Hi3 phos b standard

Purified water was obtained from an Arium Pro UV ultrapure water system (Sartorius Stedim Biotech, Goettingen, Germany). LC-MS grade water containing 0.1% (V/V) formic acid (FA) and LC-MS grade acetonitrile containing 0.1% (V/V) FA were obtained from Fisher Scientific (Dublin, Ireland). All reagents and materials required for digestion such as Thermo Scientific™ SMART Digest™ trypsin kit, Thermo Scientific™ KingFisher Deepwell 96-well plates, and 12-tip combs were obtained from Thermo Fisher Scientific (Sunnyvale, CA, USA). DL-dithiothreitol (DTT) (≥98%), iodoacetamide (IAA), Trizma® base, phosphate-buffered saline (PBS), and glycine were purchased from Sigma-Aldrich (Wicklow, Ireland). Hi3 Phos B standard for label-free Hi3 quantitation of detected HCPs was obtained from Waters (Wexford, Ireland). A commercially available IgG1, rituximab, was provided by the Pharmacy Unit of the University Hospital of San Cecilio in Granada, Spain.

A sample of seeds (n = 100 grain) was weighed after discarding any shriveled seeds and the average grain weight found to be 49.47 mg. Three plump seeds per extraction were selected to be representative and had a weight similar to the average weight for the total seed sample (±7.13 mg). Three seeds were extracted per type of buffer and each extract analyzed in triplicate by LC-MS using the QTOF (giving pooled data from 27 analyses from nine seeds) and in duplicate using the LTQ (giving pooled from 18 analyses from nine seeds). Three technical replicates of the MS acquisition were used to calculate the mean protein abundances and allow statistical analysis to be carried out in measurements. A summary of the experimental workflow can be found in Supplementary Material, Figure S1.
Controls implemented include the use of LeuEnk during the detector set up of the mass spectrometer and Hi3 PhosB standard (Waters, Wilmslow, UK) was used as a standard for all sample preparation and verifying instrument performance. Samples were randomized for analysis and blank injections of MilliQ water were carried out every three injections.
Variation between protein extractions was calculated as the standard deviation. Variation was also accessed on a run to run basis for all peptides residues to investigate the occurrence in the biological and technical replicates.