Fresh frozen brain tissues (n = 3–6 mice per group) were sectioned at −20°C using a Microm HM 525 cryostat (Thermo Fisher Scientific). Serial tissue sections of 10 μm thickness were thaw-mounted directly onto stainless steel MALDI plates and stored in −80 °C. Prior to analysis, the plate was dried under vacuum for two minutes to remove residual moisture. The plate was then submerged in cold 50 mM ammonium formate for 20 s, the dried again. Matrix application was done using a home-built sublimation apparatus. Here, 1,5-Diaminonaphthalene (DAN) was sublimed on the MALDI plate to obtain the matrix density (0.18–0.22 mg/cm2). Mass spectrometry imaging was performed using a model 4800 Plus MALDI- TOF/TOF Analyzer (Sciex) equipped with a 200 Hz Nd-YAG (355 nm) pulsed laser. Data were acquired in the negative ion reflectron mode. The number of laser shots per pixel was set to 50 and the raster distance between each pixel was set to 100 μm using the 4800 Imaging Tool v. 3.2 (https://ms-imaging.org/wp/4000-series-imaging). The acquired data were processed using MSiReader where MS images were normalized by their total ion current (TIC) (Alexandrov, 2012 (link)). Lipid assignments were made by comparing accurate mass measurements to the LIPID MAPS database.
4800 plus maldi tof tof analyzer
Manufactured by AB Sciex
Sourced in United States
The 4800 Plus MALDI TOF/TOF Analyzer is a mass spectrometry instrument manufactured by AB Sciex. It is designed to perform matrix-assisted laser desorption/ionization (MALDI) time-of-flight (TOF) analysis and tandem mass spectrometry (MS/MS) analysis.
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Lab products found in correlation
α2 3 6 8 9 neua, by New England Biolabs (2 mentions)
Calmix 5 opti tof high resolution tis calibration insert, by AB Sciex (2 mentions)
2 4 6 trihydroxyacetophenone monohydrate, by Merck Group (2 mentions)
Pngase f, by New England Biolabs (2 mentions)
Ziptip, by Merck Group (2 mentions)
4800 maldi tof tof analyzer, by AB Sciex (2 mentions)
Pierce bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Procise model 492 edman micro sequencer, by Thermo Fisher Scientific (2 mentions)
Model 140c pth amino acid analyzer, by Thermo Fisher Scientific (2 mentions)
Microm hm 525, by Thermo Fisher Scientific (1 mentions)
Mascot software v 2, by Matrix Science (1 mentions)
Opti tof 384 well insert, by AB Sciex (1 mentions)
Mascot software, by Matrix Science (1 mentions)
Ziptip pipette tip, by Merck Group (1 mentions)
Trypsin gold, by Promega (1 mentions)
Md 4010, by Jasco (1 mentions)
Mini protean 3 electrophoresis apparatus, by Bio-Rad (1 mentions)
Pu 4180, by Jasco (1 mentions)
Ft ir 4100, by Jasco (1 mentions)
Microtof 2, by Bruker (1 mentions)
28 protocols using 4800 plus maldi tof tof analyzer
1
MALDI-TOF Imaging of Mouse Brain Lipids
2
Silver Staining and MALDI-TOF Protein Identification
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After silver staining of equal amounts of protein lysate from EVs, representative bands were selected and excised from the gel and washed with 50% acetonitrile in 50 mM ammonium bicarbonate. Reduction and alkylation were sequentially performed with 25 mM DTT at 56ºC and 55 mM IAA at RT in the dark, both for 20 min. Proteins were digested with 20 ng of trypsin for 3 h at 37ºC in the presence of 0.01% surfactant (Promega). The resulting peptides were analysed on a MALDI mass spectrometer (4800 Plus MALDI TOF/TOF Analyzer, SCIEX) as described in [43 ]. Proteins were identified by Peptide Mass Fingerprint using the Mascot software v2.6.1 (Matrix Science, London, UK). Protein searches were performed against the UniProt protein sequence database for the Homo sapiens and Bos taurus taxonomic selections (2017_11). The established search parameters were: maximum of two missed trypsin cleavages, cysteine carbamidomethylation (fixed modification) and methionine oxidation (variable modification). The peptide tolerance was 25 ppm.
