An Aquatic Microbial Metaproteomics Workflow: From Cells to Tryptic Peptides Suitable for Tandem Mass Spectrometry-based Analysis

Summary

This protocol is for the extraction and concentration of protein and DNA from microbial biomass collected from seawater, followed by the generation of tryptic peptides suitable for tandem mass spectrometry-based proteomic analysis.

Abstract

Meta-omic technologies such as metagenomics, metatranscriptomics and metaproteomics can aid in the understanding of microbial community structure and metabolism. Although powerful, metagenomics alone can only elucidate functional potential. On the other hand, metaproteomics enables the description of the expressed in situ metabolism and function of a community. Here we describe a protocol for cell lysis, protein and DNA isolation, as well as peptide digestion and extraction from marine microbial cells collected on a cartridge filter unit (such as the Sterivex filter unit) and preserved in an RNA stabilization solution (like RNAlater). In mass spectrometry-based proteomics studies, the identification of peptides and proteins is performed by comparing peptide tandem mass spectra to a database of translated nucleotide sequences. Including the metagenome of a sample in the search database increases the number of peptides and proteins that can be identified from the mass spectra. Hence, in this protocol DNA is isolated from the same filter, which can be used subsequently for metagenomic analysis.

Introduction

Microorganisms are ubiquitous and play essential roles in Earth’s biogeochemical cycles ¹. Currently, there are numerous molecular approaches available for characterizing microbial community structure and function. Most common is the analysis of 16S rRNA gene sequences PCR-amplified from environmental DNA ^2–4. A disadvantage of 16S rRNA gene analysis is that it only provides information on phylogenetic identity and community structure, with little information on metabolic function. In contrast, approaches such as metagenomics, metatranscriptomics and metaproteomics provide information on community structure and m....

Protocol

1. Prepare Reagents

1. Prepare SDS-extraction solution: 0.1 M Tris-HCl pH 7.5, 5 % glycerol, 10 mM EDTA and 1 % SDS. Filter-sterilize using a 0.22 µm filter and store at 4 °C.
2. Prepare stock reagents needed for the polyacrylamide gel.
   1. Prepare 1.5 M Tris-HCL pH 8.8. Filter-sterilize using a 0.22 µm filter and store at room temperature.
   2. 0.5 M Tris-HCL pH 6.8. Filter-sterilize using a 0.22 µm filter and store at room temperature.
   3. Prepare 10 % SDS. Filter sterilize using a 0.22 µm filter and store at room temperature.
3. Prepare solutions needed for in-gel trypsin digest and ....

Results

As a demonstration, we performed the protocol on two seawater samples collected from the surface and the chlorophyll maximum of the coastal ocean in Northern Canada. While at sea, 6-7 L of seawater was passed through a 3 µm GF/D prefilter, then microbial cells were collected onto a 0.22 µm cartridge filter unit following the protocol of Walsh et al. ²⁰. Cells were immediately stored in an RNA stabilization solution until further processing. Upon returning to the lab, we performed the protocol as it is.......

Discussion

Sample preservation is key to metaproteomic studies and previous work demonstrated that an RNA stabilization solution is a useful storage buffer for storing cells prior to protein extraction ²⁸. Ideally, samples would be preserved in situ to negate shifts in protein expression during handling ^33,34. In fact, in situ sampling and fixation technologies have been developed, which allow for the autonomous collection and preservation of samples by ship-deployed instruments. However, acc.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Sterivex -GP 0.22 μm filter unit	Millipore	SVGP01050	Sampling
RNAlater Stabilization Solution	Ambion	AM7021	Sampling
Tris	Bio Basic	77-86-1 or TB0196-500G	Protein Extraction/ SDS PAGE gel
DTT	Sigma-Aldrich	D0632-1G	Protein Extraction
SDS	Bio Basic	15-21-3	Protein Extraction/ SDS PAGE gel
EDTA	Bio Basic	6381-92-6	Protein Extraction
Glycerol	Fisher Scientific	56-81-5	Protein Extraction
10K Amicon Filter	Millipore	UFC801024	Protein Extraction
Methanol	Sigma-Aldrich	179337-4L	Protein Precipitation
Acetone	Fisher Scientific	67-64-1	Protein Precipitation
MPC Protein Precipitation reagent	Epicenter	mmP03750	DNA Precipitation
2-Propanol	Fisher Scientific	67-63-0	DNA Precipitation
Qubit dsDNA BR Assay kit	Life Technologies	Q32850	DNA Quantification
Qubit Protein Assay kit	Life Technologies	Q33211	Protein Quantification
Sucrose	Bio Basic	57-50-1	SDS PAGE gel
TEMED	Bio Rad	161-0800	SDS PAGE gel
APS	Bio Rad	161-0700	SDS PAGE gel
30% Acrylamide	Bio Rad	161-0158	SDS PAGE gel
SimplyBlue SafeStain	Invitrogen	LC6060	SDS PAGE gel
Glycine	Bio Rad	161-0718	SDS PAGE gel
B-mercaptoethanol	Bio Basic	60-24-2	SDS PAGE gel
Laemmli Sample Buffer	Bio Rad	161-0737	SDS PAGE gel
Precision Plus Protein Kaleidoscope Ladder	Bio Rad	161-0375EDU	SDS PAGE gel
Acetonitrile	VWR	CABDH6044-4	In-gel Trypsin digest
NH4HCO3	Bio Basic	1066-33-7	In-gel Trypsin digest
DTT	Sigma-Aldrich	D0632-1G	In-gel Trypsin digest
Formic Acid	Sigma-Aldrich	F0507-500ML	In-gel Trypsin digest
HPLC grade H2O	Sigma-Aldrich	270733-4L	In-gel Trypsin digest
Iodoacetamide	Bio Basic	144-48-9	In-gel Trypsin digest
Trypsin	Promega	V5111	In-gel Trypsin digest
Protein LoBind Tube 1.5 ml	Eppendorf	22431081	In-gel Trypsin digest
2 ml ROBO vial 9 mm	Candian Life Science	VT009/C395SB	In-gel Trypsin digest
PP BM insert, No spring	Candian Life Science	4025P-631	In-gel Trypsin digest