The predicted molecular weight of the expressed fusion protein was confirmed using MALDI-TOF mass spectrometry. Purified pG_EAK was diluted 10 times to a final concentration of 5.46 μM, with 40 μL of this diluted solution combined with 10 μL of 0.5% TFA MilliQ-water. This protein solution was directly applied to target of Applied Biosystems Voyager DE-STR MALDI-TOF mass spectrometer.
Voyager de str maldi tof mass spectrometer
Manufactured by Thermo Fisher Scientific
Sourced in United States
The Voyager DE-STR MALDI-TOF mass spectrometer is a laboratory instrument designed for the analysis of biomolecules, such as proteins and peptides. It utilizes matrix-assisted laser desorption/ionization (MALDI) technology coupled with time-of-flight (TOF) mass spectrometry to provide accurate mass measurements of these analytes.
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Lab products found in correlation
200 μm optical fibers, by OceanOptics (2 mentions)
Nns450 sem, by Thermo Fisher Scientific (2 mentions)
Hl 2000 tungsten halogen light, by OceanOptics (2 mentions)
Mono q 5 50 gl, by GE Healthcare (1 mentions)
Ammonium bicarbonate, by Promega (1 mentions)
Data explorer software, by Thermo Fisher Scientific (1 mentions)
Tm 1000 tabletop sem, by Hitachi (1 mentions)
Cary 50 uv vis spectrophotometer, by Agilent Technologies (1 mentions)
Data explorer software package, by Thermo Fisher Scientific (1 mentions)
P10 c18 ziptips, by Merck Group (1 mentions)
Qstar mass spectrometer, by Thermo Fisher Scientific (1 mentions)
1200 series reverse phase hplc, by Agilent Technologies (1 mentions)
Synapt g2 hdms q tof mass spectrometer, by Waters Corporation (1 mentions)
Mascot search engine program, by Matrix Science (1 mentions)
Sequazyme peptide mass standard kit, by Thermo Fisher Scientific (1 mentions)
Trypsin, by Promega (1 mentions)
16 protocols using voyager de str maldi tof mass spectrometer
1
MALDI-TOF Protein Mass Spectrometry
2
Protein Identification and Domain Analysis
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The identity of full-length recombinant lymphostatin was confirmed by in-gel protein digest and peptide analysis. Excised gel-bands were incubated at a porcine trypsin:lymphostatin ratio of ∼1:30, in 50 mm ammonium bicarbonate overnight at 32 °C (Promega). Peptides were identified by matrix-assisted laser desorption ionization (MALDI) mass spectroscopy on a Voyager DE-STR MALDI-TOF mass spectrometer (Applied Biosystems) using an α-cyano-4-hydroxycinnamic acid matrix. The spectral data were processed using Data Explorer software (Applied Biosystems) and the MASCOT NCBInr database searched against the peptide mass map (Matrix Science). To investigate the domain structure of lymphostatin, purified protein was incubated with trypsin at a ratio of 375:1, at 21 °C, to give limited digestion. Aliquots were removed at 1, 2, 3, and 4 h and the reaction stopped by boiling samples adjusted with 2 mm EDTA and 2 mm PMSF in SDS-PAGE loading buffer. Digest products were separated by SDS-PAGE and individual bands were subjected to in-gel tryptic digestion and MALDI-TOF mass spectroscopy as described above. Peptide masses were compared with the sequence of full-length rLifA using GPMAW 9.2 software, mass tolerance 50 ppm (19 (link)). Fragment F1 was purified to homogeneity from other digest products by ion-exchange chromatography (Mono-Q 5/50 GL; GE Healthcare) as described above.
3
MALDI-TOF Analysis of Aesculin Glycoside
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Purified 3-O-β-d -glycosyl aesculin in water was mixed 1:1 (v/v) with 2,5-dihydroxybenzoic acid (1 mg/ml). The mixed solution (1 μl) was spotted onto a stainless steel plate and dried at room temperature. The mass spectra were obtained in the positive linear mode with delayed extraction (average of 150 laser shots) with a 65-kV acceleration voltage by using a Voyager DE-STR MALDI-TOF mass spectrometer (Applied Biosystems, Foster City, CA).