3
Quantification of PEG-Peptide Conjugates by MALDI-TOF MS
4
Protein Identification by MALDI-TOF Mass Spectrometry
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Following silver staining, the major visible band was excised from the gel, washed with 50% acetonitrile in 50 mM ammonium bicarbonate, digested for 3 h with 20 ng of trypsin at 37 °C, and analyzed on a MALDI mass spectrometer (4800 Plus MALDI TOF/TOF Analyzer, SCIEX) as described in [61 (link)]. Proteins were identified by Peptide Mass Fingerprint using the Mascot software v2.5.1 (Matrix Science, London, UK). Protein searches were performed against the UniProt protein sequence database for the Homo sapiens taxonomic selection (2017_08, UP000005640 reviewed proteome, canonical proteins). The established search parameters were: up two missed cleavages allowed, cysteine carbamidomethylation as a fixed modification and methionine oxidation as a variable modification. The peptide tolerance was 20 ppm. Protein scores greater than 56 were considered to be significant (p < 0.05).
5
MALDI-TOF/TOF Bioactive Peptide Identification
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Peptides from SPH, DSPI and DSPH were detected and acquired by a MALDI-TOF/TOF mass spectrometer (4800 Plus MALDI TOF/TOF Analyzer, SCIEX, Framingham, MA). Each spectrum was the accumulated sum of at least 2000 laser shots within the ion range at m/z 700–5000. The samples were co-crystallized with HCCA matrix (4-hydroxycinnamic-α-cyano acid), in a stainless steel MALDI sample plate (Opti-TOF 384- Well insert, AB SCIEX, Framingham, MA) at room temperature. MS data was acquired in reflector positive mode. Some of the ion peptide peaks were further selected for MS/MS sequencing analysis. Combined Peptide Mass Fingerprint (PMF) + MS/MS analysis was performed with the Mascot software (Matrix Science, UK) using the UniProt protein sequencing database for Glycine max taxonomic selection. Methionine oxidation was selected as a variable modification.
To determine the bioactivity, all the identified peptides were searched against a database available online, BIOPEP (Minkiewicz et al., 2008 (link)). For SPH peptides which coincided with those reported in our previous study (Coscueta et al., 2016 (link)), the frequency of bioactive fragments occurrence in peptide sequence (A) was analysed: where a is the number of fragments with a given activity in a peptide sequence, and N is the number of amino acid residues of the peptide (Dziuba, Iwaniak, & Minkiewicz, 2003 ).
To determine the bioactivity, all the identified peptides were searched against a database available online, BIOPEP (Minkiewicz et al., 2008 (link)). For SPH peptides which coincided with those reported in our previous study (Coscueta et al., 2016 (link)), the frequency of bioactive fragments occurrence in peptide sequence (A) was analysed: where a is the number of fragments with a given activity in a peptide sequence, and N is the number of amino acid residues of the peptide (Dziuba, Iwaniak, & Minkiewicz, 2003 ).
6
MALDI-TOF MS Protein Identification
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Protein spots of interest from 2DE gel were manually excised using a clean razor blade and in-gel protein digestion was performed using Trypsin Gold (Promega, Massachusetts, USA) according to manufacturer's procedure. The extracted peptides were purified and concentrated using ZipTip® pipette tips (Millipore Corporation, Massachusetts, USA) following the manufacturer's instructions. Eluted peptides in 2.5 μl of 70 % acetonitrile (ACN)/0.1 % trifluoroacetic acid containing 10 mg/ml α-cyano-4-hydroxycinnamic acid were spotted directly onto MALDI plate for subsequent MALDI-TOF MS analysis by 4800 Plus MALDI TOF/TOF™ Analyzer (AB SCIEX, Massachusetts, USA). MS/MS scans were analyzed using Mascot Server (http://www.matrixscience.com ) to search against the NCBInr protein database (ftp://ftp.ncbi.nlm.nih.gov/blast/db/ ); choosing fungi as the taxonomic category. The following search parameters for sequence query were implemented: complete carbamidomethylation of cysteines and/or oxidation of methionines, unrestricted protein mass (monoisotopic mass values), peptide mass tolerance of ± 100 ppm, fragment mass tolerance of ± 0.2 Da, and maximum of one missed cleavage allowed. Protein scores are derived from ions scores as a non-probabilistic basis for ranking protein hits.
