An analytical workflow based on liquid chromatography, trapped ion mobility spectrometry, and time-of-flight mass spectrometry (LC-TIMS-ToF MS/MS) for high confidence and highly reproducible “bottom-up” analysis of histone modifications and identification based on principal parameters (retention time [RT], collision cross section [CCS], and accurate mass-to-charge [m/z] ratio).
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.
In eukaryotic cells, DNA is packaged as chromatin into functional units called nucleosomes. These units are composed of an octamer of four core histones (two each of H2A, H2B, H3, and H4)1,2,3,4. Histones are amongst the most abundant and highly conserved proteins in eukaryotes, which are largely responsible for gene regulation5. Histone posttranslational modifications (PTMs) play a large role in the regulation of chromatin dynamics and rigger various biological processes such as DNA transcription, replication, and repair6. PTMs occur primarily on the accessible surface of the N-terminal regions of histones that are in contact with DNA3,7. However, tail and core modifications influence chromatin structure, altering inter-nucleosome interactions and recruiting specific proteins3,8.
A current challenge during liquid chromatography-mass spectrometry (LC-MS)-based proteomics is the potential co-elution of analytes of interest. In the case of data-dependent analyses (DDA), this translates into the potential loss of several precursor ions during the MS/MS acquisition process9. Time-of-flight (ToF) instruments acquire spectra at very high frequency9,10 (up to tens of kHz)11; this makes them capable of rapidly scanning the total precursor ions within a complex sample (MS1), thus promising optimal sensitivity and MS/MS sequencing rates (up to 100 Hz)9 and making them ideal for biological sample analysis10. Nevertheless, the sensitivity available at these high scan rates is limited by the MS/MS rate9. The addition of trapped ion mobility spectrometry (TIMS) in combination with an orthogonal quadrupole time-of-flight (qToF) mass spectrometer was used to mitigate these limitations. In TIMS, all precursor ions are accumulated in tandem and eluted as a function of their mobility, rather than selecting single precursor masses with a quadrupole9. Parallel accumulation-serial fragmentation (PASEF) allows for hundreds of MS/MS events per second without any loss of sensitivity9.
The principal aim of this work was to show the recent developments of DDA using parallel accumulation in the mobility trap followed by sequential fragmentation and collision-induced dissociation (CID). Histone PTMs were confidently assigned based on their retention times (RTs), mobilities, and fragmentation patterns.
NOTE: Histone samples were extracted using a method adapted from Bhanu et al. (2020)12.
1. Sample preparation
2. TIMS software interface
3. LC-TIMS-PASEF-ToF MS/MS
4. Data analysis
A bottom-up proteomic workflow (Figure 7) typically involves the following: extraction of the target protein(s) from a crude sample, followed by quantifying the concentration of the protein(s), and then fractionation, usually by gel electrophoresis or liquid chromatography. After fractionation, the proteins are digested using a proteolytic enzyme (often trypsin), and finally, mass spectrometric analysis of the resulting peptides and protein identification using an established database18. Sequence information is derived from precursor ions within the mass-to-charge (m/z) range indicated, which are subjected to collision-induced dissociation (CID), producing fragmentation patterns to be identified and sequenced using a database19 (Figure 8).
For this work, the principal goal was to develop and apply an LC-TIMS-PASEF-ToF MS/MS DDA method following the steps described previously in the protocol section. Determining the positionality of posttranslational modifications on isomeric and isobaric peptides has presented a particular challenge regarding identification and spectrum interpretation. In this study, recombinant human histone standards and HeLa S3 cells were used as samples.
Histone PTM analysis of human histone standards via ESI-TIMS-PASEF-ToF MS/MS yielded mid- to large-sized peptides (3-30 amino acids in length) detected with as many as 5 charges per peptide. The propionylation procedure was successful in producing longer, more informative peptides than those commonly produced by Tryptic digestion. Upon data analysis, peptides were identified in variously modified states. As an advantage, the TIMS-based method differentiated some positional isomeric peptides carrying the same PTMs. For example, two isomeric species may overlap in retention time and m/z; however, the two signals could be separated in the mobility domain (Figure 9).
