Trypsin lys c mix

Sample preparation was performed using a Filter-Aided-Sample preparation-Protocol (FASP) according to Wiśniewski et al. (2009) (link) and Wiśniewski et al. (2016) (link) as described in Ramires et al. (2022) (link). In brief, 30 µg of protein were digested on a 10 kDa Pall Nanosep centrifugal device (Cytiva, MA, USA).
After reduction with dithiothreitol and alkylation with iodoacetamide, proteins were digested using Trypsin-LysC-Mix from Promega (WI, USA). In order to stop the trypsin digestion, 5% trifluoroacetic acid was added. Peptides were extracted in three rounds each of 50 µL 50 mM Tris followed by centrifugation. Peptides were acidified using trifluoroacetic acid to a pH below 2. After C18 clean-up with spin columns (Pierce, Thermo Fisher), peptides were separated on a nanoRSLC system equipped with a 25 cm Acclaim PepMap C18 column (Thermo Fisher) and analysed on a high-resolution Q Exactive HF Orbitrap mass spectrometer as described in Gutiérrez et al. (2019) (link).

Gel pieces were excised from the gel lanes and proteolytic digestion with Trypsin/Lys-C mix (V5073, Promega, Southampton, United Kingdom) was performed as described (Shevchenko et al., 2006 (link)). The gel pieces were covered with 50 mM NH₄HCO₃ / 50% ACN and shaken for 10 min. This step was repeated with 100% acetonitrile and finally dried in a speed vac. Samples were reduced with 10 mM DTT in 50 mM NH₄HCO₃ at 56 °C for 1 hr and alkylated with 50 mM iodoacetamide in 50 mM NH₄HCO₃ at room temperature without light for 45 min. The gels were covered with 50 mM NH₄HCO₃ and 100% ACN and shaken for 10 min. These steps were repeated and samples were dried in a speed vac. Trypsin/Lys-C buffer was added to the sample according to manufacturer’s instructions and incubated for 45 min on ice.
Next 30 μl 25 mM NH₄HCO₃ was added and samples were incubated at 37 °C for 16 hr. The gel pieces were covered with 20 mM NH₄HCO₃ and shaken for 10 min. Supernatant with peptides was collected. Next, the gels were covered with 50% ACN / 5% FA and shaken for 20 min. These steps were repeated and peptides were dried in a speed vac. Samples for glycopeptide enrichment were digested in-solution according to Queiroz et al., 2019 (link). Samples were reduced and alkylated in 10 mM DTT and 50 mM iodoacetamide. Proteins were digested in final concentration of 2.5 μg Trypsin/Lys-C buffer for 16 hr at 37 °C.