title: An Aquatic Microbial Metaproteomics Workflow: From Cells to Tryptic Peptides Suitable for Tandem Mass Spectrometry-based 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.
authors.affiliation: Department of Biology, Concordia University; d_colat@live.concordia.ca.
authors.forename: David
authors.initials: D
authors.lastname: Colatriano
authors.affiliation: Department of Biology, Concordia University.
authors.forename: David A
authors.initials: DA
authors.lastname: Walsh
mesh_terms: D041963:Bacteroidetes / Q000737:chemistry; D018533:Biomass; D004247:DNA / Q000032:analysis / Q000737:chemistry / Q000302:isolation & purification; D056186:Metagenomics / Q000379:methods; D009842:Oligopeptides / Q000032:analysis / Q000737:chemistry* / Q000302:isolation & purification; D020560:Proteobacteria / Q000737:chemistry; D040901:Proteomics / Q000379:methods*; D012623:Seawater / Q000382:microbiology; D053719:Tandem Mass Spectrometry / Q000379:methods*; D014357:Trypsin / Q000032:analysis / Q000737:chemistry*; D057188:Workflow
chemical_list: D009842:Oligopeptides; D004247:DNA; D014357:Trypsin