The corresponding fragmentation spectra for the peptides shown in Figure 9 were annotated by proteomic analysis software using the appropriate FASTA files. In Figure 9A, the unmodified peptide is seen with three propionyl (+56.03) groups (on the N-terminal, lysine 18, and lysine 23). In Figure 9B, the peptide is observed with an acetyl group (+42.02) on lysine 18 and two propionyl groups (one at the N-terminal and one on lysine 23). Finally, in Figure 9C the peptide is seen with an acetylation observed on lysine 23 and two propionyl groups (on the N-terminal and lysine 18). As published previously, the PASEF advantage could be used for increasing sequencing speed and sensitivity by targeting the same feature repeatedly9. This allows the user to obtain more structural information from biological samples. In this case, this is applied to the type and position of PTMs occurring on each histone.
Posttranslational modification analysis can also be represented visually as a sequence coverage plot, as seen in Figure 10. In Figure 10A, the histone H3 standard which has been propionylated prior to digestion, presents with longer peptides than would have otherwise resulted, denoted by the blue lines. Histones extracted from HeLa S3 cells were processed in the same fashion, as represented by Figure 10B. Several PTMs were indicated, including many different patterns at the same amino acid positions. This is to be expected from biological samples. Of note, the few gray lines in Figure 10B denote peptides that were identified ambiguously due to the lack of an MS2 resulting from the low intensity.
Figure 1: Schematic representation and production of stage tips. (A–G) Step-by-step guide on the manufacture of a C18-silica disk stage tip. Please click here to view a larger version of this figure.
Figure 2: Data processing: 20210804 Propionylated Standard Histones Mix_QC Peptide. Before starting to process the data, make sure to prepare the theoretical list of possible charge states and their fragmentations (1550.9013; 775.9543; 517.6386; etc.) to extract those values from the base peak chromatogram (BPC +All MS). After extracting each peptide, make sure it looks like the analysis list shown in the figure. The peak 775.9543 was selected as an example. On the right side of the figure, three graphs are shown: the first corresponds to the chromatogram (intensity vs. time graph), the second to the mobilogram, and the third to the mass spectrum with PASEF fragmentation included. Please click here to view a larger version of this figure.
Figure 3: timsControl protocol steps 1-4. The figure shows the first four steps of the timsControl procedure. On the upper left-hand side, click the Instrument button to turn on and off the connection between the instrument and the software. Before executing any task, one must ensure that the software is in the operating mode. Finally, verify that the TIMS parameters are correct. Please click here to view a larger version of this figure.
Figure 4: Source parameters. In this case, Syringe Hamilton 500 µL was used only for TuneMix. Verify that the other parameters remain correct. Please click here to view a larger version of this figure.
Figure 5: Mass-to-charge (m/z) calibration. Select Calibrate until a score of 100% has been obtained in the bottom left panel Calibration Mode. Please click here to view a larger version of this figure.
Figure 6: Mobility calibration. Select Calibrate until a score of at least 98.5% has been obtained in the bottom left panel Calibration Mode. Please click here to view a larger version of this figure.
Figure 7: Typical bottom-up proteomics workflow. Step-by-step of the bottom-up procedures from sample preparation to identification9. Please click here to view a larger version of this figure.
Figure 8: Retention time, isotopic pattern, and H3 18-26 mobility profiles. (A) Unmodified propionylated, (B) K23Ac peptide propionylated in the other two positions, and (C) K18Ac propionylated in the other two positions. Notice the advantages of the mobility separation for the case of the structural isomers shown in panels B and C. Please click here to view a larger version of this figure.
