Multiplexed Isobaric Quantitative Cross-Linking Reveals Drug-Induced Interactome Changes in Breast Cancer Cells

The study of protein structures and
interactions is critical to
understand their function. Chemical cross-linking of proteins with
mass spectrometry (XL-MS) is a rapidly developing structural biology
technique able to provide valuable insight into protein conformations
and interactions, even as they exist within their native cellular
environment. Quantitative analysis of cross-links can reveal protein
conformational and interaction changes that occur as a result of altered
biological states, environmental conditions, or pharmacological perturbations.
Our laboratory recently developed an isobaric quantitative protein
interaction reporter (iqPIR) cross-linking strategy for comparative
interactome studies. This strategy relies on isotope encoded chemical
cross-linkers that have the same molecular mass yet produce unique
and specific isotope signatures upon fragmentation in the mass spectrometer
which can be used for quantitative analysis of cross-linked peptides.
The initial set of iqPIR molecules allowed for binary comparisons.
Here, we describe the in vivo application of an extended
set of six iqPIR reagents (6-plex iqPIR), allowing multiplexed quantitative
interactome analysis of up to six biological samples in a single LC–MS
acquisition. Multiplexed iqPIR is demonstrated on MCF-7 breast cancer
cells treated with five different Hsp90 inhibitors revealing large
scale protein conformational and interaction changes specific to the
molecular class of the inhibitors.