Metal Affinity Enrichment Increases the Range and Depth of Proteome Identification for Extracellular Microbial Proteins

Many key proteins, such as those involved in cellular
signaling
or transcription, are difficult to measure in microbial proteomic
experiments due to the interfering presence of more abundant, dominant
proteins. In an effort to enhance the identification of previously
undetected proteins, as well as provide a methodology for selective
enrichment, we evaluated and optimized immobilized metal affinity
chromatography (IMAC) coupled with mass spectrometric characterization
of extracellular proteins from an extremophilic microbial community.
Seven different metals were tested for IMAC enrichment. The combined
results added ∼20% greater proteomic depth to the extracellular
proteome. Although this IMAC enrichment could not be conducted at
the physiological pH of the environmental system, this approach did
yield a reproducible and specific enrichment of groups of proteins
with functions potentially vital to the community, thereby providing
a more extensive biochemical characterization. Notably, 40 unknown
proteins previously annotated as “hypothetical” were
enriched and identified for the first time. Examples of identified
proteins includes a predicted TonB signal sensing protein homologous
to other known TonB proteins and a protein with a COXG domain previously
identified in many chemolithoautotrophic microbes as having a function
in the oxidation of CO.