Deep Proteome Mapping of Mouse Kidney Based on OFFGel Prefractionation Reveals Remarkable Protein Post- Translational Modifications

Performing a comprehensive
nonbiased proteome analysis is an extraordinary
challenge due to sample complexity and wide dynamic range, especially
in eukaryotic tissues. Thus, prefractionation steps conducted prior
to mass spectrometric analysis are critically important to reduce
complex biological matrices and allow in-depth analysis. Here we demonstrated
the use of OFFGel prefractionation to identify more low abundant and
hydrophobic proteins than in a nonfractionated sample. Moreover, OFFGel
prefractionation of a kidney protein sample was able to unveil protein
functional relevance by detecting PTMs, especially when prefractionation
was augmented with a targeted enrichment strategy such as TiO2 phospho-enrichment. The OFFGel-TiO2 combination
used in this study was comparable to other global phosphoproteomics
approaches (SCX-TiO2, ERLIC-TiO2, or HILIC-TiO2). The detailed mouse kidney proteome with the phosphopeptide
enrichment presented here serves as a useful platform for a better
understanding of how the renal protein modification machinery works
and, ultimately, will contribute to our understanding of pathological
processes as well as normal physiological renal functions.