Digested peptides solution was desalted using ZipTip µ-C18 Pipette Tips (Millipore). Peptides were analyzed by an Orbitrap Fusion Tribrid mass spectrometer (Thermo Fisher Scientific, San Jose, CA) equipped with a Thermo Scientific EASY-Spray nanoelectrospray ion source and coupled to an Easy nano-LC Proxeon 1000 system (Thermo Fisher Scientific, San Jose, CA). Chromatographic separation of peptides was performed with the following parameters: pre-column Acclaim PepMap100 (2 cm, 75 μm i.d., 3 μm, 100 Å), column LC EASY-Spray C18 column (75 cm, 75 μm i.d., 2 μm, 100 Å), 300 nl/min flow, gradient rising from 95% solvent A (water, 0.1% formic acid) to 35% B (100% acetonitrile, 0.1% formic acid) in 98 min. Peptides were analyzed in the orbitrap cell in full ion scan mode at a resolution of 120000 (at m/z 400) within a mass range of m/z 350–1550. Fragments were obtained with a Higher-energy Collisional Dissociation (HCD) activation with a collisional energy of 27%, and a quadrupole isolation width of 1.6 Da. MS/MS data were acquired in the linear ion trap in top-speed mode, with a dynamic exclusion of 50 s and a repeat duration of 60 s. The maximum ion accumulation times were set to 250 ms for MS acquisition and 60 ms for MS/MS acquisition in parallelization mode.
Easy nano lc proxeon 1000 system
Manufactured by Thermo Fisher Scientific
The Easy nano-LC Proxeon 1000 system is a compact and automated liquid chromatography instrument designed for nano-scale separations. It features a low-flow gradient pump, an auto-sampler, and a column oven. The system is suitable for a variety of applications that require high-resolution separations of complex samples, such as proteomics and metabolomics studies.
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2 protocols using easy nano lc proxeon 1000 system
1
Orbitrap Mass Spectrometry for Peptide Analysis
2
Proteomic Analysis of Glutathionylation
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All digests of protein extracts were analyzed using an LTQ Velos Orbitrap equipped with an EASY-Spray nanoelectrospray ion source coupled to an Easy nano-LC Proxeon 1000 system (all devices were purchased from Thermo Fisher Scientific, San Jose, CA, U.S.A.) following chromatographic separation of the peptides. All the experimental conditions for LC-MS/MSMS acquisitions were recently described [47] (link). MS/MS data were processed using an in-house Mascot search server (Matrix Science, Boston, MA, U.S.A., version 2.4.1). The mass tolerance was set to 7 ppm for precursor ions and 0.5 Da for fragments. The following variable modifications were allowed for identification of glutathionylated proteins: oxidation (M), phosphorylation (STY), acetylation (K, N-term), and deamidation (N, Q). In a second round, glutathionylated cysteines were identified by taking into account NEM and MMTS modifications and HPDP-biotin derivatization as variable modifications. The maximum number of missed trypsin cleavages was limited to two. MS/MS data were used to search against the Uniprot database restricted to the S. cerevisiae taxonomy.
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