TAGZyme Kit

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Publication protocol

NAT oligopeptide-substrate recovery and RP-HPLC based separation. Peptides starting with pyroglutamate were unblocked prior to the second SCX fractionation step. Here, 25 µl of pGAPase (25 U/ml) (TAGZyme kit, Qiagen, Hilden, Germany) was activated for 10 min at 37°C by addition of 1 µl of 50 mM EDTA (pH 8.0), 1 µl of 800 mM NaCl, and 11 µl of freshly prepared 50 mM cysteamine-HCl. 25 µl of Qcyclase (50 U/ml, TAGZyme) was then added to the pGAPase solution. The dried peptides were re-dissolved in 212 µl of buffer containing 16 mM NaCl, 0.5 mM EDTA, 3 mM cysteamine, and 50 µM aprotinin. The activated pGAPase and Q-cyclase mixture was added to the peptide sample and the mixture (275 µl total volume) was incubated for 60 min at 37°C. 275 µl acetonitrile was then added and the sample was acidified to pH 3 using a 1% TFA stock solution in 50% acetonitrile. The sample was further diluted with 10 mM sodium phosphate in 50% acetonitrile to a final volume of 1 ml. SCX enrichment of Nα-blocked N-terminal peptides was performed as described [48] (SCX fractionation 2). The SCX fraction containing the newly blocked N-terminal peptides was vacuum dried and re-dissolved in 100 µl of HPLC solvent A. To prevent oxidation of methionine between the primary and secondary RP-HPLC separations (and thus unwanted segregation of methionyl peptides [51], methionines were uniformly oxidized to sulfoxides prior to the primary RP-HPLC run by adding 2 µl of 30% (w/v) H2O2 (final concentration of 0.06%) for 30 min at 30°C. This peptide mixture was injected onto a RP-column (Zorbax 300SB-C18 Narrowbore, 2.1 mm (internal diameter)×150 mm length, 5 µm particles, Agilent Technologies) and the RP-HPLC separation was performed as described previously [48]. Fractions of 30 s wide were collected from 20 to 80 min after sample injection (120 fractions). To reduce LC-MS/MS analysis time, fractions eluting 12 min apart were pooled, vacuum dried and re-dissolved in 40 µl of 2% acetonitrile. In total, 24 pooled fractions per setup were subjected to LC-MS/MS analysis (see below).

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