Quadrupole ion trap mass spectrometer

All protein digests were measured immediately after proteolytic digestion by LC‐MS. Mass analysis was carried out on a Bruker AmaZon (Billerica, MA) quadrupole ion trap mass spectrometer equipped with an electrospray ionization source. Typically, the electrospray needle voltage was kept at ∼4 kV, and the capillary temperature was set at 220 °C. Either collision‐induced dissociation (CID) or electron transfer dissociation (ETD) was applied to identify the position of the labels.
HPLC separation was conducted by a Thermo Scientific (Waltham, MA) Ultimate 3000 HPLC with a Thermo Acclaim PepMap RSLC C₁₈ column (300 μm × 15 cm, 2 μm particle size). Peptide mixtures from the proteolytic digests were eluted using a gradient of acetonitrile containing 20% water and 0.1% formic acid that increased from 5% to 45% for 45 min at a flow rate of 4 μl/min.

To quench the CL reaction of intact proteins and remove excess CL reagents and buffer salts, a Thermo Scientific Ultimate 3000 HPLC system (Thermo Scientific, Tewksbury, MA) with an OPTI-TRAP C4 reverse phase column (1 × 8 mm) was used. The protein was eluted using an acetonitrile gradient that increases from 1 to 99% over 12 min at a flow rate of 0.2 mL/min. The labeled protein was collected for proteolytic digestion or intact protein MS characterization. Size-exclusion chromatography (SEC) was used to purify the β2m dimer after formation, and the details of this separation can be found in the SI.
Mass spectral analyses of the HPLC separated intact protein samples from the covalent labeling experiments were acquired on a Bruker AmaZon (Billerica, MA) quadrupole ion trap mass spectrometer equipped with an electrospray ionization source. The electrospray needle voltage was kept at 4 kV, and the capillary temperature was set to 250 °C.