Anchor chip maldi plate

Protein structures may lead to anomalously fast migration in SDS-PAGE (Jong et al. 1978 (link), Rath et al. 2009 (link)). To determine the exact molecular mass, purified RiD was analyzed on a matrix-assisted laser desorption/ionization time-of-flight Autoflex III mass spectrometer (Bruker Daltonics, Germany). Samples were prepared by mixing equal volumes of 0.1 % trifluoroacetic acid (TFA), acetonitrile (1:1) and the protein solution. A 2 µL portion of the above sample was mixed with 2 µL of freshly prepared α-cyano-4-hydroxycinnamic acid (HCCA) matrix in 50 % acetonitrile and 1 % TFA (1:1), and 2 µL was spotted on the target Anchor Chip MALDI plate (Bruker Daltonics, Germany). The protein was also digested with trypsin (Promega, Madison, USA) according to the in solution digestion protocol of Mann (2006 (link)). The spectra obtained were analyzed with Flex Analysis Software (version 2.4, Bruker Daltonics, Germany) for deletion of matrix peaks and tryptic autolysis peaks. Processed spectra were then searched using MS Biotools (version 3.2) program against the taxonomy Viridiplantae (Green plants).

N-glycans were extracted from slides as described previously and dried by vacuum centrifugation [20 (link)]. The ethyl esterification protocol, including the modification and enrichment, was adapted from Reiding etal. [22 (link)]. Briefly, 2 µL water and 40 µL 0.25 M HOBt/EDC were added to dried glycans followed by incubation at 37 °C for 1 h. 40 µL acetonitrile was added and the mixture was placed at −20 °C for 20 min. Glycans were enriched using cotton-HILIC tips according to Selman etal. [26 (link)]. Briefly, cotton wool composed of 100% cotton (Assured, Rio Rancho, NM, USA) was inserted in 20 µL tips and equilibrated with 10 µL water three times followed by 10 µL 85% ACN three times. Samples were loaded and unbound material removed by washing three times with 10 µL 85% acetonitrile with and without 1% TFA, respectively. Tip-bound glycans were eluted in 10 µL water. Enriched and modified glycans were spotted on an Anchorchip MALDI plate (Bruker Daltonics) with 2,5-dihydroxybenzoic acid (DHB) at a concentration of 5 mg/mL in 50% ACN/50% water/1 mM NaOH. Ethanol was then used for recrystallization.

One µL of prepared peptide solutions were prespotted on an Anchor Chip MALDI plate (Bruker, Bremen, Germany). When the protein samples were dried, their surfaces were covered by 1 μL of α-cyano-4-hydroxycinnamic acid matrix (HCCA, Bruker, Bremen, Germany). Peptide standard solution (Peptide Calibration Standard II, Bruker, Bremen, Germany) was also spotted and covered by matrix on calibration spots. Mass spectra were recorded in active positive reflector mode within the 700–4000 m/z range using an Ultraflextreme MALDI TOF/TOF (Bruker, Bremen, Germany) spectrometer and the flexControl 3.3 (Bruker, Bremen, Germany) software. Collected spectra were smoothed and baseline corrected. Generated in flexAnalysis 3.0 software (Bruker, Bremen, Germany), peak list for the signal-to-noise ratio of > 3 was transferred to BioTools 3.2 (Bruker, Bremen, Germany), and compared to Mascot 2.2 software (Matrix Science, Boston, MA, USA) using the Swiss-Prot database (www.uniprot.org (accessed on 15 June 2021)) restricted to “mammalia” taxonomy with maximum error 0.3 Da and carbamidomethylation of cysteine as obligatory modification. Results with a Mascot score above 61 were considered statistically significant (p ≤ 0.05); otherwise, the fragment ion spectra of chosen peptides were obtained using the LIFT mode and combine in the aim of MALDI TOF/TOF identification.