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

• Published in: Journal of proteome research Vol. 23; no. 9; pp. 3867 - 3876
• Main Authors: Fuller, Cassandra N., Valadares Tose, Lilian, Vitorino, Francisca N. L., Bhanu, Natarajan V., Panczyk, Erin M., Park, Melvin A., Garcia, Benjamin A., Fernandez-Lima, Francisco
• Format: Journal Article
• Language: English
• Published: United States American Chemical Society 06-09-2024
• Subjects: Amino Acid Sequence; Cell Line, Tumor; Chromatography, Liquid - methods; Histones - analysis; Histones - chemistry; Humans; Ion Mobility Spectrometry - methods; Molecular Sequence Data; Protein Processing, Post-Translational; Proteomics - methods; Reproducibility of Results; Tandem Mass Spectrometry - methods; histones; tandem mass spectrometry; nano liquid chromatography; bottom-up proteomics; post-translational modifications; trapped ion mobility spectrometry; parallel accumulation-serial fragmentation
• ISSN: 1535-3893; 1535-3907; 1535-3907
• DOI: 10.1021/acs.jproteome.4c00177

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.