Evaluation of Acquisition Modes for the Quantitative Analysis of Cross-Linked Peptides by Targeted and Untargeted Mass Spectrometry
2020
Cross-linking
mass spectrometry (XL-MS) is a structural biology technique that can provide
insights into the structure and interactions of proteins and their complexes,
especially those that cannot be easily assessed by other methods. Quantitative
XL-MS has the potential to probe the structural and temporal dynamics of
protein complexes; however, it requires further development. Until recently,
quantitative XL-MS has largely relied upon isotopic labeling and data dependent
acquisition (DDA) methods, limiting the number of biological samples that can
be studied in a single experiment. Here, the acquisition modes available on an
ion mobility (IM) enabled QToF mass spectrometer are evaluated for the
quantitation of cross-linked peptides, eliminating the need for isotopic labels
and thus expanding the number of comparable studies that can be conducted in
parallel. Workflows were optimized using metabolite and peptide standards
analyzed in biological matrices, facilitating modelling of the data and addressing
linearity issues, which allow for significant increases in dynamic range. Evaluation
of the DDA acquisition method commonly used in XL-MS studies indicated
consistency issues between technical replicates and reduced performance in quantitative
metrics. On the contrary, data independent acquisition (DIA) and parallel
reaction monitoring (PRM) modes proved more robust for analyte quantitation.
Mobility enabled modes exhibited an improvement in sensitivity due to the added
dimension of separation, and a simultaneous reduction in dynamic range, which
was largely recovered by correction methods. Hi[3] and probabilistic
quantitation methods were successfully applied to the DIA data, determining the
molar amounts of cross-linked peptides relative to their linear counterparts.
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