Collision-Induced
Unfolding, Tandem MS, Bottom-up
Proteomics, and Interactomics for Identification of Protein Complexes
in Native Surface Mass Spectrometry
posted on 2023-12-14, 15:06authored byRaul A. Villacob, Neda Feizi, Sarah C. Beno, Touradj Solouki
Endogenously occurring
salts and nonvolatile matrix components
in untreated biological surfaces can suppress protein ionization and
promote adduct formation, challenging protein identification. Characterization
of labile proteins within biological specimens is particularly demanding
because additional purification or sample treatment steps can be time-intensive
and can disrupt noncovalent interactions. It is demonstrated that
the combined use of collision-induced unfolding, tandem mass spectrometry,
and bottom-up proteomics improves protein characterization in native
surface mass spectrometry (NSMS). This multiprong analysis is achieved
by acquiring NSMS, MS/MS, ion mobility (IM), and bottom-up proteomics
data from a single surface extracted sample. The validity of this
multiprong approach was confirmed by the successful characterization
of nine surface-deposited proteins, with molecular weights ranging
from 8 to 147 kDa, in two separate mixtures. Bottom-up proteomics
provided a list of proteins to match against observed proteins in
NSMS and their detected subunits in tandem MS. The method was applied
to characterize endogenous proteins from untreated chicken liver samples.
The subcapsular liver sampling for NSMS analysis allowed for the detection
of endogenous proteins with molecular weights of up to ∼220
kDa. Moreover, using IM-MS, collision cross sections and collision-induced
unfolding pathways of enzymatic proteins and protein complexes of
up to 145 kDa were obtained.