Maldi tof

Immobilization of the BTLA protein in a microcolumn and the affinity test were performed according to the procedure described in our previous studies [30 (link)]. The mass spectroscopy (MS) measurements were conducted using a MALDI-TOF/TOF 5800 (AB Sciex, Germany) instrument. α-Cyano-4-hydroxycinnamic acid (CHCA) was used as the matrix (10 mg/mL, Sigma-Aldrich, Saint Louis, MO, USA). The measurements were conducted in reflector positive mass mode with previous mass calibration with a commercial standard peptide mixture (The Peptide Mass Standards Kit for Calibration of AB SCIEX MALDI-TOF™ Instruments).

A single PE crystal was solubilized
in water and analyzed by SDS-PAGE. For in-gel hydrolysis, SDS-PAGE
bands were excised from the gel lane, destained by consecutive cycles
of 0.1 M NH₄HCO₃ at pH 8.0 and acetonitrile
(ACN), followed by reduction (10 mM DTT in 100 mM NH₄HCO₃, 45 min, at 56 °C) and alkylation (55 mM IAM in 100
mM NH₄HCO₃, 30 min, at room temperature). The
gel pieces were washed with 0.1 M NH₄HCO₃ of
pH 8.0 and ACN and subjected to the enzymatic hydrolysis by covering
them with 40 μL sequencing grade modified trypsin (10 ng/μL
trypsin; 10 mM NH₄HCO₃) overnight at 37 °C.
Peptide mixtures were eluted, vacuum-dried, and resuspended in 2%
ACN acidified with 0.1% HCOOH. Tryptic peptide mixtures were analyzed
by MALDI-TOF (AB SCIEX, Milan, Italy) to reveal the amino acid sequence
of the three phycoerythrin chains.