µL of a 1:1 ammonium bicarbonate 0.1/trifluoroethanol solution. For each 1 µg of protein content, 300 fmols of both heavy-isotope labelled peptides were added (SpikeTides^TM TQL: STELLIR for H3; LLLPGELAK for H2B were prepared as heavy-isotopically labelled proteotypic peptides that terminate with a C-terminal heavy Arg: ¹³C6,¹⁵N4; Lys: ¹³C6,¹⁵N2) (JPT Peptide Technologies, Berlin, Germany).
Then cysteine residues were reduced by adding 10 mM of D-L‐dithiothreitol at 56 °C for 30 min. The sulfhydryl groups were alkylated with 14 mM of iodoacetamide in the dark at RT for 30 min. The excess was neutralised with 10 mM of D-L‐dithiothreitol in 50 mM of ammonium bicarbonate at a final volume of 100 µL for 30 min at RT. Samples were subjected to trypsin digestion with 1 μg of sequencing grade-modified trypsin (TCPK Trypsin-ABSciex) overnight at 37 °C. The reaction was stopped with trifluoroacetic acid (TFA) at a final concentration of 1%. Samples were dried using speedvac and were resuspended in 50 µL of 0.1% TFA. Peptides were concentrated and purified using ZipTip C18 Tips (Merck Millipore, Darmstadt, Germany). Finally, samples were diluted in 2% acetonitrile (ACN) and 0.1% formic acid (FA) for injections.

After trypsin digestion, each peptides samples was desalted using a Strata X SPE column, dried, and resuspended in 25 μL 500 mM TEAB, and labeled with an 8-plex iTRAQ kit. Each dried and labeled peptide sample was reconstituted using High Performance Liquid Chromatography (HPLC) solution A (2% acetonitrile [can], pH 10) and fractionated by high-pH reverse-phase HPLC on a Waters Bridge Peptide BEH C18 (130 Å, 3.5 μm, 4.6 × 250 mm). Loaded peptides were eluted with 2% to 98% acetonitrile gradient buffer solution at pH 10 in 60 fractions at a speed of 0.5 ml/min over 88 min. A total of 20 fractions were combined and each fraction was desalted by using ZipTip C18 tips (Merck Millipore, Ziptip Pipette Tips 10μL). Sample fractions were dried on a vacuum concentrator and stored at -20°C pending MS analyses.

SFTI-derived peptides were synthesized and analyzed as described previously (29 (link)). Subsequently, cyclization experiments were carried out using 500 μm of the respective linear peptide and 0.5 μm AtLEGβ in a buffer composed of 100 mm NaCl and 50 mm Tris, Bis-Tris, citric acid, pH 4.0 or pH 6.0. Reactions were incubated at 30 °C for 12 h. Subsequently, the reactions were desalted using ZipTip C₁₈ tips (Merck Millipore) and analyzed by MALDI-TOF-MS (Autoflex, Bruker Daltonics, matrix, α-cyano-4-hydroxycin-namic acid).

The plasma (1 µL) was diluted in 20 µL of a 1:1 100 mM ammonium bicarbonate/trifluoroethanol solution. Cysteine residues were then reduced for 30 min at 56 °C by adding 10 mM D-L-dithiothreitol (DTT). Sulfhydryl groups were alkylated in the dark at RT for 30 min with 14 mM IAA and the excess IAA was neutralized for 30 min at RT with 10 mM DTT with 50 mMammonium bicarbonate in a final volume of 100 µL. The sample was digested overnight at 37 °C with 1 μg of sequencing grade-modified trypsin (TCPK Trypsin-ABSciex) and the reaction was stopped with TFA at a final concentration of 1%. Samples were dried in a Speed Vac, resuspended in 50 µL of TFA (0.1%), and the peptides were concentrated and purified using ZipTip C18 Tips (Merck Millipore, Darmstadt, Germany). Finally, the samples were spiked with a mix of heavy-labeled peptides in 2% ACN and 0.1% FA for injection.

Microbial fractions were extracted by multiple steps of centrifuge/resuspension as detailed in a previous protocol [24 (link)]. Then, the recovered microbial cells were subjected to the extraction of the metaproteomes and in-solution trypsin (Promega, MA, USA) digestion as per previously employed procedures [57 (link)].
The recovered peptides were purified and desalted using Zip-Tip C18 tips (Millipore, Billerica, MA, USA) and dried in a SpeedVac (Eppendorf, Milan, Italy) until the LC-MS/MS measurements.

0.1 U SVP or 0.005 U BSP enzymes were applied to 200 pmol DNA/Pt adduct for 5 min at room temperature and then enzyme was deactivated though incubation at 95 °C for 5 mins. DNA in the solution was recovered by Ziptip C18 tips (Milipore, USA), and eluted by the matrix mixture for MALDI-TOF MS.