Empore

For GC–MS analysis a protocol according to Weckwerth et al. was used (Weckwerth et al. 2004 (link)). Deep frozen plant material was ground to a fine powder using a mortar and pestle under constant adding of liquid nitrogen. About 45 mg of each replicate was transferred to pre-cooled reaction tubes. For the extraction process, 1 ml of ice cold extraction mixture (methanol:chloroform:water, 5:2:1, v:v:v) was subsequently added. Additionally, 10 μl of internal ¹³C₆-Sorbitol standard were added into each tube. Tubes were vortexed for several seconds and incubated on ice for 8 min to achieve a good extraction. Hereupon, the samples were centrifuged for 4 min at 14,000×g, separating the soluble compounds from remaining cell structure components. For phase separation, the supernatant was then carried over into a new tube containing 500 μl deionized water and 200 μl chloroform. After 2 min of centrifugation at 14,000×g, the water/methanol phase, containing the polar metabolites, was separated from the subjacent chloroform phase and completely dried out overnight.
Samples were derivatised by dissolving the dried pellet in 20 μl of a 40 mg methoxyamine hydrochloride per 1 ml pyridine solution and incubation on a thermoshaker at 30 °C for 90 min. After adding of 80 μL of N-methyl-N-trimethylsilyltrifluoroacetamid (MSTFA), the mixture was again incubated at 37 °C for 30 min with strong shaking.
A solution of even-numbered alkanes (Decane C10, Dodecane C12, Tetradecane C14, Hexadecane C16, Octadecane C18, Eicosane C20, Docosane C22, Tetracosane C24, Hexacosane C26, Octacosane C28, Triacontane C30, Dotriacontane C32, Tetratriacontane C34, Hexatriacontane C36, Octatriacontane C38, Tetracontane C40) was spiked into the derivatized sample before GC–MS analysis in order to infer the retention time and create the retention index.
For LC–MS analysis, frozen plant leaf material was ground as for GC–MS sample preparation, followed by addition of 1 ml pre-chilled 80/20 v:v MeOH/H₂O extraction solution containing each 1 μg of the internal standards Ampicillin and Chloramphenicol per 50 mg of fresh weight. Samples were hereupon centrifuged at 15,000×g for 15 min and the supernatant was placed into a new tube and completely dried out overnight. The resulting pellet was then dissolved in 100 μl of a 50/50 v:v MeOH/H₂O solution and centrifuged again for 15 min at 20,000×g. The remaining supernatant was then filtered through a STAGE tip (Empore/Disk C18, diameter 47 mm) into a vial with a micro insert tip. Before analysis lipid components were removed by adding 500 µl of chloroform, centrifugation and separation of the non-polar-phase to avoid contamination of the ESI ion transfer capillary.

25 µg of purified AGA, GUSB CTSD, and GAA were dissolved in 50 mM ammonium bicarbonate (AmBic) buffer (pH 7.4) and further reduced with 10 mM dithiothreitol (DTT) at 60°C for 45 min on shaker, followed by alkylation with 20 mM iodoacetamide (IAA) at 25°C for 30 min in darkness. AGA, GUSB, CTSD were subjected to proteolytic digestion with chymotrypsin (1:40 enzyme-substrate ratio), while GAA was digested in gel with trypsin (1:25 enzyme-substrate ratio) after SDS-PAGE separation. The reaction was quenched with 1 µL trifluoroacetic acid (TFA) and the digested sample was desalted by custom-made modified StageTip colums with three layers of C18 and two layers of C8 membrane (3 M Empore disks, Sigma-Aldrich). Samples were eluted with two steps of 50 µL 50% methanol in 0.1% formic acid. Final sample was aliqoted in two equal parts. The first aliquot was placed into a glass insert (Agilent), dried completely in SpeedVac (Eppendorf) and further re-dissolved in 50 µL 0.1% formic acid (FA) and submitted for nLC-MS analysis. The second aliqout was placed inside an Eppendorf tube, dried completely using SpeedVac, and then re-dissolved in 50 µL of 50 mM AmBic buffer (pH 7.4) and incubated with PNGase F (1U per sample) for 12 h with shaking at 37°C. Samples treated with PNGase F were desalted and dried using the same methods mentioned above for the first aliqout and submitted for nLC-MS/MS analysis.

The samples were enriched for phosphopeptides by Hydroxy Acid Modified Metal Oxide Chromatography (HAMMOC) [109 (link)]. Per 500 ug peptide digest a 200 ul tip with an Octyl C8 membrane (Empore) and 2.5 mg Titansphere TiO₂ 10 μm (GL Sciences) were used. The tips were preconditioned with 20/80/0.1 (v/v/v) milliQ/ACN/TFA (solution A) and equilibrated with 300 mg/ml DL lactic acid (Fluka Analytical) in solution A. Peptide samples were mixed 1:1 with 300 mg/ml DL lactic acid in solution A and loaded on the tips. The tips were washed with 300 mg/ml DL lactic acid in solution A and solution A. 100 ul 20% phosphoric acid (Sigma-Aldrich) was put in collection tubes and phosphopeptides were eluted with 50 ul 0.5% piperidine (Actu-All Chemicals) followed by 50 ul 5% piperidine. Peptides were desalted on 200 ul tips with an SDB-XC membrane (Empore). Tips were preconditioned with solution A and equilibrated with 0.1% TFA. Samples were loaded on the tips and the tips were washed with 0.1% TFA. Peptides were eluted with solution A and lyophilized in a CHRIST RVC-2-18 CDplus.

A total of 3.8 μg raBSA (BSA digest in ice water) was loaded onto an SDS-PAGE to separate intact raBSA from impurities and nicked raBSA. The raBSA band was cut out and washed twice with 500 μL H₂O followed by two times incubation of 15 min with 50 μL 50% acetonitrile. Then, 50 μL of 100% acetonitrile was added and incubated for 15 min before the reaction buffer was added to a 1:1 ratio. The liquid was removed after 15 min and lyophilized until the gel plug was dry. Matured protease (600 nM) was added to the dried plugs and incubated for 10 min. Thirty μL reaction buffer was added and incubated for 1.5 h. Triton X-100 was excluded as detergents are not compatible with MS. All incubation was performed at RT except protease digestions, which were allowed to proceed at 37°C. The reaction was quenched with 7.1% formic acid. The resulting peptides from protease digestion were micro-purified using C18 column material (Empore™) in P10 pipette tips (Sarstedt). The column material was activated with solvent B (99.9% acetonitrile and 0.1% formic acid) and then equilibrated with solvent A (0.1% formic acid). Peptides were then loaded and washed in solvent A before being eluted with 70% solvent B and dried for MS. The experiment was carried out in triplicates.