TY - JOUR
T1 - Bottom-up Histone Post-translational Modification Analysis using Liquid Chromatography, Trapped Ion Mobility Spectrometry, and Tandem Mass Spectrometry
AU - Fuller, Cassandra N.
AU - Valadares Tose, Lilian
AU - Vitorino, Francisca N.L.
AU - Bhanu, Natarajan V.
AU - Panczyk, Erin M.
AU - Park, Melvin A.
AU - Garcia, Benjamin A.
AU - Fernandez-Lima, Francisco
N1 - Publisher Copyright:
© 2024 American Chemical Society.
PY - 2024/9/6
Y1 - 2024/9/6
N2 - 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.
AB - 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.
KW - bottom-up proteomics
KW - histones
KW - nano liquid chromatography
KW - parallel accumulation-serial fragmentation
KW - post-translational modifications
KW - tandem mass spectrometry
KW - trapped ion mobility spectrometry
UR - http://www.scopus.com/inward/record.url?scp=85201899340&partnerID=8YFLogxK
U2 - 10.1021/acs.jproteome.4c00177
DO - 10.1021/acs.jproteome.4c00177
M3 - Article
C2 - 39177337
AN - SCOPUS:85201899340
SN - 1535-3893
VL - 23
SP - 3867
EP - 3876
JO - Journal of Proteome Research
JF - Journal of Proteome Research
IS - 9
ER -