7
Enzymatic Release and Analysis of HIV-1 gp145 N-Glycans
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Enzymatic release of N-linked glycans from HIV-1 gp145 was carried out following the method of Küster et al. [32 (link)] with minor modifications. Briefly, excised bands containing 20 μg of gp145 were incubated with 5000 units of PNGase F (NEB) for 24 hrs. at 37°C [32 (link)]. For the analysis of oligosaccharides containing sialic acid, 20 μg of gp145 in solution were incubated with 50 units of α2–3,6,8,9 NEUA (NEB) for 24 hrs. at 37°C, prior to PNGase F digestion. Subsequently, glycans were extracted into LC-MS water in an ultrasonic bath. The extracted glycans were then dried in a vacuum concentrator and dissolved with water. For MALDI-ToF analysis, glycans were co-crystallized with a solution of 50% acetonitrile/50% 20 mM ammonium citrate containing 134 mM of 2′,4′,6′-Trihydroxyacetophenone monohydrate (Sigma-Aldrich). MALDI-ToF MS analysis of the N-glycans was performed in the reflector positive and negative ion mode using a 4800 Plus MALDI ToF/ToF Analyzer (AB Sciex) that was calibrated externally using the Calmix 5 Opti-ToF High-Resolution TIS Calibration Insert (AB Sciex). N-glycan assignments were carried out using GlycoWorkbench software and confirmed by MALDI-ToF post-source decay spectra (S2 Fig ) [33 (link)].
8
MALDI-TOF Proteomic Identification
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The bands of interest were cut from the gel and chopped into 1x1x1 mm pieces. The pieces were destained, and DTT and iodoacetamide were applied to reduce and block cysteines. The samples were trypsinized as described previously [57 (link)]. The dried-droplet method of sample preparation was employed and spectra were acquired in a 4800 Plus MALDI TOF/TOF analyzer (AB Sciex). Data were analyzed using an in-house running Mascot server 2.2.07 and matched against a database composed of the following background databases: baculovirus expression system, insect SF9 cell proteome database of common contaminants, and MPyV VP1 sequences. Cysteine carbamidomethylation was set as a fixed modification, and methionine oxidation and N, Q deamination were set as variable modifications. One missed cleavage site was allowed. Precursor accuracy was set to 50 ppm and the accuracy for MS/MS spectra to 0.25 Da.
9
MALDI-TOF/TOF Proteomic Workflow
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MALDI data was acquired using a 4800 Plus MALDI TOF/TOF Analyzer (AB Sciex) with the 4000 Series Explorer software version 3.5.3. The instrument was calibrated using the AB Sciex Mass Standards Kit. Data was first acquired in the MS positive-ion reflector mode at a fixed laser energy of 3200. A reflector interpretation method was created to run the MS-MS 1kv positive acquisition method in batch mode on all spots. The MS-MS was performed at 4200 fixed laser intensity on the 50 strongest precursor peaks discovered during the reflector mode acquisition. The exclusion list of common CHCA, trypsin and gel contaminant peaks was also included as a parameter in the interpretation method to improve for the selection of protein-specific precursor peaks. MS-MS data was analyzed with ProteinPilot software version 4.0.8 (AB Sciex) based on the paragon algorithm. MS-MS data was searched against the most recent Homo sapiens protein database from Uniprot (Swiss-Prot/TrEMBL annotated protein sequences).
10
MALDI-TOF Mass Spectrometry of Biomolecules
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