Figure 9: Example of MS/MS fragmentation peptide sequencing using PASEF. Fragment spectra were obtained from proteomic analysis software for the H3 peptide with amino acid positions 18-26. (A) unmodified propionylated, (B) K23Ac peptide propionylated in the other two positions, and (C) K18Ac propionylated in the other two positions. Please click here to view a larger version of this figure.
Figure 10: Example of a visual histone PTM analysis summary. Results of observed peptides and PTMs from (A) an H3 standard and (B) H3 from HeLa S3 cells. Please click here to view a larger version of this figure.
Table 1: Standard and HeLa S3 peptide LC-TIMS-ToF MS/MS characteristics. Target and observed peptide list, including experimental properties (i.e., retention time, m/z, 1/Ko, and LC peak areas). Please click here to download this Table.
Histones are basic proteins that regulate chromatin structure by interacting with DNA in the form of octamers consisting of the four core histones (two each of H2A, H2B, H3, and H4)20. Histones contain numerous lysine and arginine residues, which are readily modified, leading to extensive PTMs that alter the chromatin chemistry by influencing histone function or by binding to other cellular proteins21. PTMs can elicit biological responses by working in tandem, with specific groups of PTMs having been reported in several diseases, most notably, several types of cancer22.
When DNA damage is recognized at the cellular level, it is instantly followed by the action of a complex signaling cascade where lesions are marked, followed by the coordination of cell cycle progression and activation of the required repair pathways. In addition, DNA damage induces various modifications, such as acetyl and methyl adducts, which facilitate protein recruitment23. The great variety of PTMs that are involved in DNA lesions leads to the question of how these molecular mechanisms regulate their coexistence and what the functional importance of defending the integrity of the genome through an extremely complex integrated network is. For example, lysine 9 trimethylation of histone H3 (H3K9me3) has been linked to different pathologies in various diseases24. For reasons such as this, it is necessary to develop instrumental analytical methodologies that allow the complete characterization of these modifications at the cellular level23.
Analysis of the HeLa S3 histone extractions using manual data analysis software and proteomic analysis software revealed PTMs, including acetylation (+42.01 Da), methyl-propionylation (+70.04 Da), dimethylation (+28.03 Da), and trimethylation (+42.05) for several histone proteins. Additionally, the PASEF-based MS/MS method was able to differentiate some positional isomeric peptides carrying the same PTMs.
In the introduction, the advantages of coupling LC-TIMS-ToF MS/MS in the study of PTMs to show the recent developments of DDA using parallel accumulation in the mobility trap followed by sequential fragmentation and collision-induced dissociation are briefly described. The main idea is to establish a methodology that allows for the resolution of signals coming from different peptides and that, up until now, classical techniques have not been able to resolve. The derivatization process using propionic anhydride prevents the cleavage of lysine C-terminals by Trypsin, generating longer, more informative peptides. Peptides with the same m/z and retention time were able to be identified by their fragmentation patterns, but it was also seen that some of these species could be separated in the mobility domain using this LC-TIMS-PASEF-ToF MS/MS method.
To better understand this, Figure 8 represents three main characteristics of any molecule, thus allowing the identification of a compound, whether they are intact proteins, lipids, or peptides (in this case, histone H3 18-26), to name a few examples. These characteristics include the retention time (min) of a compound in the chromatographic column, the mass-charge ratio (m/z) of each compound, and the mobility (1/Ko) that these compounds present when they interact with the drift gas. In Figure 8A, the unmodified H3 peptide 18-26 is shown to have an RT of 28.15 min and that it presents two bands in its mobility spectrum, indicating that it has at least two conformations, a result that is suspected to be a result of the two lysines (18 and 23) that have been propionylated following the previously described protocol. The following spectra (Figure 8B,C) show the same peptide (H3 18-26) but varying the position of the acetylation group (42.02) between B, K18Ac and C, K23Ac. These two isomers (K18Ac and K23Ac) have been identified through the mobilogram, as they present with different spatial distributions, which results in different interactions with the gas in the TIMS cell. The importance of this method lies in the possibility of identifying and studying in more detail the different PTMs that have been associated with different diseases through, for example, DNA damage.
