Bottom-up Histone Post-translational Modification Analysis using Liquid Chromatography, Trapped Ion Mobility Spectrometry, and Tandem Mass Spectrometry

The amino acid position within a histone sequence and
the chemical
nature of post-translational modifications (PTMs) are essential for
elucidating the “Histone Code”. Previous work has shown
that PTMs induce specific biological responses and are good candidates
as biomarkers for diagnostics. Here, we evaluate the analytical advantages
of trapped ion mobility (TIMS) with parallel accumulation-serial fragmentation
(PASEF) and tandem mass spectrometry (MS/MS) for bottom-up proteomics
of model cancer cells. The study also considered the use of nanoliquid
chromatography (LC) and traditional methods: LC-TIMS-PASEF-ToF MS/MS
vs nLC-TIMS-PASEF-ToF MS/MS vs nLC-MS/MS. The addition of TIMS and
PASEF-MS/MS increased the number of detected peptides due to the added
separation dimension. All three methods showed high reproducibility
and low RSD in the MS domain (<5 ppm). While the LC, nLC and TIMS
separations showed small RSD across samples, the accurate mobility
(1/K0) measurements (<0.6% RSD) increased the confidence
of peptide assignments. Trends were observed in the retention time
and mobility concerning the number and type of PTMs (e.g., ac, me1–3) and their corresponding unmodified, propionylated
peptide that aided in peptide assignment. Mobility separation permitted
the annotation of coeluting structural and positional isomers and
compared with nLC-MS/MS showed several advantages due to reduced chemical
noise.