An automated spot picker was used to recover the spots of interest from the 2D gels to corresponding siliconized Eppendorf tubes. The in-gel proteolytic digestion was performed following a protocol from Shevchenko et al. [11 ]. For MALDI-TOF/TOF MS, the peptides were mixed with an equal volume of MALDI matrix (10 mg·mL−1α-cyano-4-hydroxycinnamic acid (Sigma) saturated with 50% acetonitrile/0.1% formic acid) and spotted onto the MALDI sample plates. MS measurements were carried out on an ABI 4700 Proteomics Analyzer with delayed ion extraction (Applied Biosystems, Foster City, CA). The MS/MS setting was 2 kV positive mode (CID on) and 5 monoisotopic precursors selected (S/N > 200). The calibration was performed using the calibration mixture 1 of 4,700 Proteomics Analyzer calibration mixtures (Applied Biosystems, Foster City, CA). The spectra were calibrated externally using P14R and insulin chain B oxidized from bovine pancreas (Sigma). Autolytic peaks of trypsin served as internal standards for mass calibration. All PMFs obtained were used to search the NCBInr database using Mascot Daemon (Matrix Science, London, UK) as a client attached to the Mascot search protocol. The database searches had peptide mass tolerance set at approximately ±0.1 Da and one missed cleavage site. The protein spots were annotated by searching gene ontology (GO) (http://www.geneontology.org/ ).
Maldi matrix
Manufactured by Merck Group
Sourced in Germany, Macao, United States, United Kingdom
MALDI matrix is a chemical compound used in matrix-assisted laser desorption/ionization (MALDI) mass spectrometry. It functions as a carrier and facilitator for the analyte, enabling efficient ionization and detection of the sample.
Automatically generated - may contain errors
Lab products found in correlation
α cyano 4 hydroxycinnamic acid, by Merck Group (2 mentions)
Abi 4700 proteomics analyzer, by Thermo Fisher Scientific (1 mentions)
Insulin b chain, by Merck Group (1 mentions)
Mascot daemon, by Matrix Science (1 mentions)
Citrinin, by Merck Group (1 mentions)
Iron free ferrichrome, by Santa Cruz Biotechnology (1 mentions)
Amberlite xad 16, by Merck Group (1 mentions)
Sephadex lh 20, by Merck Group (1 mentions)
Trypsinogen, by Merck Group (1 mentions)
Apomyoglobin, by Merck Group (1 mentions)
Hematoxylin stain, by Merck Group (1 mentions)
Acetic acid, by Thermo Fisher Scientific (1 mentions)
Methanol, by Thermo Fisher Scientific (1 mentions)
Chloroform, by Thermo Fisher Scientific (1 mentions)
Mtp anchorchip maldi target, by Bruker (1 mentions)
Insulin, by Merck Group (1 mentions)
Glycerol, by Merck Group (1 mentions)
Cytochrome c, by Merck Group (1 mentions)
Ubiquitin, by Merck Group (1 mentions)
Hematoxylin and eosin h e staining kit, by Thermo Fisher Scientific (1 mentions)
7 protocols using maldi matrix
1
Proteomic Analysis via MALDI-TOF/TOF
2
Comprehensive Chemical Sourcing for Research
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All
chemicals used for YES and M1 media,
Universal MALDI matrix, citrinin, sephadex-LH20, and Amberlite XAD-16
were purchased from Sigma-Aldrich except for Instant Ocean aquarium
salts (Pentair Aquatic Eco-Systems, Inc.). J. T. Baker organic solvents
were purchased from Avantor Performance Materials, Inc. Iron-free
ferrichrome was purchased from Santa Cruz Biotechnology Inc.
chemicals used for YES and M1 media,
Universal MALDI matrix, citrinin, sephadex-LH20, and Amberlite XAD-16
were purchased from Sigma-Aldrich except for Instant Ocean aquarium
salts (Pentair Aquatic Eco-Systems, Inc.). J. T. Baker organic solvents
were purchased from Avantor Performance Materials, Inc. Iron-free
ferrichrome was purchased from Santa Cruz Biotechnology Inc.
3
Sample Preparation for MALDI-TOF
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Chemicals, cell culture media and buffers, for sample preparation, solvents and the MALDI matrix 2,5-dihydroxybenzoic acid (DHB), were (unless stated otherwise) obtained in the highest commercially available purity from Sigma-Aldrich Chemie GmbH (Taufkirchen, Germany) and used as supplied.
