Peptide mixtures were analyzed by LC-MS MS using the LC/MSD Trap XCT Ultra (Agilent Technologies, CA, USA) equipped with a 1100 HPLC system and a chip cube (Agilent Technologies, CA, USA) as previously described [24 (link)]. Briefly, after loading, peptide mixture (8 μl in 0,2 % HCOOH) was pre-concentrated, washed at 4 μl/min in 40 nl enrichment column (Agilent Technologies chip) and separated on a RP-C18 column (75 μm × 43 mm) at a flow rate of 200 nl/min with a linear gradient of eluent B (2 % formic acid in acetonitryl) in eluent A (2 % formic acid) from 5 to 60 % in 60 min. Peptides were analyzed using data-dependent acquisition of MS scan (400–2000 m/z) followed by MS/MS scans of the three most abundant ions. Dynamic exclusion was used to acquire a more complete survey of the peptides. A permanent exclusion list of the most frequent peptide contaminants was included in the acquisition as previously described [25 (link)].
Lc msd trap xct ultra
Manufactured by Agilent Technologies
Sourced in United States
The LC/MSD Trap XCT Ultra is a liquid chromatography mass spectrometry (LC/MS) system designed for high-performance biomolecular analysis. It combines liquid chromatography with an ion trap mass spectrometer to provide accurate mass detection and identification of chemical compounds.
Automatically generated - may contain errors
Lab products found in correlation
1100 hplc system, by Agilent Technologies (4 mentions)
Chip cube, by Agilent Technologies (3 mentions)
4800 proteomics analyzer, by Thermo Fisher Scientific (2 mentions)
4800 plus maldi tof tof analyzer, by Thermo Fisher Scientific (2 mentions)
Ettan spot picker, by GE Healthcare (1 mentions)
Mascot software, by Matrix Science (1 mentions)
4 protocols using lc msd trap xct ultra
1
Peptide Analysis by LC-MS/MS
2
Mass Spectrometry-based Protein Identification
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The spots of interest were excised, hydrolyzed and the peptide mixtures analyzed by mass spectrometry, MALDI-MS and LC-MS/MS using respectively 4800 Plus MALDI TOF/TOF™ Analyzer, Applied Biosystems 4800 Proteomics Analyzer (Applied Biosystems, Framingham, MA, USA) and a LC/MSD Trap XCT Ultra (Agilent Technologies, Palo Alto, CA, USA) equipped with a 1100 HPLC system and a chip cube (Agilent Technologies). MALDI spectra were acquired in the positive ion reflector mode using delayed extraction in the mass range between 800 and 4000 Da. LC-MSMS analysis was performed using data-dependent acquisition of one MS scan followed by MS/MS scans of the three most abundant ions in each MS scan. Raw data analyses were converted into a Mascot format text to identify proteins using Matrix Science software. The protein search considered the following parameters: non-redundant protein sequence database (NCBInr), specificity of the proteolytic enzyme used for the hydrolysis (trypsin), taxonomic category of the sample, no protein molecular weight was considered, up to one missed cleavage, cysteines as S-carbamidomethylcysteines, unmodified N- and C-terminal ends, methionines both unmodified and oxidized, putative pyro-Glu formation by Gln, precursor peptide maximum mass tolerance of 200 ppm, and a maximum fragment mass tolerance of 200 ppm.
3
Protein Identification by Mass Spectrometry
4
Proteome Identification by LC-MS/MS
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For protein identification, a semipreparative gel was prepared by loading 0.5 mg of unlabeled protein extracts and stained as previously described64 (link). Protein spots were picked using an Ettan Spot Picker (GE Healthcare), washed in 50 mM ammonium bicarbonate and 50% acetonitrile and then hydrolyzed with trypsin as described65 (link),66 (link). Peptide mixtures were analyzed by liquid chromatography-tandem mass spectrometry (LC-MS/MS) using the LC/MSD Trap XCT Ultra (Agilent Technologies, Palo Alto, CA, USA) equipped with an 1100 HPLC system and a chip cube (Agilent Technologies) as previously described17 (link). Mascot software (Matrix Science, London, UK) was used for protein identification against NCBI database (version of February 2015) containing 61,078,976 sequences and using the following standard parameters: Homo Sapiens; one missed cleavage; carboxyamidomethylation of Cys, partial Met oxidation and putative modification of Gln to pyro-Glu, mass tolerance of 300 ppm on precursor ions and 0.6 Da on the product ions, individual ion scores > 44.
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