Anchorchip 384 bc maldi target plate

A 15.9 μg of PSA glycoprotein sample was dissolved in 500 μL of 0.1% TFA using VivaSpin 500 column (molecular weight cut-off limited to 10 kDa) (Sartorius Stedim, DE). Briefly, 7.6 mg of 2,5-DHAP was dissolved in 375 μL of ethanol and 125 μL of 18 mg/mL aqueous solution of diammonium hydrogen citrate was added. The intact glycoprotein sample (1 μL) was spotted onto an AnchorChip 384 BC MALDI target plate (Bruker Daltonics, MA, USA), pre-mixed with 1 μL 0.1% TFA, overlaid with 1 μL of matrix 2,5-DHAP solution and left to dry by air to enable the crystallisation. The mass spectrometric measurement of intact PSA was performed on an UltrafleXtreme (Bruker Daltonics, MA, USA) in a linear positive ion mode. Calibration of the mass spectra was carried out with a Protein calibration standard II from Bruker Daltonics. 3000 laser shots were collected into one spectrum and the ions were recorded in the range between 20 000 and 50 000 m/z. The acquired raw spectra were processed by the FlexAnalysis and ProteinScape 3.0 software (Bruker Daltonics, MA, USA).

Peptides from tryptic digestion of 2 μg of PSA were loaded onto a trap column (Acclaim PepMap100 C18, 75 μm x 20 mm, Dionex, CA, USA) and separated with a C18 column (Acclaim PepMap C18, 75 μm x 150 mm, Dionex) on UltiMate 3000 RSLCnano system (Dionex, CA, USA). A sample was injected and a linear 30 min gradient (10–55% B) was used for peptide separation. Two mobile phases were used where A was 0.05% TFA (v/v) and B was 80% ACN (v/v) with 0.05% TFA. Fractions were spotted on AnchorChip 384 BC MALDI target plate (Bruker Daltonics, MA, USA) together with 4-HCCA solution as a MALDI matrix. The fractionated samples were analysed with a MALDI TOF/TOF (UltrafleXtreme, Bruker Daltonics, MA, USA) instrument operated in the positive ion mode. Fragmentation data were searched by the Mascot search engine (Version 2.4.1, Matrix Science, UK) against SwissProt protein sequence database (version 2015_09) limited to Homo sapiens taxonomy with allowed oxidation of methionines and carbamidomethylation of cysteines as variable modifications and expected semitrypsin cleavage.