5800 maldi tof tof

All reagents were purchased from company sources and were ready for use. ¹H NMR spectra of the materials were measured using a BRUKER AVANCEIIIHD400 with deuterated chloroform as the solvent and tetramethylsilane (TMS) as the internal standard. The UV absorption was measured by a JASCO V-570 spectrophotometer. The instruments for mass spectrometry analysis of materials were AB SCIEX MALDI-TOF/TOF 5800. Cyclic voltammetric curves of materials were measured using the CHI 660C instrument. Measurement of the response of nonlinear optical (NLO) characteristics uses a Z-scan technique (NLO-Z), where the laser pulse is chosen to be 21 ps.

The experiments were performed as previously described and summarized here^{48 (link)–51 (link)}. Hep3B cells (1 × 10⁶) were lysed in 1 ml of lysis buffer (50 mM HEPES, pH 7.4, 150 mM NaCl, 0.1% Triton X-100, 1 mM EDTA, 10 μg/ml, aprotinin, 10 μg/ml leupeptin, 1 mM phenylmethylsulfonyl fluoride). Lysates were clarified by centrifugation at 16,000 × g for 10 min at 4 °C. Biotinylated peptides were binded to Streptavidin in spin columns. For immunoprecipitation, 0.4 ml lysate was incubated with 0.2 mg of biotin-VI-17 or biotin-AI-17 at 4 °C for 2 h. Spin columns were washed three times with 250 µl of lysis buffer containing 0.25 M NaCl. The precipitates were captured by 250 µl of Elution Buffer and were analyzed by standard immunoblot procedures. The immunoprecipitated proteins were subjected to SDS-PAGE and visualized by SimplyBlue SafeStain (Life technologies). Each band was trypsinized and subjected to MALDI-TOF MS analysis with MALDI-TOF/TOF5800 (AB SCIEX, Tokyo, Japan). These experiments were repeated thrice and identified proteins in at least two experiments were considered as candidates for peptide-binding partners. Precipitated proteins were analyzed by Western blotting.

For N‐glycan detection, ~500 μg of protein extract from AAV2 vectors was reduced and alkylated and digested. Samples were then treated with glycosidase enzyme PNGase F for deglycosylation at 37 °C for 24 h and purified by C18 cartridge. For O‐glycan detection, samples were prepared by permethylation using methyl iodide. Phase separation was carried out with chloroform and the samples were recovered and purified by C18 cartridge. Samples for both N‐glycan and O‐glycan analysis were step eluted with 15%, 35%, 50%, and 75% Acetonitrile (ACN) using SEP PAK C 18 columns, then dried in Speed Vac and resuspended in 10 μL of methanol and DHB mixture analyzed using MALDI‐TOF/TOF 5800 (AB Sciex) in positive mode.

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).

In-gel digestion on the selected gel spots was performed using XL-TrypKit (APRO Science Inc., Tokushima, Japan) according to the manufacturer’s instructions. Matrix-assisted laser desorption ionization time of flight/ time of flight of mass spectrophotometer (MALDI-TOF/TOF MS) analyses were carried out using MALDI-TOF/TOF 5800 (AB sciex), as described previously [23 (link)]. MS spectra were acquired from m/z 800 to 4000, and each spectrum was obtained by accumulating 800 laser shots. Bradykinin fragment 2–9 (m/z 904.47), angiotensin I (m/z 1296.69), Glu¹-fibrinopeptide B (m/z 1570.68), ACTH fragment 1–17 (m/z 2093.09), ACTH fragment 18–39 (m/z 2465.20), and ACTH fragment 7–38 (m/z 3657.93) were used for external calibration. MS/MS spectra were automatically acquired from the intensive precursor ions (S/N > 50). Protein identification by MS/MS ion search (MIS) was performed using ProteinPilot software (ver.4.5; AB sciex). Search parameters were as follows: database, uniprot_sprot_can+iso_20100622; species, none; Cys alkylation, iodoacetamide; digestion, trypsin; and special factors, Gel-based-ID, max missed cleavage, 1. Protein identification was considered to be correct based on the following selection criteria: protein having at least 2 peptides with an ion score above 95% confidence; and protein with protein score (ProtScore) > 1.3 (unused, p < 0.05, 95% confidence).

For MALDI‐TOF, 25 µg of PapA2 protein subjected to trypsin digestion was injected into a MALDI‐TOF/TOF 5800 (AB Sciex, USA) and the fragments were identified from SwissProt.

The purified hFGF21 (20 µg) was alkylated with 10 mM iodoacetamide (IAA) for 20 min at room temperature in the dark. Trypsin (1.4 µg) was then added to the sample and the reaction was left at 37°C overnight to yield non-reduced sample. For the preparation of reduced sample, the protein was incubated with 10 mM DTT for 10 min at 95°C prior to alkylation and trypsinisation. All the samples were desalted, concentrated by Ziptip C18 (Millipore, Billerica, MA) and then mixed with matrix α-Cyano-4-hydroxycinnamic acid. The analysis was conducted using MALDI-TOF/TOF™ 5800 (AB SCIEX, Framingham, MA) at the Korea Basic Science Institute (Seoul, Korea).