Improved Protein Coverage in Bottom-Up Proteomes Analysis Using Fluoroalcohol-Mediated Supramolecular Biphasic Systems With Mixed Amphiphiles for Sample Extraction, Fractionation, and Enrichment

A new
class of supramolecular biphasic systems containing fluoroalcohol-induced
coacervates (FAiC) provides concomitant fractionation of complex protein
mixtures, high solubilizing power for extraction of various types
of proteins, especially those with high hydrophobicity (such as membrane
proteins), and enrichment of low-abundance proteins. Subsequently,
the use of FAiC biphasic systems (BPS) in the bottom-up proteomics
workflow resulted in significantly higher coverage for the whole proteome,
various subproteomes, especially those embedded or associated with
membranes, post-translationally modified proteins, and low-abundance
proteins (LAPs) as compared to the conventional methodologies. In
this work, we used a new type of FAiC-BPS composed of mixed amphiphiles,
a zwitterionic surfactant 3-(N,N-dimethylmyristyl ammonia) propane sulfonate (DMMAPS), a quaternary
ammonium salt (QUATS), and hexafluoroisopropanol (HFIP) as the coacervator
for extraction, fractionation, and enrichment of yeast proteome in
bottom-up proteomics. The coverage of the lower-abundance proteins
(abundance below 2000 molecules/cell) improved by more than 100% using
DMMAPS and DMMAPS + QUATS systems as compared to the conventional
methods using urea or detergent solutions for protein solubilization.
Additionally, these coacervate systems show increased coverage of
integral membrane proteins and proteins with α-helices by up
to 24 and 555%, respectively.