When fragmentation data are sparse, identifying a modification at a specific residue is challenging because two or more dissimilar modifications could occur simultaneously at (or near) the same residues and may be understood as a single modification25. This could be resolved by ensuring that the unmodified peptide has been identified, especially by using a standard to confirm or deny the presence of a single modification rather than multiple modifications (Table 1).
To avoid excessive contamination or extractions of impure histones, it is important to check the quality of the reagents before use. For example, if the NIB buffer solution is stored and used in bulk, ensure that the solution is clear with no outward appearance of turbidity or abnormal presentation. Turbidity may be the result of bacterial growth, which would contaminate samples and could result in a mixture of histones and bacterial proteins. In addition, it is recommended to prepare fresh calibration curves for assays, such as the BCA or Bradford assay used to determine protein concentration, ensuring that the protein used for the calibration curve is not expired or degraded.
This method can be extended to other types of cells or organisms, for example, mosquitoes. In the case of whole or partial organisms, selecting an appropriate number of organisms is especially important to ensure that the final histone concentration is suitable for analysis.
Also, as a general guideline for mass spectrometer maintenance, the front end should be cleaned periodically to prevent buildup on the instrument and contamination between runs. This cleaning should include the curtain, orifice plate, and quadrupole, as required.
Generally, when an LC is used, it is necessary to take into consideration preparing fresh mobile phase(s) each week using MS-grade solvents. It is good practice to keep dedicated pipettes and glassware for mobile phase preparation and to purge the LC lines whenever new solutions are placed on the system. Guard and separation columns should usually be replaced every 100-200 injections and 500-1500 injections, respectively26. Be sure to inject blanks before and after running a batch of samples. If there are a large number of samples within a given batch, one may also consider running a blank at various intervals within the batch.
The protocol provides a PASEF-based DDA workflow for detecting histone PTMs and differentiation of isobaric and isomeric species based on ion mobility.
This protocol requires extensive sample preparation, and overall experimental sample preparation time should be accounted for. On average, the sample preparation protocol requires 2-3 business days to complete. Additionally, differences between laboratories and instrument versions can affect the overall sensitivity of the analysis.
Very few proteomic data analysis software have been deemed adequate for use in analyzing histones via bottom-up methods without manual adjustment or correction27,28,29. Results should (at least at first) be confirmed using manual analysis, which is also time-consuming. If analytical software is used, it should have MS/MS annotation capabilities, which are generally easy to confirm or reject.
It is also worth mentioning that it is impossible to separate isomers through mass spectrometry unless a TIMS cell is inserted and mobility values are used; for example, the positions of histone modifications can be determined using fragmentation patterns (PASEF).
The authors have nothing to disclose.
This material is based upon work supported by the National Science Foundation under Grant No. HRD-1547798 and Grant No. HRD-2111661. These NSF Grants were awarded to Florida International University as part of the Centers of Research Excellence in Science and Technology (CREST) Program. This is contribution number 1672 from the Institute of Environment, a Preeminent Program at Florida International University. Additional support was provided by the National Institute of Health under Grant No. R21AI135469 to Francisco Fernandez-Lima and Grant No. R01HD106051 to Benjamin A. Garcia, as well as by the National Science Foundation under Grant No. CHE-2127882 to Benjamin A. Garcia. The authors would like to acknowledge the initial support of Dr. Mario Gomez Hernandez during initial method developments.
