Spots from SYPRO-stained gilthead seabream skin mucus 2-DE gels (n = 6) were picked, excised and subjected to in-gel reduction, alkylation, and tryptic digestion using 2–10 ng/μl trypsin (V511A; Promega) as described elsewhere [66 (link)]. Peptide mixtures containing 0.1% formic acid were loaded onto a nanoACQUITY UltraPerformance LC (Waters), containing a 5 μm Symmetry C18 Trap column (180 μm × 20 mm; Waters) in front of a 1.7 μm BEH130 C18 analytical column (100 μm × 100 mm; Waters). Peptides were separated with a gradient of 5–95% acetonitrile, 0.1% formic acid, with a flow of 0.4 μl min−1 eluted to a Q-TOF Ultima mass spectrometer (Micromass/Waters). The samples were run in data dependent tandem mass spectrophotometry (MC/MC) mode. Peak lists were generated from MS/MS by Mascot Distiller Workstation and submitted to MASCOT search engine (version 2.5.1) and searched against NCBInr with the following parameters: maximum one missed cleavage by trypsin, peptide mass tolerance 100 ppm, MS/MS ion tolerance set to 0.1 Da, carbamidomethylation of cysteine selected as fixed modification and methionine oxidation as variable modification. Protein hits not satisfying a significance threshold (p < 0.05) or with low sequence coverage were further searched against Swissprot and vertebrate EST (expressed sequence tags) databases, taxonomy Actinopterygii.
Beh 130 c18 analytical column
Manufactured by Waters Corporation
The BEH 130 C18 analytical column is a high-performance liquid chromatography (HPLC) column designed for the separation and analysis of a wide range of analytes. The column features a 1.7 μm particle size and a 130 Å pore size, providing efficient separation and high-resolution chromatography. The C18 bonded phase offers excellent retention and selectivity for a variety of organic compounds.
Automatically generated - may contain errors
Lab products found in correlation
Synapt g2 hdms mass spectrometer, by Waters Corporation (2 mentions)
Nanoacquity ultraperformance lc system, by Waters Corporation (2 mentions)
Nanoacquity ultra performance lc, by Waters Corporation (1 mentions)
Symmetry c18 trap column, by Waters Corporation (1 mentions)
Trypsin, by Promega (1 mentions)
Sequencing grade trypsin, by Merck Group (1 mentions)
Rapigest surfactant, by Waters Corporation (1 mentions)
Proteinlynx global server software, by Waters Corporation (1 mentions)
Symmetry c18, by Waters Corporation (1 mentions)
4 protocols using beh 130 c18 analytical column
1
Gilthead Seabream Skin Mucus Proteome
2
Proteomic Analysis of Photosystem I
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The Liquid Chromatography–Tandem Mass Spectrometry (LC-MS-MS) was performed on the native and the His6-PsaD-PSI samples, as described in Krupnik et al. (2013) [57 (link)]. Briefly, the PSI samples were precipitated in acetone and dissolved in 0.1% (w/v) RapiGestTM surfactant (Waters, Milford, MA, USA) in the presence of 50 mM ammonium bicarbonate. Following the reduction and alkylation of the Cys groups, the proteins were digested using sequencing grade trypsin (Sigma-Aldrich) overnight at 30 °C. Digested peptides were separated using a nanoAcquity Ultra Performance LC system connected to a SYNAPT G2 HDMS mass spectrometer (Waters). Peptides were loaded onto a Symmetry® C18 (5 μm; 180 μm × 20 mm) trap column (Waters). The specifically captured peptides were then separated on a BEH 130 C18 analytical column (1.7 µm; 75 µm × 200 mm) (Waters). The elution was performed with a linear gradient of acetonitrile in 0.1% formic acid. Each sample was analyzed at least three times. Protein identification was performed using a ProteinLynx Global SERVER software (PLGS; version 2.2.5; Waters, Milford, MA, USA).
