Rlys c

Dried GFP-trap eluates were solubilized in 50 μl of 50 mM triethylammonium bicarbonate (TEAB) in 50:50 (vol/vol) trifluoroethanol (TFE) to water. Protein thiols were reduced and carbamidomethylated in one step (thermomixer at 70°C, 500 rpm, 30 min) by addition of 500 nmol tris(2-carboxyethyl)phosphine (TCEP) and 625 nmol 2-chloroacetamide (each solubilized in 100 mM aqueous TEAB). Subsequently, the protein solution was concentrated to a final volume of approximately 5 μl using a vacuum concentrator and diluted with 45 μl 100 mM TEAB to gain a final volume of 50 μl. Tryptic cleavages were performed using 1 μg rLys C (Promega number [no.] V1671) with incubation for 2 h at 37°C, and subsequently, 2 μg Trypsin Gold (Promega no. V5280) was added and the mixture was incubated for a further 16 h at 37°C. GluC cleavages were performed using 2 μg GluC (Promega no. V1651) and incubating for 18°C at 37°C. Peptides were dried and resolubilized in 25 μl 0.05% trifluoroacetic acid and 2% acetonitrile using a water bath sonicator (15 min). Finally, the samples were filtered through 0.2-μm spin filters (Merck Millipore Ultrafree-MC, hydrophilic polytetrafluoroethylene [PTFE] membrane, catalog no. UFC30LG25) at 18,000 × g for 15 min, and the filtrate was transferred into high-performance liquid chromatography (HPLC) vials.

Samples were treated with a protocol previously described^{31 (link)}. In order to perform protein extraction and digestion, the micro-samples were placed in separate Protein Lo-Bind tubes (Eppendorf, Germany) and, along with a blank control, incubated for 2 h at 80 °C with 100 μL of an aqueous buffer containing: 2 M guanidinium chloride (GuHCl), 10 mM tris(2-carboxyethyl)phosphine (TCEP), 20 mM chloroacetamide (CAA), and 100 mM trisaminomethane (Tris). The pH was adjusted to around 8.0 using NH₄OH 10% when needed. Proteins were digested under agitation for 2 h at 37 °C in-solution with 0.2 μg rLysC (Promega, Sweden). Samples were then diluted to a final concentration of 0.6 M GuHCl using 25 mM Tris in 10% acetonitrile (ACN) in water, and digested overnight under agitation at 37 °C with 0.8 μg Trypsin (Promega, Sweden). Samples were then acidified to pH 2 using 10% trifluoroacetic acid (TFA) to quench digestion. The resulting peptides were collected on in-house made C18 extraction stage-tips, as previously described^{50 (link)}. Stage-tips were stored at − 18 °C until mass spectrometry analysis.

An amount of 50 μg of total protein were solubilized into 80 μL with lysis buffer. Subsequently, samples were reduced in 50 mM TCEP (Sigma ‐ 646547) for 1H followed by alkylation in 50mM iodoacetamide (Sigma ‐ I114) for 1H in the dark. Proteins were first digested with 1 μg rLys-C (Promega ‐ V1671) for 3H at 25°C and a second digestion was performed with 1 μg Sequencing Grade Modified Trypsin (Promega ‐ V5111) for 16H at 37°C. Incubation time and temperature control for reduction, alkylation and digestions were performed with an Eppendorf ThermoMixer® C equipped with a ThermoTop®. The digestion was stop with formic acid (FA) at 1% final and peptides were desalted on reversed phase C18 Sep-Pak Cartridge (Waters ‐ WAT054955). Peptides were eluted 2 times with Acetonitrile (ACN) 50%, FA 0.1% and 1 time with ACN 80%, FA 0.1%. Finally, samples were dried in vacuum centrifuge and resuspended with ACN 2% / FA 0.1%.