Nanoacquity gradient uplc pump system

An UPLC HSS T3 column (Waters, Milford, MA, USA) and an UPLC Symmetry C18 trapping column (Waters, Milford, MA, USA) for LC as well as a PicoTip Emitter (SilicaTip, 10 mm i.d., New Objective, Woburn, MA, USA) were used in combination with the nanoACQUITY gradient UPLC pump system (Waters, Milford, MA, USA) coupled to a LTQ Orbitrap Elite mass spectrometer (Thermo Fisher Scientific Inc., Waltham, MA, USA). The peptides were eluted with a 105-min gradient of 2% to 85% acetonitrile with 0.1% formic acid at a flow rate of 400 nL/min (0–5 min: 2%; 5–10 min: 2–5%; 10–71 min: 5–30%; 72–77 min: 85%; 77–105 min: 2%). The LTQ Orbitrap Elite was operated via instrument method files of Xcalibur (Rev. 2.1.0) in positive ion mode. The linear ion trap and Orbitrap were operated in parallel, i.e., during a full MS scan on the Orbitrap in the range of 150–2000 m/z at a resolution of 240,000 MS/MS spectra of the 20 most intense precursors were detected in the ion trap using the rapid scan mode. The relative collision energy for collision-induced dissociation (CID) was set to 35%. Dynamic exclusion was enabled with a repeat count of 1- and 45-s exclusion duration window. Singly charged and ions of unknown charge state were rejected from MS/MS.

In this dataset, previously published by Cerletti et al.^{43 (link)}, the proteomes of two halophilic archaea, H. volcanii H26 and N. magadii ATCC 43099, during exponential and stationary growth were compared. Cultures were grown at 42 °C, shaking at 200 rpm, in rich medium (MGM and Tindall medium, respectively) and membrane and cytoplasm fractions were obtained. Protein samples were processed, digested with trypsin, and subjected to LC-ESI-MS/MS using a nanoACQUITY gradient UPLC pump system (Waters) and an LTQ Orbitrap Elite mass spectrometer. Proteins were originally identified and quantified with MaxQuant (version 1.5.3.17)^{83 (link)} using the LFQ algorithm searching against the HaloLex H. volcanii DS2 proteome (version 24-SEP-2013; 10.5281/zenodo.3565581) and the HaloLex N. magadii ATCC 43099 proteome (January 2017; 10.5281/zenodo.3571186; contains 4295 entries, while 4023 were claimed). While N. magadii samples were included in the reanalysis for comparative purposes, only the raw data corresponding to samples from H. volcanii were used for the combined ArcPP dataset, comprising three and six biological replicates for the cytoplasm and membrane fractions, respectively.