Masslynx data system

Protein bands of interest were sliced from a Coomassie-G250 stained SDS-PAGE gel and subjected to in-gel reduction (10 mM dithiothreitol), alkylation (55 mM iodacetamide), and overnight trypsin digestion at 37 °C (Promega). Tryptic digests were dried, redissolved in 3% acetonitrile and 0.1% formic acid solution, and submitted to LC-MS/MS using Q-TOF micro mass equipment (Waters Corporation, Milford, MA, USA).
Electrospray voltage was set at 3500 V, source temperature at 80 °C, and cone voltage at 30 V. Instrument control and data acquisition were conducted using a MassLynx data system (Version 4.1, Waters), and experiments were performed by scanning from a mass-to-charge ratio (m/z) of 400–2000 using a scan time of 1 s, applied during the whole chromatographic process. Data-dependent MS/MS acquisitions (DDA) were performed on precursors with charge states of 2, 3, or 4 over a range of 50–2000 m/z and under a 2 m/z window. A maximum of 3 ions were selected for MS/MS from a single MS survey obtained by collision-induced dissociation (CID). Exported MS data were analyzed in Mascot MS/MS Ions Search, with parameters described in Figure S3.

Initial analysis of Fc-peptide products was carried out by reversed-phase HPLC/MS of the intact or reduced (5 mM DTT, 55 °C, 30 min) product on the system described below. However, mass accuracy and measurement precision were insufficient to ensure unambiguous determination of the state of the peptide carboxy terminus. Consequently, samples were digested with HRV3C protease (PreScission, GE Healthcare Bio-Sciences, Pittsburgh, PA). Digestions of Fc-peptide were performed for 5 h at 5 °C in a buffer consisting of 20 mM TrisHCl, pH 7.0, 150 mM NaCl, 1 mM DTT using one unit of enzyme for up to 100 μg of protein. Polypeptides resulting from the digestion were chromatographically resolved on a Waters (Milford, MA) Acquity UPLC with an Agilent (Santa Clara, CA) PLRP-S column (2.1 × 50 mm, 5 μ d_p, 1000 Å) using a multi-linear gradient. Buffer A was 0.1 % formic acid, and buffer B was 0.1 % formic acid in acetonitrile. The peptides were mass analyzed on a Waters API US mass spectrometer scanned from m/z 500–3000 at a rate of 1 Hz. The peptide m/z data was deconvoluted to the single-charge mass domain by the MaxEnt3 algorithm implemented in the Waters MassLynx data system.

Electrospray ionization [ESI; negative (−) and positive modes (+)] and matrix-assisted laser desorption/ionization (MALDI; +) mass spectrometry (MS) were performed on a Waters Synapt G2-Si ToF mass spectrometer (Waters Corporation, United States). MassLynx data system (Waters Corporation, United States) provided instrument control, data acquisition, and data processing. For all three analyses, sorghum polyphenol extracts were prepared to a concentration of 0.1 mg/ml in 50% (v/v) aq. methanol. Sorghum polyphenol extracts were prepared, run, and analyzed in triplicate and three different extracts per variety were analyzed. For ESI, capillary voltage was 2.2 kV, source temperature was 100°C, and desolvation temperature was 280°C. Solutions were injected at a flow rate of 5 μl min⁻¹. ESI – tandem MS (MS²) was performed on specific ions produced by ESI-MS in the negative mode. For MALDI sample preparation, the matrix chemical alpha-cyano-4-hydroxycinnamic acid [CHCA; 5 mg/ml in methanol with 0.1% formic acid (v/v)] was mixed with the prepared extracts in a 1:1 ratio. From this mixture, 1 μl was spotted onto a steel MALDI plate for analysis. All spectra were measured from 50 to 1,500 Da for each analysis type.