Peptide standard 2

Coomassie-stained protein bands were excised and prepared as described [38 (link)]. Briefly, excised and chopped bands were washed, destained and digested by trypsin (Trypsin Gold, Mass Spectrometry Grade, Promega, Mannheim, Germany) overnight. The ZipTip (C₁₈, Millipore Corporation, Billerica, MA, US) eluates of the obtained tryptic fragments were mixed 1:1 (v/v) with a 12 mg/mL α-cyano-4-hydroxycinnamic acid matrix (Bruker Daltonics, Bremen, Germany), dissolved in a 2:1 (v/v) mixture of 100% acetonitrile/0.3% TFA, and spotted on the target. Tryptic mass fingerprinting was performed as described previously [39 (link)] using a Reflex III (Bruker Daltonics, Bremen, Germany) in reflector mode, while applying an acceleration voltage of 20 kV. External mass calibration was performed with peptide standard II (Bruker Daltonics, Bremen, Germany). Mascot Peptide Mass Fingerprint (http://matrixscience.com) and NCBInr database were used to identify digested fragments. For database search the following filters were applied: taxonomy on other green plants, peptide tolerance of ± 0.3 Da and up to one allowed missed cleavage. Variable modifications were the oxidation of methionine residues and N-terminal acetylation.

Tryptically digested SHBG was analyzed at both reducing and nonreducing conditions and chymotryptically digested SHBG at reducing conditions.
Purified SHBG was mixed 1 : 1 (v/v) with digestion buffer [2% (w/v) sodium deoxycholate in 100 mm triethylammonium bicarbonate] and incubated 10 min at 99 °C. For the samples to be reduced, tris(2‐carboxyethyl)phosphine was added in a 1 : 25 (w/w) reagent‐to‐protein ratio after cooling below 37 °C and incubated 30 min at 37 °C, whereafter iodoacetamide was added in a 1 : 10 (w/w) ratio and the sample incubated for 20 min at 37 °C in the dark. Trypsin or chymotrypsin was added in a 1 : 50 (w/w) ratio and the samples incubated overnight at 37 °C.
To precipitate sodium deoxycholate, formic acid was added to a concentration of 2.0% and the sample incubated at room temperature for 5 min. The samples were then centrifuged at 13 000 g for 20 min at 4 °C. The supernatant was desalted in a column with a C18 disc (Empore™, Oxford, PA, USA) and R2 and R3 Poros beads (Applied Biosystems, Foster City, CA, USA) and eluted with 10 µL 80% acetonitrile and 0.1% formic acid.
A total of 0.5 μL desalted protein [or peptide standard II (Bruker Daltonik, Bremen, Germany)] was deposited on an AnchorChip™ MALDI target plate followed by 0.5 μL DHB matrix (Thermo Fisher) (40 mg·mL⁻¹ DHB in 80% acetonitrile and 0.1% formic acid).