Autoflex speed maldi tof tof mass spectrometer

The samples were analyzed by SDS-PAGE using Novex 10–20% Tricine precast gels and Tris/Tricine SDS Running buffer (100 mM Tris base; 100 mM Tricine; 0.1% SDS; pH = 8.3). 20 µL of each sample were mixed with 5 µL of gel loading buffer (1.66 M Tris; 44.4% glycerol; 9.3% SDS; 9.3% ß-mercaptoethanol; 0.093% Coomassie Blue). 10 µL of each mixture were loaded, and the gel was run at 70 V for 15 min, followed by 120 V for 1 h. The gel was stained with InstantBlue^TM overnight for optimal band detection. Gel bands of interest were excised and subjected to in-gel digestion. Gel pieces (1 × 1 mm²) were washed twice in 50 mM ammonium bicarbonate (NH₄HCO₃)/50% acetonitrile (ACN), followed by dehydrated in 100% ACN. Digestion was performed by adding 50 mM NH₄HCO₃ with 12 ng/µL sequencing-grade modified trypsin (Promega, Madison, WI, USA), incubation on ice for 4 h before overnight incubation at 37 °C. The next day trifluoroacetic acid (TFA) was added to a final concentration of 0.5% and the peptide containing solution above the gel pieces was withdrawn and used for further analysis. Mass spectrometry analysis was performed in reflector positive mode on an Autoflex Speed MALDI TOF/TOF mass spectrometer (Bruker Daltonics, Bremen, Germany). All peptide mass spectra were externally calibrated using Bruker Peptide Calibration Standard II.

Matrix Assisted Laser Desorption Ionization Time of Flight (MALDI TOF) MS data was acquired on Bruker Daltonics (Billerica MA) Autoflex Speed MALDI TOF-TOF mass spectrometer using FlexControl v.3.4 software and low mass range data acquisition methods supplied by the manufacturer. Unless otherwise indicated, mass spectra were acquired in the positive linear mode, 750–7000 m/z mass range using the laser repetition rate of 2 kHz. Between 2000 and 10,000 single shot spectra were collected from individual microarray spots using the random walk method. The instrument laser power was typically between 20–50%, laser attenuator offset 30%, attenuator range 20%. The voltage settings were 19.50 kV (ion source 1), 18.35 kV (ion source 2) and 6.0 kV (lens). The pulsed ion extraction was 130 ns. The detector gain voltage was 4.0X or 2910 V.

Assays were performed using the same basic mixture as described above, except that 1 mM of an adapted form of the EA2 peptide with an C-terminal arginine rather than lysine residue (PTTDSTTPAPTTR) was employed. The products were analysed using a Bruker Autoflex Speed MALDI ToF/ToF mass spectrometer (equipped with a Smartbeam™-II laser) in positive mode; the samples were first dried under vacuum on a steel plate and then 6-aza-2-thiothymine (ATT) was applied twice as matrix. Using the LIFT mode, fragmentation of parent ions was performed by laser-induced dissociation and 4000 shots were sampled. MS/MS spectra were processed with the manufacturer's software (Bruker Flexanalysis 3.3.80) using the SNAP algorithm with a signal/noise threshold of 3 (four-times smoothed).

The spectra obtained with MALDI-TOF mass spectrometry were acquired in positive mode by a reflected Auto Flex Speed MALDI-TOF/TOF mass spectrometer (Bruker Daltonics, Bremen, Germany). Data were acquired in a mass range from 700–900 m/z with 1500 laser shots in different oocyte regions. The laser was applied until all signs had disappeared in the region of interest due to sample desorption. The laser intensity was standardized at 40% for the spectrum acquisition in all the samples. Spectra were centered and aligned using mMass 5.5.0 software [30 (link)]. The most intense ions upon the detection of peaks corresponding to isotopic distributions were considered as the starting point corresponding to the lipid ions. For the experiments, a total of 10–15 oocytes per group of fresh oocytes from the four different systems were used; no vitrified and warmed oocytes were evaluated.

Mass spectrometric analysis of pantocin wh-1 was performed through an ABI4800 MALDI-TOF device with a 337 nm nitrogen laser for desorption/ionization. The saturated matrix solution used for MALDI-TOF MS was α-cyano-4-hydroxycinnamic acid (CHCA) in 50% aqueous acetonitrile containing 0.1% v/v TFA [28 (link),29 (link)]. Positive ion detection and linear mode were used for MALDI-TOF mass spectra. Proteolytic digestion of pantocin wh-1 was monitored with a Bruker Autoflex Speed MALDI-TOF/TOF mass spectrometer. The sample preparation method was the same as that outlined above.

The differentially expressed protein spots were excised from the gel manually and washed with double-distilled water twice. The gel slices were destained, dehydrated, and digested with trypsin. The digested protein peptides were analysed over a mass range of 800–4,000Da using an Autoflex speed™ MALDI-TOF-TOF mass spectrometer (Bruker Daltonics, Bremen, Germany). Subsequently, the obtained PMF data were searched against the NCBI nr database and Swiss-Port database using MASCOT software (Mascot Wizard 1.2.0, Matrix Science Ltd., www.matrixscience.com). The parameters were set as follows: carbamidomethylation of cysteine and oxidation of methionine; peptide charge state of +1 and peptide mass tolerance of 0.5Da; a maximum of one for missed cleavages and monoisotopic.

Purified samples of Hjc_15-6, produced using complex (LB) medium or defined (mAT) medium as described previously, were analysed by mass spectrometry (MS). The protein sample concentration was adjusted to 5 mg ml⁻¹ and it was frozen in liquid nitrogen and stored frozen at −80°C prior to MS analysis. As a precaution, the samples were spun down after thawing on ice.
MS spectra were acquired using an Autoflex Speed MALDI–TOF/TOF mass spectrometer (Bruker Daltonics) in positive linear mode. 0.5 µl matrix solution consisting of 5 mg ml⁻¹ α-cyano-4-hydroxycinnamic acid, 80%(v/v) aceto­nitrile, 0.1%(w/v) trifluoroacetic acid (TFA) was added to 1 µl Hjc_15-6 sample on a MALDI stainless-steel plate. A total of 5000 laser shots were collected per spectrum and were calibrated using the Protein I calibration standard (Bruker Daltonics) containing six internal standard proteins (insulin, m/z 5734.52; cytochrome c, m/z 6181.05; myoglobin, m/z 8476.66; ubiquitin I, m/z 8565.76; cytochrome c, m/z 12 360.97; myoglobin, m/z 16 952.31).