Characterization of Proteoform Post-Translational Modifications by Top-Down and Bottom-Up Mass Spectrometry in Conjunction with Annotations

Many proteoforms
can be produced from a gene due to genetic mutations,
alternative splicing, post-translational modifications (PTMs), and
other variations. PTMs in proteoforms play critical roles in cell
signaling, protein degradation, and other biological processes. Mass
spectrometry (MS) is the primary technique for investigating PTMs
in proteoforms, and two alternative MS approaches, top-down and bottom-up,
have complementary strengths. The combination of the two approaches
has the potential to increase the sensitivity and accuracy in PTM
identification and characterization. In addition, protein and PTM
knowledge bases, such as UniProt, provide valuable information for
PTM characterization and verification. Here, we present a software
pipeline PTM-TBA (PTM characterization by Top-down and Bottom-up MS
and Annotations) for identifying and localizing PTMs in proteoforms
by integrating top-down and bottom-up MS as well as PTM annotations.
We assessed PTM-TBA using a technical triplicate of bottom-up and
top-down MS data of SW480 cells. On average, database search of the
top-down MS data identified 2000 mass shifts, 814.5 (40.7%) of which
were matched to 11 common PTMs and 423 of which were localized. Of
the mass shifts identified by top-down MS, PTM-TBA verified 435 mass
shifts using the bottom-up MS data and UniProt annotations.