4
Characterization of Nanoparticle Samples
5
Limited Proteolysis of Actin-Myosin Complexes
6
MALDI-TOF/MS Analysis of Differentially Expressed Proteins
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For the identification of differentially expressed proteins, MALDI-TOF/MS analysis of selected spots was performed as previously described (33 (link)). Selected spots sliced from the gel were stained with CBB and then digested with trypsin. Extracted peptides were subjected to MALDI-TOF/MS analysis on a Voyager DE-STR MALDI-TOF mass spectrometer (Applied Biosystems, Foster City, CA, USA). All acquired spectra of samples were processed using Voyager™ 5.1 software (Applied Biosystems) in the default mode. Averages of 500 spectra were obtained for each sample, and scans were performed twice. Spectra were calibrated automatically upon acquisition using an external 3-point calibration. Peaks were manually assigned using the DATA Explorer™ software package (Applied Biosystems), and spectra were used to search against non-redundant protein sequence databases available online (SWISS-PROT and/or NCBInr Data Bank). The peptide mass fingerprinting data were applied to ProFound and MASCOT search engines (http://prowl.rockefeller.edu/ ; http://www.matrixscience.com/search_form_select.html ) for protein identification based on ProFound and MASCOT scores. The Z score of ProFound is the distance to the population mean in units of the standard deviation.
7
PSMA Enzymatic Digest Analysis
8
MALDI-TOF Protein Identification Protocol
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Five protein spots chosen for excision were carried out using the solution-phase nitrocellulose method. Briefly, α-cyano-4-hydro-xycinnamic acid (40 mg/ml) and nitrocellulose (20 mg/ml) were prepared separately in acetone, and mixed with 2-propanol at a ratio of 2:1:1. Then, 2 μl of the mixture was added to 2 μl of the peptide sample solution, and 1 μl of each solution was spotted on a MALDI plate for 5 min. The plates were then washed with 5% formic acid, followed by water. Dried spots were analyzed using a Voyager-DE STR MALDI-TOF mass spectrometer (Applied Biosystems). Proteins were identified by peptide mass finger printing using the program Aldente (http://www.expasy.org/ ).
9
Spectroscopic and Microscopic Analysis of Samples
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Absorbance spectra were collected using a USB 2000 + UV-Vis spectrometer with illumination from a HL-2000 Tungsten-Halogen light source guided through 200 μm optical fibers (Ocean Optics, Dunedin, FL). Mass spectra were acquired as an average of 60 laser shots using a Voyager-DE STR MALDI-TOF mass spectrometer (Applied Biosystems, Framingham, MA) operating in positive reflector mode at an accelerating voltage of 20 kV. Scanning electron microscopy (SEM) was performed on an FEI NNS450 SEM (Hillsboro, OR) in CFAMM at UC Riverside. For SEM analysis, all samples were sputtered with a Pt/Pd mixture for 30 s to enhance contrast and prevent titania sample charging. Atomic force microscopy was conducted on a Veeco Dimension 5000 (Santa Barbara, CA) under tapping mode at a scan rate of 1 Hz.
10
Protein Identification by Mass Spectrometry
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The protein spots of interest were destained and then subjected to in-gel digestion by TPCK-trypsin for 12 h at 37°C. The tryptic peptides were purified by ZipTip C18 tips before the MS analysis. Most of the tryptic peptide samples were analyzed using a Voyager DE STR MALDI TOF mass spectrometer (Applied Biosystems). A saturated CHCA solution was used as a matrix, which was mixed with peptide samples and then loaded on the sample plate. Besides MALDI TOF MS, nano-ESI-MS/MS was performed on some of the tryptic peptide samples by using a QSTAR mass spectrometer (Applied Biosystems). The tryptic peptide sample was loaded onto a PicoTip emitter and then ionized through an external nanoelectrospray ion source. The ions with multiple charge states were manually selected for MS/MS analysis to obtain the data for their fragment ions. Both MS and MS/MS data were searched against the human subset in the SwissProt database using the MASCOT software to identify the protein spots.
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