3
Peptide Profiling by LC-MS/MS
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The resulting
peptide mixtures
were analyzed by reversed-phase chromatography using a Waters nanoAcquity
UPLC pump coupled online to an LTQ/Orbitrap mass spectrometer. Peptides
were loaded onto a Symmetry 180 μm × 2 cm C18 trap column,
which was washed and placed in-line with a Waters BEH130 C18 analytical
column (75 μm × 25 cm). As the peptides elute off the column
they are ionized into the gas phase of the mass spectrometer. The
peptide ion masses are measured with resolution of 60 000 at m/z 400 by the Orbitrap, followed by MS/MS
fragmentation of the six most intense precursors with charge state
≥2 above an intensity threshold of 10 000 in the LTQ
ion trap. Dynamic exclusion was used to avoid repeat sequencing, with
a repeat count of one and exclusion duration of 30 s for precursors
within 10 ppm. The MS/MS spectra were extracted and searched against
the human Uniprot (release 2013_01) using the MASCOT search engine,
version 2.2 (Matrix Science) with a mass tolerance on precursor ions
of 25 ppm and 0.5 Da for fragment ions. The following variable modifications
were considered: carbamidomethyl-Cys, methionine oxidation, and pyro-glutamic
acid for N-terminal Gln. Peptide identifications were accepted at
a 1% FDR threshold based on a reversed protein database search. Protein
assembly and label-free quantification by spectral counting was performed
using Isoform Resolver.51 (link)
peptide mixtures
were analyzed by reversed-phase chromatography using a Waters nanoAcquity
UPLC pump coupled online to an LTQ/Orbitrap mass spectrometer. Peptides
were loaded onto a Symmetry 180 μm × 2 cm C18 trap column,
which was washed and placed in-line with a Waters BEH130 C18 analytical
column (75 μm × 25 cm). As the peptides elute off the column
they are ionized into the gas phase of the mass spectrometer. The
peptide ion masses are measured with resolution of 60 000 at m/z 400 by the Orbitrap, followed by MS/MS
fragmentation of the six most intense precursors with charge state
≥2 above an intensity threshold of 10 000 in the LTQ
ion trap. Dynamic exclusion was used to avoid repeat sequencing, with
a repeat count of one and exclusion duration of 30 s for precursors
within 10 ppm. The MS/MS spectra were extracted and searched against
the human Uniprot (release 2013_01) using the MASCOT search engine,
version 2.2 (Matrix Science) with a mass tolerance on precursor ions
of 25 ppm and 0.5 Da for fragment ions. The following variable modifications
were considered: carbamidomethyl-Cys, methionine oxidation, and pyro-glutamic
acid for N-terminal Gln. Peptide identifications were accepted at
a 1% FDR threshold based on a reversed protein database search. Protein
assembly and label-free quantification by spectral counting was performed
using Isoform Resolver.51 (link)
4
Silver-stained Protein Identification by Mass Spectrometry
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Silver-stained bands were excised, de-stained with 15 mM potassium hexacyanoferrate (III), 50 mM sodium thiosulphate solution, and subjected to in-gel protease digestion using trypsin in 100 mM ammonium bicarbonate overnight at 30 °C. After extraction from gel pieces, peptides were dried in a SpeedVac concentrator, dissolved in 1% (v/v) trifluoroacetic acid, 5% (v/v) acetonitrile mixture, and separated using a nanoAcquity Ultra Performance LC system connected to a Synapt G2 HDMS mass spectrometer (Waters). Peptides were trapped on Symmetry® C18 (5 μm; 180 μm × 20 mm) column (Waters) and separated on a BEH 130 C18 analytical column (1.7 μm; 75 μm × 200 mm) (Waters). The elution was performed with a linear gradient of acetonitrile in 0.1% formic acid. Protein identification was performed using ProteinLynx Global Server (PLGS version 2.4) software (Waters, Milford, MA, USA).
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