Abstract
Proteomic tools identify constituents of complex mixtures, often delivering long lists of identified proteins. The high-throughput methods excel at matching tandem mass spectrometry data to spectra predicted from sequence databases. Unassigned mass spectra are ignored, but could, in principle, provide valuable information on unanticipated modifications and improve protein annotations while consuming limited quantities of material. Strategies to “mine” information from these discards are presented, along with discussion of features that, when present, provide strong support for modifications. In this study we mined LC-MS/MS datasets of proteolytically-digested concanavalin A pull down fractions from Methanosarcina mazei Gö1 cell lysates. Analyses identified 154 proteins. Many of the observed proteins displayed post-translationally modified forms, including O-formylated and methyl-esterified segments that appear biologically relevant (i.e., not artifacts of sample handling). Interesting cleavages and modifications (e.g., S-cyanylation and trimethylation) were observed near catalytic sites of methanogenesis enzymes. Of 31 Methanosarcina protein N-termini recovered by concanavalin A binding or from a previous study, only M. mazei S-layer protein MM1976 and its M. acetivorans C2A orthologue, MA0829, underwent signal peptide excision. Experimental results contrast with predictions from algorithms SignalP 3.0 and Exprot, which were found to over-predict the presence of signal peptides. Proteins MM0002, MM0716, MM1364, and MM1976 were found to be glycosylated, and employing chromatography tailored specifically for glycopeptides will likely reveal more. This study supplements limited, existing experimental datasets of mature archaeal N-termini, including presence or absence of signal peptides, translation initiation sites, and other processing. Methanosarcina surface and membrane proteins are richly modified.
Highlights
Knowledge about Archaea and their proteins is limited, making their characterization important
Peptide tandem mass spectrometry (MS/MS) datasets from previous investigations of Methanosarina S-layer and surface-exposed proteins (Francoleon et al, 2009; Rohlin et al, 2012), as well as from mixtures recovered by concanavalin A binding were selected for further analysis, motivated by interest in how protein modifications can impact organisms’ interactions with their environment and with other organisms
We identified 154 proteins from concanavalin A pull down fractions and cell surface labeling
Summary
Knowledge about Archaea and their proteins is limited, making their characterization important. Tools are available to identify proteins at high throughput, while bioinformatic analyses can overlay existing knowledge onto this kingdom. Protein modifications unique to these organisms and/or rare in well-studied microbes may elude us, primarily because high throughput proteomic methods focus on matching peptide fragment data to what can be anticipated, primarily from the genome sequence. Progress in understanding archaeal cell surface structures has been hindered by the limited availability of experimental results, and any new protein modifications that are revealed may hint at function. Peptide tandem mass spectrometry (MS/MS) datasets from previous investigations of Methanosarina S-layer and surface-exposed proteins (Francoleon et al, 2009; Rohlin et al, 2012), as well as from mixtures recovered by concanavalin A binding were selected for further analysis, motivated by interest in how protein modifications can impact organisms’ interactions with their environment and with other organisms.
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
