Dataset: Metaproteome
Deployment: KN192-05

Microorganisms were concentrated from large volume (100–200 l) surface seawater samples (5–8 m) collected from a surface pump. We targeted the < 0.8 um size fraction to obtain the same bacterioplankton community used for GOS shotgun sequencing (Rusch et al., 2007). All concentrations were performed in a 50 l polystyrene reservoir using a Pellicon 2 cassette tangential flow filtration system equipped with one 30kD Biomax Polyethersulfone cassette (Millipore Corporation, Billerica, MA, USA). Seawater was continuously added to the concentration reservoir until cell densities reached ~10^8 cells/ml. In all, 100–200 ml of cell concentrate was obtained in 1.5–3h. Concentrated cells were flash frozen in liquid nitrogen and stored at -80 C until further processing on shore.

A cell pellet was prepared from each tangential flow filtration concentrated sample by centrifugation at 4 1C for 60 min (17 000 g). The supernatant was discarded and cell pellets were resuspended in 3 ml of 20 m Tris buffer pH 7.4. Crude extracts were prepared by passing the cells through a French pressure mini cell at 8000 lb inch–2 two times and subsequent centrifugation at 4 1C for 30 min (18 000 g). Crude pellet and soluble fractions were separated and pellets were rinsed with 100ml 20mM Tris buffer pH 7.4. Highly enriched membrane material for proteomic analyses was obtained from soluble cell fractions by centrifugation at 4 1C for an additional 60 min (104 000 g). Membrane- enriched material was isolated and rinsed with 100 ml 20 mM Tris buffer pH 7.4.