4
MALDI-TOF MS Protein Analysis
5
Mass Spectrometry Tissue Imaging
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Analytical grade solvents, acetonitrile (ACN) and water (H2O) were purchased from Fisher Scientific (Pittsburgh, PA, USA). Histological grade xylene and ethanol, and the MALDI matrix, alpha-cyano-4-hydroxycinnamic acid (CHCA) were purchased from Sigma Aldrich (St. Louis, MO). Hematoxylin and eosin (H&E) staining kit, citraconic anhydride for antigen retrieval and positively charged glass slides were purchased from Thermo Scientific (San Jose, CA, USA). Recombinant Peptide N-Glycosidase F (PNGase F) was obtained from the laboratory of Dr. Richard Drake (Charleston, NC, USA) and the commercial version, PNGase F PRIME™, was purchased from N-zyme Scientifics (Doylestown, PA, USA).
6
MALDI-TOF Bacterial Identification Protocol
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The isolated and purified bacterial cultures were identified using a MALDI-TOF Biotyper mass spectrometry (Brucker Daltonics, Bremen, Germany).
A small amount of a purified culture was re-suspended in 300 μL of distilled water. Subsequently, 900 μL of 99.8% ethanol (Centralchem, Bratislava, Slovakia) was administered and the mixture was centrifuged (920× g, 2 min, 20 °C). The resulting pellet was allowed to dry freely and then mixed vigorously with 30 μL of 70% formic acid (Sigma-Aldrich, St. Louis, MO, USA) and 30 μL of acetonitrile (Sigma-Aldrich, St. Louis, MO, USA). The samples were subsequently centrifuged at 1096× g at 20 °C for 2 min. Then, 1 μL of the supernatant was placed on the MALDI identification plate, allowed to dry freely and subsequently covered with a working solution of MALDI matrix, composed of acetonitrile, ultrapure water, trifluoroacetic acid, and cinnamic acid (Sigma-Aldrich, St. Louis, MO, USA). Identification of the isolates was carried out with a Microflex LT instrument and the flexControl software version 3.4. The spectra obtained by MALDI-TOF were linked with the MALDI Biotyper Bruker Taxonomy database (Bruker Daltonics, Bremen, Germany) [10 (link)].
A small amount of a purified culture was re-suspended in 300 μL of distilled water. Subsequently, 900 μL of 99.8% ethanol (Centralchem, Bratislava, Slovakia) was administered and the mixture was centrifuged (920× g, 2 min, 20 °C). The resulting pellet was allowed to dry freely and then mixed vigorously with 30 μL of 70% formic acid (Sigma-Aldrich, St. Louis, MO, USA) and 30 μL of acetonitrile (Sigma-Aldrich, St. Louis, MO, USA). The samples were subsequently centrifuged at 1096× g at 20 °C for 2 min. Then, 1 μL of the supernatant was placed on the MALDI identification plate, allowed to dry freely and subsequently covered with a working solution of MALDI matrix, composed of acetonitrile, ultrapure water, trifluoroacetic acid, and cinnamic acid (Sigma-Aldrich, St. Louis, MO, USA). Identification of the isolates was carried out with a Microflex LT instrument and the flexControl software version 3.4. The spectra obtained by MALDI-TOF were linked with the MALDI Biotyper Bruker Taxonomy database (Bruker Daltonics, Bremen, Germany) [10 (link)].
7
MALDI Matrix Application and Data Acquisition
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Sections were coated with MALDI matrix (N-(1-naphthyl) ethylenediamine dihydrochloride monomethanolate (NEDC, Sigma Aldrich, UK), 7 mg/mL in 1:1 methanol: deionised water) using a SunCollect (SunChrom Friedrichsdor, Germany) sprayer. Slides were coated with 6 layers of NEDC matrix, spraying each layer at 3.5 µL/min. Mass spectra was collected on a Bruker Autoflex III fitted with a "Smartbeam" Nd:YAG laser (355 nm, 1 KHz) (Bruker, Germany). Image acquisition was performed at a pixel size of 30 µm × 30 µm in "Raster Image" mode with mass range of 400-1200 Da.
Prior to analysis the instrument was calibrated using the Bruker prepared mtp standard target peptide calibration mix 2.
Prior to analysis the instrument was calibrated using the Bruker prepared mtp standard target peptide calibration mix 2.
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