Optimized Extraction Method To Remove Humic Acid Interferences from Soil Samples Prior to Microbial Proteome Measurements

The
microbial composition and their activities in soil environments
play a critical role in organic matter transformation and nutrient
cycling. Liquid chromatography coupled to high-performance mass spectrometry
provides a powerful approach to characterize soil microbiomes; however,
the limited microbial biomass and the presence of abundant interferences
in soil samples present major challenges to proteome extraction and
subsequent MS measurement. To this end, we have designed an experimental
method to improve microbial proteome measurement by removing the soil-borne
humic substances coextraction from soils. Our approach employs an in situ detergent-based microbial lysis/TCA precipitation
coupled to an additional cleanup step involving acidified precipitation
and filtering at the peptide level to remove most of the humic acid
interferences prior to proteolytic peptide measurement. The novelty
of this approach is an integration to exploit two different characteristics
of humic acids: (1) Humic acids are insoluble in acidic solution but
should not be removed at the protein level, as undesirable protein
removal may also occur. Rather it is better to leave the humics acids
in the samples until the peptide level, at which point the significant
differential solubility of humic acids versus peptides at low pH can
be exploited very efficiently. (2) Most of the humic acids have larger
molecule weights than the peptides. Therefore, filtering a pH 2 to
3 peptide solution with a 10 kDa filter will remove most of the humic
acids. This method is easily interfaced with normal proteolytic processing
approaches and provides a reliable and straightforward protein extraction
method that efficiently removes soil-borne humic substances without
inducing proteome sample loss or biasing protein identification in
mass spectrometry. In general, this humic acid removal step is universal
and can be adopted by any workflow to effectively remove humic acids
to avoid them negatively competing with peptides for binding with
reversed-phase resin or ionization in the electrospray.