Ultraflex 3 tof tof instrument

To bind the RIPs specifically to the particles, and thus prevent crosslinking reactions, the toxins were first provided with the azide click ligand NHS-PEG₁₂-N₃ for a copper free strain-promoted alkyne-azide cycloaddition. Therefore, and to study the conjugation efficiency of the NHS-PEG₁₂-N₃ linker to the toxins, three different molar ratios (1.5, 3.0, 20 mol_linker/mol_toxin) were employed. 0.3 mg of the respective RIP was mixed with the correspondent amount of the PEG linker and incubated for 3 h at 20 °C and 800 rpm in Dulbecco’s phosphate-buffered saline buffer. The conjugates were washed three times with the same buffer by means of Amicon centrifugal filter devices (10 kDa; Merck Millipore, Burlington, MA, USA). Successful conjugation was confirmed by matrix-assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF-MS), which was conducted on an Ultraflex-III TOF/TOF instrument (Bruker Daltonics, Billerica, MA, USA) equipped with a 200 Hz N₂-laser with 337 nm wavelength (pulse energy of 150 µJ) and 3 ns pulse width, and operated in the positive linear mode.

The synthesized alkyl vinyl sulfone was solubilized in methanol and mixed with 0.125 mM carbonate buffer (pH 8.3) to a final concentration of 2 mg ml⁻¹. The solution of the alkyl vinyl sulfone was mixed with the purified rPP2A (2 mg ml⁻¹) in carbonate buffer (0.125 mM carbonate, pH 8.3) and kept at 4°C in an orbital shaker for 12 h. Thereafter, free reactive groups were blocked with a molar excess of glycine in carbonate buffer at room temperature for 4 h. The conjugates were dialysed for 12 h against a solution of ammonium acetate (0.1 M, pH 7) and then lyophilized.
Mass spectrometer analyses were performed at Laboratorio de Espectrometría de Masas (SIdI), Faculty of Sciences, UAM, Madrid (Spain) in a Ultraflex III TOF/TOF instrument (Bruker) that uses an NdYAG laser (emission, 355 nm; accelerating voltage, 25 kV). The sample (0.5 mg of lipopeptide in 0.2 ml of 1,1,1,3,3,3 hexafluoruro-2-propanol) and matrix solution (10 mg of α-ciano-4-hydroxycinnamic acid (ACC) in 1 ml of acetonitrile, 0.1% TFA in water 3 : 1 v/v), were mixed in a proportion 1 : 20, and then applied to a metal sample plate for MS analysis. The lipopeptide mass was measured at approximately 1000 Da absorption laser intensity.

N-glycan extraction and purification of neutral and acidic N-glycans, and the mass spectrometric analysis was performed as earlier described^{18 (link),19 (link)}. Briefly, the glycans were enzymatically detached by N-glycosidase F (PNGase F) digestion from the deparaffinized tissue specimens (Glyko; ProZyme Inc., Hayward, CA). After the digestion, the N-glycans were purified by solid-phase extraction in 96-well format. The Bruker Ultraflex III TOF/TOF instrument (Bruker Daltonics Inc, Bremen, Germany) was used to perform matrix-assisted laser desorption-ionization time-of-flight (MALDI-TOF) mass spectrometry to the glycan samples. The mass spectrometry detected acidic N-glycans in negative ion reflector mode primarily as [M− H]¯ ions and neutral N-glycans in positive ion reflector mode primarily as [M+ Na]⁺ ions. Satomaa et al.^{20 (link)} and Saarinen et al.^{19 (link)} have previously described how the raw data was processed into presented glycan profiles. All the MS data are presented in Supplementary Tables S1 and S2.

Mass spectrometry was performed by the LMB Mass Spectrometry Facility. Briefly, peptides from in situ trypsin digestion were extracted in 2% formic acid/2% acetonitrile mix. Digests were analyzed by nano-scale capillary LC-MS/MS using an Ultimate U3000 HPLC and C18 Acclaim PepMap100 nanoViper (Thermo Scientific Dionex). LC-MS/MS data were searched against a protein database (UniProt KB) with the Mascot search engine program (Matrix Science). MS/MS data were validated using the Scaffold program (Proteome Software). MALDI-TOF mass spectrometric measurements were carried out in positive ion mode on an Ultraflex III TOF-TOF instrument (Bruker Daltonik), using sinapinic acid (Sigma) as matrix.

Samples were prepared as described previously [10 (link)] with some modifications. Briefly, a modified Lowry protein assay was used to measure total protein concentrations in the individual and pooled sample in each group [25 ]. Dried pooled samples were reconstituted in acetonitrile (ACN) with 5% (v/v) trifluoroacetic acid (TFA) before mixing with an equal volume of MALDI matrix (10 mg/mL α-cyano-4-hydroxycinnamic acid in 100% ACN containing 5% TFA). Sixteen replicates were spotted on to MALDI target plates (Bruker Daltonics, Billerica, MA, USA). Mass spectra were obtained using an Ultraflex III TOF/TOF instrument (Bruker Daltonics) in a linear positive mode within a mass range of 1000 to 20,000 Da. An external calibration was performed using a Proteo-Mass Peptide and Protein MALDI-MS Calibration Kit (Sigma Aldrich, St. Louis, MO, USA). A total of 500 laser shots at 50 Hz were used to generate each spectrum. Peptide mass spectra were processed using flexAnalysis v. 3.3 software, whereas the PMF and PCA of target mass spectra between 1000 and 20,000 Da were analyzed using ClinProTools v. 3.0 software [26 (link)–28 (link)]. The reliability and the accuracy of the candidate peak selection were evaluated by > 90% recognition capability and cross-validation values [28 (link)].