-80 °C Freezer | |||
1x Phosphate Buffered Saline (PBS), pH 7.4 | Thermo Fisher Scientific | 10010023 | Animal Origin-Free |
1 mL Pipette Tips | Thermo Fisher Scientific | 94060710 | Finntip Flex 1000 μL, nonsterile, nonfiltered, racked tips |
1.5 mL Microcentrifuge Tubes | Thermo Fisher Scientific | 14-282-300 | Use these tubes for the simple and safe processing of sample volumes up to 1.5 mL |
10 µL Pipette Tips | Thermo Fisher Scientific | 94060100 | Finntip Flex, 10 μL, nonsterile, non-filtered, racked |
10% NP-40 | Thermo Fisher Scientific | 28324 | NP-40 Surfact-Amps Detergent Solution |
10x Dulbecco’s PBS without Ca2+/Mg2+ | (Mediatech) | MT21031CM | |
15 mL Conical Tubes | Corning | 352196 | Falcon Conical Centrifuge Tubes |
200 µL Gel-Loading Pipette Tips | Thermo Fisher Scientific | 02-707-138 | Fisherbrand Gel-Loading Tips, 1–200 μL |
200 µL Pipette Tips | Thermo Fisher Scientific | 94060310 | Finntip Flex 200μL, nonsterile, nonfiltered, racked tips |
2x Laemmli Sample Buffer | Bio-Rad | 1610737 | Premixed protein sample buffer for SDS-PAGE |
50 mL Conical Tubes | Corning | 352070 | Falcon Conical Centrifuge Tubes |
96-well flat bottom plate | Thermo Fisher Scientific | 12565501 | |
96-well plate, V-Bottom 600 μL | Axygen | P-DW-500-C-S | |
Acetone | Sigma Aldrich | 179124 | ACS reagent, ≥99.5% |
Acetonitrile (ACN) | Thermo Fisher Scientific | A998 | HPLC, Fisher Chemical |
Acetonitrile with 0.1% Formic acid (v/v), LC/MS Grade | Thermo Fisher Scientific | LS120 | Optima LC/MS Grade, Thermo Scientific |
AEBSF | Thermo Fisher Scientific | 328110500 | AEBSF hydrochloride, 98% |
Ammonium bicarbonate, NH4HCO3 | Sigma Aldrich | 09830 | BioUltra, ≥99.5% (T) |
Ammonium hydroxide solution, NH4OH | Sigma Aldrich | AX1303 | Meets ACS Specifications, Meets Reagent Specifications for testing USP/NF monographs GR ACS |
Argon (Ar) | Airgas | AR HP 300 | |
BEH C18 HPLC column | Waters | 186003625 | XBridge Peptide BEH C18 Column, 300 Å, 5 µm, 4.6 mm X 250 mm, 1K–15K |
Bovine Serum Albumin (BSA) | Sigma Aldrich | A7906 | Heat shock fraction, pH 7, ≥98% |
Calcium chloride, CaCl2 | Sigma Aldrich | C4901 | Anhydrous, powder, ≥97% |
Cell dissociation buffer | Thermo Fisher Scientific | 13151014 | |
Ceramic scoring wafer | Restek | 20116 | |
Compass DataAnalysis 6.0 | Bruker Datonics | ||
Compass HyStar 6.2 | Bruker Daltonics | ||
Compass IsotopePattern | Bruker Daltonics | ||
Compass timsControl 4.1 | Bruker Daltonics | ||
Coomassie Brilliant Blue R-250 | Bio-Rad | 1610436 | |
Deep Well, 96-Well Microplate, 2.0 mL | Thermo Fisher Scientific | 89237526 | |
Disposable Cell Lifters | Thermo Fisher Scientific | 08100240 | Fisherbrand Cell Lifters; Disposable lifters quickly remove cell layers |
Disposable Pellet Pestles | Thermo Fisher Scientific | 12-141-363 | Fisherbrand Pellet Pestles; Resuspend protein and DNA pellets or grind soft tissue in microcentrifuge tubes |
