A Workflow for Ultra-rapid Analysis of Histone Post-translational Modifications with Direct-injection Mass Spectrometry

Abstract

Chromatin modifications, like histone post translational modifications (PTMs), are critical for tuning gene expression and many other aspects of cell phenotype. Liquid chromatography coupled to mass spectrometry (LC-MS) has become the most suitable method to analyze histones and histone PTMs in a large-scale manner. Selected histone PTMs have known functions, and their aberrant regulation is linked to a wide variety of diseases, including cancer. However, histone analysis is scarcely used in diagnostics, partially due to the limited throughput and not ideal reproducibility of LC-MS based analysis. We describe a workflow that allows for high-throughput sample preparation is less than a day using 96-well plates. Following preparation, samples are sprayed into MS without LC, using an automated direct injection (DI-MS) method. Each analysis provides accurate quantification for 29 peptide sequences with 45 PTMs (methylations, acetylations and phosphorylations) for a total of 151 histone marks plus 16 unmodified histone peptides for relative quantification of histone variants. This workflow allows for < 1 min MS runs and higher reproducibility and robustness due to the absence of carryover or LC-based batch effects. Finally, we describe an engineered peptide sequence used to accurately monitor the efficiency of sample preparation, which can be detected during the DI-MS run.

Keywords: Advantage over Liquid-Chromatography (LC); Chromatin; Direct injection; Histone; Mass spectrometry; Post-translational modifications (PTMs).

Figure 1.. Merits of DI-MS workflow.

Multiplexing sample preparation from extraction to derivatization/digestion to desating in a highthroughput manner. Sample preparation still requires at least one day to get histones ready for analysis. However, the workflow allows to multiplex potentially thousands of samples in a single batch, and they can then be analyzed at the rate of one per minute. Virtually, about 344 samples can be analyzed in a regular week (5-days work), when four 96-well plates are processed simultaneously. This capacity is higher than any existing diagnostics facility analyzing histone modifications. Each sample yields data with 220 relative quantifications from 29 peptides, belonging to 16 histone types, both canonical and variant, bearing single or multiple PTMs such as methylations, acetylations and phosphorylations in a total of 45 loci is obtained within a week.

Figure 2.. Histone derivatization and desalting.

A. Set up of supplies in chemical hood for rapid performance of derivatization. B. Prepare desalting stage-tips with C18 (white plug) and PGC (black particles packed as a slurry in acetonitrile using a multichannel pipette) housed in a pipette tip wafer. Note that the first row (row A) is not usually used as we normally reserve these to check BSA standard and desalting buffers during MS analysis. This stage-tip wafer can be prepared ahead of time and stored on the bench with a lid in an empty tip box. C. Positioning the stage-tip wafer over wells in a 96-well plate. Note that the four corners are tethered by tips, if all the 84 wells are not used. Desalt wafer is positioned in the wells of the microplate and is placed in a speedvac plate holder for elution.

Video 1.. Rapid derivatization by a single user

Video 2.. Fashioning in-house desalting columns

Figure 3.. Checking quality of DI-MS run by inspecting mass spectra in Xcalibur Qual Browser 3.1.

A good MS analysis showing summed intensities of all ions, both from background noise as well as sample components in the scanned mass range (top left), base peak of the mass spectrum above (bottom left) and a representative MS2 spectrum (right). In orange, #1 indicates a good run QC where a pair each of MS and MS2 have been acquired in less than 2 min. Though the method is for 2 min, as soon as two MS-MS2 events have been recorded, the nanomate stops acquisition and the leftover sample is returned to the well. In order to get this good quality spectrum, a minimum of 10 μl of peptides at 1 mg/ml concentration is needed. #2 indicates densely ionized sample without polymer adducts, #3 shows the +2 charged abundant ions.

Figure 4.. Difference between relative ratios and single PTM abundance, illustrated for H3 9-17 peptide and H3K9me2 respectively.

The single PTM abundance of H3K9me2 is calculated as: sum of relative ratios of (H3K9me2 + H3K9me2K14ac).

Figure 5.. Visualization of histone PTM analysis data.

A. Volcano plot provides group information by plotting log fold change between two conditions [x-axis, log₂(Treatment/Control)] as a function of their significance [y-axis, -log₂(0.05)]. The elongated ovals represent imputed Zscore values for expression only in treatment (red) or in control (green) conditions. Threshold for significance is 4.32 [= log₂(0.05)] and that for fold change is 1 (= log₂ of 2). The red square represents the histone marks upregulated in the treatment and the green square encompasses histone PTMs upregulated in the control. B. Post-analysis representation of “histone ratios_single PTMs” table from EpiProfile Lite. Heat map was constructed in Perseus 1.5.3.2 version using Zscore normalized values; this heat map provides sample expression levels, along with co-trending marks shown by hierarchial clustering, with samples in the columns and histone marks in rows. Hierarchial clustering was based on histone marks with samples left unclustered. The samples here display expression ranging from -2 (downregulation, green) to 3 (upregulation, red). A definite pattern of regulation (expression from red to green and vice versa) between control and treated samples is seen in two blocks of cotrending marks. C. Abundance of global acetylations (ac) and methylations (valencies me1, me2 and me3 and total me) can be represented as graph. Horizontal bar over conditions denotes significant changes by t-test: *P = 0.04, **P = 0.01 and ***P = 0.0004.