Data-Dependent Middle-Down Nano-Liquid Chromatography–Electron Capture Dissociation-Tandem Mass Spectrometry: An Application for the Analysis of Unfractionated Histones

Middle-down
mass spectrometry (MS) combined with electron capture
dissociation (ECD) represents an attractive method for characterization
of proteins and their post-translational modifications (PTMs). Coupling
online chromatographic separation with tandem mass spectrometry enables
a high-throughput analysis, while improving sensitivity of the electrosprayed
peptides and reducing sample amount requirements. However, middle-down
ECD has not been thus far coupled with online chromatographic separation.
In this work, we examine the feasibility of coupling middle-down ECD
with online nanoflow-liqiud chromatography (nano-LC) for the analysis
of large, >3 kDa, and highly modified polypeptides in a data-dependent
acquisition mode. We evaluate the effectiveness of the method by analyzing
peptides derived from Asp-N and Glu-C digestions of unfractionated
histones from calf thymus and acid-extracted histones from HeLa, MCF-7,
and Jurkat cells. Our results demonstrate that middle-down ECD is
compatible with online chromatographic separation, providing high
peptide and protein sequence coverage while allowing precise mapping
of PTM sites. The high mass accuracy, obtained by the ICR mass analyzer,
for both the precursor and product ions greatly increases confidence
in peptide identification, particularly for modified peptides. Overall,
for all samples examined, several histone variants were identified
and modification sites were successfully localized, including single,
multiple, and positional isomeric PTM sites. The vast majority of
the identified peptides were in the mass range from 3 to 9 kDa. The
data presented here highlight the feasibility and utility of nano-LC–ECD-MS/MS
for high-throughput middle-down analysis.