Dataset: Proteomics Colwellia psychrerythraea strain 34H at subzero temperatures.

Colwellia psychrerythraea strain 34H (ATCC No. BAA-681; GenBank Accession No. AF396670) used in this study was originally isolated from Arctic marine sediments (Huston et al., 2000). Cells were cultured from frozen glycerol stocks by incubating at -1∞C with daily inversion in half-strength Marine 2216 broth until early stationary growth phase (2 weeks or less). Prior to the initiation of the experiments, cells were harvested by centrifugation (2800◊g; 20 min; 4∞C). After washing with cold (-1∞C), 0.2µm filtered ASW {0.1M NaCl, 0.01M KCl, 0.06M MgCl2, 8mM MgSO47H20, 5mM TAPSO [3-(N- tris(hydroxymethyl) methylamino]-2-hydroxypro-pane-sulfonic acid], pH 7.5}, cells were resuspended in ASW (-1∞C) with 1.5g C ml-1 extracellular polysacharide substances to an OD600nm of 0.2 corresponding to an average (±standard deviation) cell count of 5.7E7 ± 1.1E7 cells ml-1. Triplicate 100µl of aliquots of cell suspensions were fixed in 2% formaldehyde, and stored at 4∞C for abundance analysis by epifluorescence microscopy as described by Junge and colleagues (2004b). For [3H]-leucine and [3H]-thymidine incubation experiments, 500µl of aliquots of the cell suspension were placed into 1.5ml Eppendorf tubes for immediate use.

Bacterial metabolic activity was measured using leucine ([3H]-leu) and thymidine ([3H]-thy) incorporation assays, following methodologies based on the analysis of bacterial activities in freshwater ice and saline ice. To determine rates of [3H]-leu incorporation and [3H]-thy incorporation in the prepared cell suspensions, time-course experiments were conducted at temperatures of -1, -5 and -10∞C and sampling occurred at 0, 1, 12 and 24 h, and 1, 2, 4 and 8 week time points. Triplicate negative (killed) controls were completed at each temperature by adding 100µl of 50% trichloroacetic acid (TCA; 4∞C) to control tubes prior to tracer addition. Methods described by Junge and colleagues (2006) were primarily followed, with the exception of modifications including: (i) samples were placed at their intended temperatures immediately after the addition of the radiolabel tracer, not flash frozen with LN2, (ii) three ethanol washes were performed during the processing stage and (iii) in addition to the [3H]-leu incorporation assay to measure metabolic activity based on protein synthesis, [3H]-thy incorporation was also measured to investigate rates of DNA synthesis.

Based on peptide concentrations, a total of 1µg of peptide digest in 10µl of 5% acetonitrile (ACN), 0.1% formic acid was sampled per LC-MS/MS analysis. Samples were separated and introduced into the mass spectrometer (MS) by reverse-phase chromatography using a 25cm long, 75µm i.d., fused silica capillary column packed with C18 particles (Magic C18AQ, 100 ≈, 5) fitted with a 2cm long, 100µm i.d. precolumn (Magic C18AQ, 200 ≈, 5). Peptides were eluted using an acidified (formic acid, 0.1% v/v) water-acetonitrile gradient (5ñ35% acetonitrile in 90 min). Mass spectrometry was performed on a Thermo Fisher QExactive. The top 20 most intense ions were selected for MS2 acquisition from precursor ion scans of 400ñ1600 m z-1. Quality control peptide mixtures were analyzed every sixth injection to monitor chromatography and MS sensitivity. Skyline was used to determine that QC standards did not deviate >10% through all analyses (Maclean et al., 2010).

Instrument(s): Thermo QExactive