μziptipc18 pipette tips

Glycopeptides were directly deglycosylated on the MALDI target by treatment with 0.2 U of PNGase F (Roche) in 50 mM NH₄HCO₃, pH 8, at 37 °C, for 1 h. Then, 2 µL of 0.1% trifluoroacetic acid was added to reaction mixtures, which were desalted on μZipTipC18 pipette tips (Millipore) before MALDI-TOF-TOF-MS analysis [61] (link).
Disulfide-containing peptides were directly reduced on the MALDI target by treatment with 10 mM mM DTT in 50 mM NH₄HCO₃, pH 8, at 37 °C, for 1 h. Then, 2 µL of 0.1% trifluoroacetic acid was added to reaction mixtures, which were desalted on μZipTipC18 pipette tips (Millipore) before MALDI-TOF-TOF-MS analysis [61] (link).

Rabbit seminal fluid OBP was resolved by SDS-PAGE, excised from the gel, triturated, in-gel reduced, S-alkylated and digested with trypsin, as previously reported [56] (link). Gel particles were extracted with 25 mM NH₄HCO₃/acetonitrile (1∶1 v/v) by sonication, and digests were concentrated. Peptide mixtures were either desalted using μZipTipC₁₈ pipette tips (Millipore) before MALDI-TOF-MS analysis, directly analyzed by nanoLC-ESI-LIT-MS/MS (see below) or simply resolved on an Easy C₁₈ column (100×0.075 mm, 3 µm) (Proxeon) using a linear gradient of acetonitrile containing 0.1% trifluoroacetic acid in aqueous 0.1% trifluoroacetic acid, at a flow rate of 300 nL/min, for 80 min. In the latter case, collected fractions were concentrated and analyzed by MALDI-TOF-MS.

Spots from 2D-E were manually excised from gels, triturated, and washed with water. Proteins were in-gel reduced, S-alkylated, and digested with trypsin, as previously reported (D'Ambrosio et al., 2008 (link)). Protein digests were subjected to a desalting/concentration step on μZipTipC18 pipette tips (Millipore Corp., Bedford, MA, USA) and then analyzed by nano-liquid chromatography (nLC)-electrospray ionization (ESI)-linear ion trap (LIT)-tandem (MS/MS) mass spectrometry, using a LTQ XL mass spectrometer (Thermo Fischer Scientific, USA) equipped with a Proxeon nanospray source connected to an Easy-nanoLC (Proxeon, Odense, Denmark). Peptide mixtures were separated on an Easy C18 column (100 × 0.075 mm, 3 μm) (Thermo, USA) using a gradient of acetonitrile containing 0.1% formic acid in aqueous 0.1% formic acid; acetonitrile was ramped from 5 to 35% over 10 min, from 35 to 95% over 2 min, and remained at 95% for 12 min, at a flow rate of 300 nL/min. Spectra were acquired in the range m/z 400–2000. Acquisition was controlled by a data-dependent product ion-scanning procedure over the 3 most abundant ions, enabling dynamic exclusion (repeat count 2; exclusion duration 1 min). The mass isolation window and collision energy were set to m/z 3 and 35%, respectively.