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Description
Introduction
Histones are heavily and variably decorated by PTMs, thereby affecting their binding to chromosomal regions. Top-down proteomics of histones is advantageous in capturing the PTM combination to obtain epigenetic status. Being rich in lysine residues hampers histone separation by RP chromatography. Capillary electrophoresis (CE) – MS has emerged as powerful alternative for histone analysis. Thus, ZipChip(CE) was benchmarked against nanoRP-LC with a C4 column.
Methods
Calf thymus histone preparation was dissolved in water to 1 mg/ml. For CE 5 ng (5 nl) were injected per analysis and separated in 6 min on a HS CE chip at a field strength of 500V / cm2 in intact antibody BGE buffer. MS and MS/MS spectra were recorded on an orbitrap Ascend set to intact protein mode using instrument templates (<30 kDa intact protein). Protein XML files of Bos Taurus histones were imported into Prosight 4.2/Proteome Discoverer 3.0 for proteoform identification. For LC, 2 µg protein were separated on C4 column in 50 min.
Results and Discussion
The HS chip separated the histones into two major peaks, whereas LC displayed broad elution without baseline separation. The preparation contained histones H1, H2a, H2b, H3, and H4, all of which were identified in the merged MS data (CID, HCD, ETD, EThCD, UVPD). In total, 216 proteoforms and 6291 PrSMs (proteoform spectral matches) were identified at high confidence with CE compared to 569 proteoforms and 35091 PrSMs with RP-LC. PrSMs displayed a most frequent cleavage efficiency of 10%. PrSM count increased from UVPD, EThCD, ETD, HCD to CID. Even though the longer analysis time and higher protein load increased identification, separation performance and speed favored CE. The fast analysis time in combination with very low sample consumption qualify CE as a convenient tool for optimization and data acquisition over a wide range of MS settings.
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