Quantitative Top-Down Proteomics by Isobaric Labeling with Thiol-Directed Tandem Mass Tags

While identification-centric
(qualitative) top-down proteomics
(TDP) has seen rapid progress in the recent past, the quantification
of intact proteoforms within complex proteomes is still challenging.
The by far mostly applied approach is label-free quantification, which,
however, provides limited multiplexing capacity, and its use in combination
with multidimensional separation is encountered with a number of problems.
Isobaric labeling, which is a standard quantification approach in
bottom-up proteomics, circumvents these limitations. Here, we introduce
the application of thiol-directed isobaric labeling for quantitative
TDP. For this purpose, we analyzed the labeling efficiency and optimized
tandem mass spectrometry parameters for optimal backbone fragmentation
for identification and reporter ion formation for quantification.
Two different separation schemes, gel-eluted liquid fraction entrapment
electrophoresis × liquid chromatography–mass spectrometry
(LC–MS) and high/low-pH LC–MS, were employed for the
analyses of either Escherichia coli (E. coli) proteomes or combined E. coli/yeast samples (two-proteome interference
model) to study potential ratio compression. While the thiol-directed
labeling introduces a bias in the quantifiable proteoforms, being
restricted to Cys-containing proteoforms, our approach showed excellent
accuracy in quantification, which is similar to that achievable in
bottom-up proteomics. For example, 876 proteoforms could be quantified
with high accuracy in an E. coli lysate.
The LC–MS data were deposited to the ProteomeXchange with the
dataset identifier PXD026310.