Gl tiptm sdb

After HAC-2D MICS-BN-PAGE, silver or CBB staining was conducted for the slab gel to detect the spots of isolated metalloprotein that had migrated off the diagonal line. The spots cut from the resulting slab gel were then subjected to in-gel tryptic digestion using the In-Gel Tryptic Digestion Kit (Thermo Fisher Scientific, Waltham, USA), according to the kit protocol. Digested sample solutions were desalted and concentrated by GL-TipTM SDB (GL Sciences, Tokyo, Japan) prior to being subjected to MALDI-TOF MS. For MALDI-TOF MS measurements, α-cyano-4-hydroxycinnamic acid (HCCA, Wako; for proteome) was employed as the matrix. As a calibration standard, a Peptide Calibration Standard II solution (Bruker Daltonics, Billerica, USA) was employed. The MALDI-TOF MS measurements by Autoflex III (Bruker Daltonics) were conducted using the reflector positive method (with molecular weight range 1–5 kDa). The protein was identified by peptide fingerprinting using publicly available databases (MASCOT, MatixScience).

Peptide samples were passed through a C₁₈ column GL-Tip TM SDB (GL Sciences Japan). The column was preconditioned by adding 100 μl of buffer B (0.1% TFA and 80% acetonitrile (ACN)) to a C₁₈ column tip and equilibrated by adding 100 μl of buffer A (0.1% TFA and 5% ACN) to the C₁₈ column tip. Then, the peptide samples were added to the C₁₈ column tip and washed by adding 100 μl of buffer A. The desalted peptides were eluted by buffer B1 (0.1% TFA and 50% ACN), and the peptide eluents were dried by using a speed vacuum at 60 °C for 3 h. The dried peptide samples were dissolved in 5 μl of buffer containing 50% ACN, 0.1% formic acid (FA) and 20 μl 0.1% FA, and these peptide samples were desalted using 10 μl ZipTip columns (Millipore USA). The ZipTip columns were washed two times with 100% ACN and 50% ACN and three times with 0.1% FA. Then, the samples were loaded into ZipTip columns and washed with 0.1% FA. The peptide samples were finally eluted with buffer (40% ACN, 0.1% FA) and subsequently dried using speed vacuum.