Dhb matrix

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).