Dataset: Peptide spectral counts (MetZyme 0.2)
Deployment: KM1128

Ocean metaproteomes were sampled and extracted as described in Saito et al., 2014. The protocols are reproduced here:

Samples were collected during the KM1128 METZYME research expedition (Metals and Enzymes in the Pacific) on the R/V Kilo Moana October 1-25, 2011 from Oahu, Hawaii to Apia, Samoa, with Carl Lamborg and Mak Saito as Chief Scientists. Microbial biomass for protein analyses was collected on vertical profiles using in situ high volume particle filtration pumps with a focus on the North Pacific and Equatorial regions. Protein samples were collected by a suite of 4 L/min and 8 L/min McLane Pumps (WTS-LV; McLane Research Laboratories Inc., Falmouth MA, USA) outfitted with custom Mini-MULVS multiple filter head systems. The 0.2-3.0 micron size fraction was collected on 142 mm filters (Supor, Pall Corp.) for analyses used in this study. The volume of water was pumped until a minimal flow rate was achieved or the allotted cast time period expired, typically ~300-500 L. Filters were sectioned immediately after pump retrieval, and protein samples (¼ filter) were stored in RNAlater reagent (Ambion, Life Technologies), which has been shown to be an effective preservative for cyanobacterial biomass (Saito et al., 2011), frozen at -80C, transported back to the laboratory on dry ice, and stored at -80C until analysis.

Total microbial protein (0.2-3.0um fraction) was extracted using detergent-based methods, described below. Total protein showed enhanced concentrations in the photic zone, particularly in the Equatorial and South Pacific portions of the transect (Fig. S1F). For protein extraction, samples were thawed and the filter and RNAlater preservative (Ambion Life Technologies) were separated. The removed preservative was spin-concentrated by a 5K MWCO membrane (Sartorius Stedim Biotech 6 mL, 5 K MWCO Vivaspin units; Goettingen, Germany), and rinsed with 0.1M Tris buffer to recover, desalt, and concentrate any suspended material. The sample filter was unfolded and placed in a larger tube to which 1% SDS extraction buffer (1% SDS, 0.1M Tris/HCL pH 7.5, 10mM EDTA) and the rinsed/desalted RNAlater fraction was added back. Each sample was incubated at room temperature for 15 minutes, heated at 95C for 10 minutes, and shaken at room temperature for 350 rpm for 1 h. The protein extract was decanted and placed in a new tube and centrifuged for 30 min x 3220 g at room temperature. The supernatants were removed and filtered through a 5 um low protein binding syringe filter (Fisher Scientific), and the filter rinsed with extraction buffer. The extracts were concentrated by 5 kD membrane centrifugation to a small volume, washed with extraction buffer, and concentrated again. Each sample was precipitated with cold 50% methanol (MeOH) 50% acetone 0.5 mM HCL for 3 days at –20C, centrifuged at 14100 x g (14500 rpm) for 30 m at 4C, decanted and dried by vacuum concentration (Thermo Savant Speedvac) for 10 min or until dry. Pellets were resuspended in 1% SDS extraction buffer and left at room temperature (RT) for 1 h to redissolve. Total protein was quantified (Bio-Rad DC protein assay, Hercules, CA) with BSA as a standard.

Extracted proteins were purified from SDS detergent, reduced, alkylated, and  trypsin digested, while embedded within a polyacrylamide tube gel, using a modified protocol from a previously published method (38). The tube gel approach allowed all proteins including membrane proteins to be solubilized by detergent and purified while immobilized in the gel matrix. A gel premix was made by combining 1 M Tris HCL (pH 7.5) and 40% Bis-acrylimide L 29:1 (Acros Organics) at a ratio of 1:3. The premix (103 ul) was combined with an extracted protein sample (usually 25 ug-200 ug), TE, 7 ul 1% APS and 3 ul of TEMED (Acros Organics) to a final volume of 200 ul. After 1 h of polymerization at RT, 200 ul of gel fix solution (50% ETOH, 10% acetic acid in LC/MS grade water) was added to the top of the gel and incubated at RT for 20 minutes. Liquid was then removed and the tube gel was transferred into a new 1.5 mL microtube.