The methods are provided in the supplemental from the Saito et al., 2014 paper. Relevant sections are duplicated here:

Research Expedition and Sample Collection
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 (Table S1). Specifically, the 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. 2011a), frozen at -80 degrees C, transported back to the laboratory on dry ice, and stored at -80 degrees C until analysis.

Dissolved trace metal samples (iron and cobalt) were collected by an internally programmed standard SBE Rosette (Seabird Electronics Inc.) user-modified to serve as a trace metal clean system with 12 8 L X-Niskin bottles (Ocean Test Equipment), 12 X-Niskin bottles were attached to the rosette per deployment) with minimal exposed metal surfaces using 5000 m of non-metallic non-conducting line. Temperature, oxygen and conductivity sensor data were collected using a SBE19plusV2 system (Seabird Electronics Inc.) attached to a CTD extension stand on the Trace Metal Rosette. All sensors were factory calibrated immediately prior to the expedition. X-Niskins were pressurized with ultra-high purity nitrogen gas and seawater was filtered through cleaned 47 mm 0.2 micron Supor membrane filters within a HEPA filtered cleanroom space aboard the ship. The volume filtered was calculated (X-Niskin volume minus small unfiltered samples) and the filters were stored in cleaned tubes and frozen for particulate metal analysis (see below).

Protein Extraction
Total microbial protein (0.2-3.0 um 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 95 degrees C 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 –20 degrees C, centrifuged at 14100 x g (14500 rpm) for 30 m at 4 degrees C, 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 (Lu et al. 2005). 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 containing 1.2 mL of gel fix solution then incubated at room temperature, 350 rpm in a Thermomixer R (Eppendorf) for 1 h. Gel fix solution was then removed and replaced with 1.2 mL destain solution (50% MeOH, 10% acetic acid in H2O) and incubated at 350 rpm, RT for 2 h. Liquid was then removed, gel cut up into 1 mm cubes and then added back to tubes containing 1 mL of 50:50 acetonitrile:25 mM ammonium bicarbonate (ambic) incubated for 1 h, 350 rpm, and at RT. Liquid was removed and replaced with fresh 50:50 acetonitrile:ambic and incubated at 16 degrees C and 350 rpm overnight. The step was repeated for 1h the following morning. Gel pieces were then dehydrated twice in 800 ul of acetonitrile for 10 min at RT and dried for 10 min in a ThermoSavant DNA110 speedvac after removing solvent. 600 ul of 10 mM DTT in 25 mM ambic was added to reduce proteins incubating at 56 degrees C, 350 rpm for 1 h. Unabsorbed DTT solution was then removed with volume measured. Gel pieces were washed with 25 mM ambic and 600 ul of 55 mM iodacetamide was added to alkylate proteins at RT, 350 rpm for 1 h. Gel cubes were then washed with 1 mL ambic for 20 minutes, 350 rpm at RT. Acetonitrile dehydrations and speedvac drying were repeated as above. Trypsin (Promega #V5280) was added in appropriate volume of 25 mM ambic to rehydrate and submerse gel pieces at a concentration of 1:20 ug trypsin:protein. Proteins were digested overnight at 350 rpm 37 degrees C. Unabsorbed solution was removed and transferred to a new tube. 50 ul of peptide extraction buffer (50% acetonitrile, 5% formic acid in water) was added to gels, incubated for 20 min at RT then centrifuged at 14,100 x g for 2 min. Supernatant was collected and combined with unabsorbed solution. The above peptide extraction step was repeated combining all supernatants. Combined protein extracts were centrifuged at 14,100 x g for 20 minutes, and supernatants transferred into a new tube and dehydrated down to approximately 10 ul-20 ul in the speedvac. Concentrated peptides were then diluted in 2% acetonitrile 0.1% formic acid in water for storage until analysis. All water used in the tube gel digestion protocol was LC/MS grade, and all plastic microtubes were ethanol rinsed and dried prior to use.

