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The "Histone Code" is comprised of specific types and positions of post-translational modifications (PTMs) which produce biological signals for gene regulation and have potential as biomarkers for medical diagnostics. Previous work has shown that electron-based fragmentation improves the sequence coverage and confidence of labile PTM position assignment. Here, we evaluated two derivatization methods (e.g., irreversible - propionylation and reversible-citraconylation) for bottom-up analysis of histone H4 4-17 proteoforms using online liquid chromatography (LC), trapped ion mobility spectrometry (TIMS), and electron-based dissociation (ExD) in tandem with mass spectrometry. Two platforms were utilized: a custom-built LC-TIMS-q-ExD-ToF MS/MS based on a Bruker Impact and a commercial μLC-EAD-ToF MS/MS SCIEX instrument. Complementary LC-TIMS preseparation of H4 4-17 0-4ac positional isomer standards showed that they can be resolved in their endogenous form, while positional isomers cannot be fully resolved in their propionylated form; online LC-ExD-MS/MS provided high sequence coverage (>90%) for all H4 4-17 (0-4ac) proteoforms in both instrumental platforms. When applied to model cancer cells treated with a histone deacetylase inhibitor (HeLa + HDACi), both derivatization methods and platforms detected and confirmed H4 4-17 (0-4ac) proteolytic peptides based on their fragmentation pattern. Moreover, a larger number of HeLa + HDACi H4 4-17 proteoforms were observed combining LC-TIMS and LC-q-ExD-ToF MS/MS due to the positional isomer preseparation in the LC-TIMS domain of citraconylated H4 4-17 (0-4ac) peptides.

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/K 0 ) 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, me 1-3 ) 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.

Histone proteins are highly abundant and conserved among eukaryotes and play a large role in gene regulation as a result of structures known as posttranslational modifications (PTMs). Identifying the position and nature of each PTM or pattern of PTMs in reference to external or genetic factors allows this information to be statistically correlated with biological responses such as DNA transcription, replication, or repair. In the present work, a high-throughput analytical protocol for the detection of histone PTMs from biological samples is described. The use of complementary liquid chromatography, trapped ion mobility spectrometry, and time-of-flight mass spectrometry (LC-TIMS-ToF MS/MS) enables the separation and PTM assignment of the most biologically relevant modifications in a single analysis. The described approach takes advantage of recent developments in dependent data acquisition (DDA) using parallel accumulation in the mobility trap, followed by sequential fragmentation and collision-induced dissociation. Histone PTMs are confidently assigned based on their retention time, mobility, and fragmentation pattern.