Dithiothreitol (DTT) | Thermo Fisher Scientific | P2325 | 1 M |
Formic acid (FA) | Sigma Aldrich | 695076 | ACS reagent, ≥96% |
Fused silica capillary 75 μm ID x 363 μm OD | (Molex (Polymicro) | TSP075375 | |
Glacial Acetic Acid | Thermo Fisher Scientific | A38S | Acetic Acid, Glacial (Certified ACS), Fisher Chemical |
Glass Pasteur Pipettes | Sigma Aldrich | BR747725-1000EA | |
High-Performance Liquid Chromatograph | Shimadzu | Shimadzu Prominence 20 HPLC UFLC System | |
Hydrochloric acid, HCl | Sigma Aldrich | 258148 | ACS reagent, 37% |
Hypercarb 30-40 μm Carbon 150–300 Å | Thermo Fisher Scientific | 60106-402 | |
Hypersep cartridge | Thermo Fisher Scientific | 60109-404 | |
LC/MS Calibration Standard, for ESI-ToF | Agilent | G1969-85000 | TuningMix |
Magnesium chloride, MgCl2 | Sigma Aldrich | M8266 | Anhydrous, ≥98% |
Methanol, for HPLC | Thermo Fisher Scientific | A454 | Optima for HPLC, Fisher Chemical |
Microcentrifuge Tube Adapters | GL Sciences | 501021514 | |
Microcystin | Thermo Fisher Scientific | 50-200-8727 | Enzo Life Sciences Microcystin-LA |
MS sample vial, LaPhaPack, Snap, 12 mm x 32 mm | LEAP PAL Parts | LAP.11190933 | |
Nanodrop | Thermo Fisher Scientific | model: ND3300 | |
Nitrogen (N2) | Airgas | NI UHP300 | |
PEAKS Studio X+ | Bioinformatic Solutions | ||
pH indicator strips, Instachek | Micro Essential Lab | JR-113 | Model: Hydrion |
Potassium chloride, KCl | Sigma Aldrich | P3911 | ACS reagent, 99.0%–100.5% |
Pressure Injection Cell | Next Advance | model: PC77 | |
Propionic Anhydride | Sigma Aldrich | 8.00608 | For synthesis |
Refrigerated Centrifuge (700–18,000 x g) | NuAire, model: Nuwind | NU-C200V | |
Reprosil-Pur 120 C18-AQ 3 μm, 3 g | ESI Source Solutions | r13.aq.0003 | |
SDS-PAGE Gels | Bio-Rad | 4569035 | Any kD precast polyacrylamide gel, 8.6 cm × 6.7 cm (W × L), for use with Mini-PROTEAN Electrophoresis Cells |
Sodium butyrate | Thermo Fisher Scientific | A11079.06 | 98+% |
Sodium chloride, NaCl | Sigma Aldrich | S9888 | ACS reagent, ≥99.0% |
SPE disk, C18 | VWR | 76333-134 | Empore SPE disk, C18, CDS Analytical, 90 mm x 0.5 mm, 12 µm |
SpeedVac+ vacuum pump and plate rotor | Savant | model: SC210A | |
Sucrose | Millipore | 1.07651 | suitable for microbiology |
Sulfuric acid, H2SO4 | Sigma Aldrich | 339741 | 99.999% |
TIMS-ToF Mass Spectrometer | Bruker Daltonics | model Tims tof ms | |
Trichloroacetic acid solution, TCA | Sigma Aldrich | T0699 | 6.1 N |
Trifluoroacetic acid (TFA) | Sigma Aldrich | 302031 | Suitable for HPLC, ≥99.0% |
Triversa Nanomate | Advion | model: TR263 | |
TrypsinProtease, MS Grade | Thermo Fisher Scientific | 90057 | |
Tube rotator | Thermo Fisher Scientific | 88881001 | |
Vortex Mixer | Thermo Fisher Scientific | 88880017 | |
Water with 0.1% Formic acid (v/v), LC/MS Grade | Thermo Fisher Scientific | LS118 | Optima LC/MS Grade, Thermo Scientific |