Targeted Protein Analyses
Biomarkers selected for this study focused on trypsin-digested peptide fragments of the proteins (tryptic peptides) that were frequently identified in metaproteome analyses with reproducible mass spectra fragmentation patterns to allow for targeted analyses by triple quadrupole mass spectrometry (Fig. S3-4). The specificity of these tryptic peptide biomarkers was determined by searching for their sequences within sequenced microbial genomes and gene databases (Fig. S9-S14). Absolute quantitation of proteins was conducted by triple quadrupole mass spectrometry using a Thermo Vantage mass spectrometer and synthetic isotope labeled peptide standards as described previously (Bertrand et al. 2013; Saito et al. 2011b). Selected peptides were chosen with an effort to minimize presence of methionines and cysteines both of which can be oxidized and create variability in analyses (Lange et al. 2008, Stahl-Zeng et al. 2007). However, in some cases tryptic peptides were identified in the metaproteome than included these amino acid residues (Peptides IDs 31and 144) with few alternatives peptides corresponding to the protein of interest. Mass spectrometry conditions were optimized for each peptide (collision energy and S-lens), and analyzed using chromatographic scheduling to increase the multiplexing capabilities and resolution for each peptide analyte. These peptide sequences and optimization conditions are presented in Table S3 (PDF). Peptide abundances were calculated as a peak ratio of the corresponding isotopically labeled internal standard. Each internal standard was examined for its linear performance on the mass spectrometer using standard curves (Figure S17). Isotopically labeled standards were obtained from JPT Peptide Technologies, which contain a C-terminal peptide tag. The tag was released by tryptic digestion prior to analysis following the manufacturer’s protocol. Chromatographic separation and mass spectrometry were performed using a Paradigm MS4 HPLC (Michrom Bioresources) coupled to a Thermo Vantage TSQ mass spectrometer (Thermo Scientific) via an Advance capillary electrospray source (Michrom Bioresources). Samples were loaded on a peptide CapTrap prior to separation on a Magic C18AQ column (0.2 x 50 mm, 3 mm particle size, 200 Å pore size, Michrom Bioresources). Chromatographic separation was done with a 45 min gradient of 5% to 35% buffer B (where buffer A was 0.1% formic acid in water, Fisher Optima and buffer B was 0.1% formic acid in acetonitrile, Fisher Optima) at 4 mL/min. Examples of methodological precision are shown in Figure S18 for triplicate analyses of two Station 6 samples.

Pigment and Nutrient Analyses
Nutrient analyses were conducted by nutrient autoanalyzer by Joe Jennings at Oregon State University as previously described (Noble et al. 2012). HPLC: Seawater samples (4 L) were filtered onto glass fibre filters (Whatman GF/F) and stored in liquid nitrogen until analysis. Samples were analyzed on an Agilent 1100 HPLC (High Performance Liquid Chromatography) system with diode array and fluorescence detection. Elution gradient and protocols were described in detail elsewhere (DiTullio et al. 2003).

References:
Bertrand, E.M. et al., Methionine synthase interreplacement in diatom cultures and communities: Implications for the persistence of B12 use by eukaryotic phytoplankton. Limnol. Oceanogr. 58, 1431 (2013). doi: 10.4319/lo.2013.58.4.1431

DiTullio, G.R., M. E. Geesey, D. R. Jones, K. L. Daly, L. Campbell, W. O. Smith Jr., Phytoplankton assemblage structure and primary productivity along 170° W in the South Pacific Ocean. Mar. Ecol. Prog. Ser. 255, 55–80 (2003). doi:10.3354/meps255055

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Noble, A.E. C. H. Lamborg, D. C. Ohnemus, P. J. Lam, T. J. Goepfert, C. I. Measures, C. H. Frame, K. L. Casciotti, G. R. DiTullio, J. Jennings, M. A. Saito, Basin-scale inputs of cobalt, iron, and manganese from the Benguela-Angola front into the South Atlantic Ocean. Limnol. Oceanogr. 57, 989–1010 (2012). doi:10.4319/lo.2012.57.4.0989

Saito, M.A., V. V. Bulygin, D. M. Moran, C. Taylor, C. Scholin, Examination of microbial proteome preservation techniques applicable to autonomous environmental sample collection. Front. Microbiol. 2, 215 (2011a). Medline doi:10.3389/fmicb.2011.00215

Saito, M.A., E. M. Bertrand, S. Dutkiewicz, V. V. Bulygin, D. M. Moran, F. M. Monteiro, M. J. Follows, F. W. Valois, J. B. Waterbury, Iron conservation by reduction of metalloenzyme inventories in the marine diazotroph Crocosphaera watsonii. Proc. Natl. Acad. Sci. U.S.A. 108, 2184–2189 (2011b). Medline doi:10.1073/pnas.1006943108

Stahl-Zeng, J., V. Lange, R. Ossola, K. Eckhardt, W. Krek, R. Aebersold, B. Domon, High sensitivity detection of plasma proteins by multiple reaction monitoring of N-glycosites. Mol. Cell. Proteomics 6, 1809–1817 (2007). Medline doi:10.1074/mcp.M700132-MCP200

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