dC ₅                                 deoxyoligonucleotide with the sequence: CCCCC

dC ₂AC ₂                          deoxyoligonucleotide with the sequence: CCACC

RNase A                         bovine pancreatic ribonuclease A

ESI-IT-MS                      electrospray ionization ion-trap mass spectrometry

nESI-Q-TOF-MS           nanoelectrospray ionization quadrupole time-of-flight mass spectrometry

RNase A+dC ₅                ligand-bound form of RNase A (with one dC ₅ ligand)

RNase A+dC ₂AC ₂         ligand-bound form of RNase A (with one dC ₂AC ₂ ligand)

RSD                                relative standard deviation

Bovine pancreatic ribonuclease A (RNase A) is an endoribonuclease (EC 3.1.27.5) that hydrolyzes RNA. It is a small single chain polypeptide (124 amino acids) containing four disulfide bridges and is known for its significant stability ¹ . RNase A has been called “the most studied enzyme of the 20 ^th century” and it has seen wide use as a model protein in biochemical and biophysical experiments ¹ . Undergraduate life-science majors often learn of RNase A as part of a biochemistry course in the context of the Nobel Prize winning protein folding experiments performed by Christian Anfinsen ² . Students may also be familiar with the need to inhibit ribonucleases when working with RNA in the lab, often accomplished with diethyl pyrocarbonate, or will have learned about the role of ribonucleases in microRNA biology ³ . Still others may recognize RNase A as an example of an enzyme that employs general acid-base catalysis as part of its chemical mechanism ⁴ . Thus, RNase A is an excellent model for undergraduate lab experiments, not only because it has been extensively studied, but also because its use presents an opportunity to reemphasize important concepts in biochemistry and biology.

The application of mass spectrometry to the analysis of biomolecules has made an enormous impact in the life sciences. Protein identification, the characterization of protein modifications, and the quantification of biomolecules using mass spectrometry are commonplace. Of these, protein identification is the most established in an undergraduate teaching lab ^{5– 10} . Numerous other biological applications of mass spectrometry have existed for many years, but some of these are arguably, less broadly appreciated, and this is especially true for undergraduates. Native mass spectrometry is an approach based on electrospray ionization, where biomolecules are sprayed from a non-denaturing solvent ¹¹ . Under such conditions, protein-ligand complexes can be maintained and a dissociation constant ( K _d) can be determined via a titration experiment ^{12– 14} .

Previously, nanoelectrospray ionization quadrupole time-of-flight mass spectrometry (nESI-Q-TOF-MS) was used to investigate ligand binding to RNase A ^{12, 15, 16} . These studies used nESI ionization for its superior sensitivity and relied on the TOF mass analyzer for its high mass range ^{12, 15, 16} . In Zhang et al., free RNase A and the ligand-bound forms of RNase A populated three charge states (+8, +7, and +6) at pH 6.6, with most of the signal (~90%) coming from the +7 charge state, which exceeded m/z 2000 in the ligand-bound forms ¹² . Similarity, in Sundqvist et al., focus was placed on the +7 charge state of free RNase A and its ligand-bound forms ¹⁵ . In contrast, Yin et al. reported the most abundant charge state of free and ligand-bound forms of RNase A to be +8 at pH 6.6 ¹⁶ . Unfortunately, California State University-Chico does not own a nESI-Q-TOF-MS as employed by each of these research groups. Instead, we have an electrospray ionization ion-trap mass spectrometer (ESI-IT-MS), which by comparison to nESI-Q-TOF-MS, offers a lower sensitivity and mass range (50–2000 m/z). Consequently, at the outset of this preliminary investigation, it was recognized that observation of the +7 and +6 charge states of ligand-bound RNase A would not be possible with our instrument.

This work was an attempt to develop a biochemistry lab experience that would introduce undergraduate life-science majors to the use of mass spectrometry for the analysis of protein-ligand interactions. Two deoxyoligonucleotides, CCCCC (dC ₅) and CCACC (dC ₂AC ₂), were investigated for their ability to bind RNase A. Titration experiments were performed using a fixed RNase A concentration and variable deoxyoligonucleotide concentrations. Samples at equilibrium were infused directly into our ESI-IT-MS under native conditions. The relative simplicity of the sample preparation and instrument operation (by direct infusion) were viewed as desirable features for an undergraduate teaching lab. Data analysis was also straightforward. Herein is described the results of this preliminary investigation. This work differentiates itself from the abovementioned RNase A ligand binding studies (using mass spectrometry) by the experimental conditions employed, which includes the identity of the investigated ligands and the type of mass spectrometer used ^{12, 15, 16} .

Table 1 indicates that samples contained an overall [RNase A] of 40.9 μM. Relatively low signal intensities observed for the +8 charge state of free and ligand-bound forms of RNase A necessitated this concentration, which was higher than the 5–20 μM RNase A used by others in nESI-Q-TOF-MS experiments ^{12, 15, 16} . Table 2 and Table 3 present predicted m/z values for free RNase A and the ligand-bound forms of RNase A (RNase A+dC ₅ and RNase A+dC ₂AC ₂) with multiple P _i adducts, which correlated well with observed m/z values ( Figure 1). Upon increasing the concentration of dC ₅, the total ion abundance of free RNase A was found to decrease in intensity while the total ion abundance of RNase+dC ₅ was found to increase in intensity, which suggested 1:1 stoichiometry for the dC ₅:RNase A interaction ( Figure 1A–E). Similar results were seen for the titration using dC ₂AC ₂ ( Figure 1F–J). Table 4 presents total ion abundance data for free RNase A in samples that contained no added deoxyoligonucleotide. Total ion abundance data for free RNase A across six replicates gave a RSD of 16.4% ( Table 4). Table 5 contains total ion abundance data for free RNase A and the ligand-bound forms of RNase A in samples that contained various concentrations of dC ₅ or dC ₂AC ₂. Total ion abundance data across three replicates at each [deoxyoligonucleotide] exhibited RSD values of approximately 20% or less ( Table 5). A plot of the total ion abundance for free RNase A, RNase A+dC ₅, and RNase A+dC ₂AC ₂ as a function of [deoxyoligonucleotide] is shown in Figure 2. The total ion abundance for RNase A+dC ₅ and RNase A+dC ₂AC ₂ increased until 20 μM deoxyoligonucleotide, but decreased at 40 μM ( Figure 2). Table 6 presents the calculated total ion abundance ratio ( R) and dissociation constant ( K _d) at each [deoxyoligonucleotide]. Samples containing <40 μM deoxyoligonucleotide unexpectedly produced negative K _d values ( Table 6). By contrast, Table 6 shows that samples containing 40 μM deoxyoligonucleotide produced consistent positive values where the average K _d for dC ₅ was 2.2 ± 0.1 μM and the average K _d for dC ₂AC ₂ was 1.0 ± 0.1 μM.

This preliminary work demonstrates the potential and pitfalls of a LCQ ESI-IT-MS instrument to investigate protein-ligand interactions in an undergraduate teaching lab. Even though dC ₅ and dC ₂AC ₂ binding to RNase A are clearly illustrated in Figure 1, the presence of the P _i adducts complicated the mass spectra and broadened the signals for free RNase A and the ligand-bound forms of RNase A. In-source collision-induced dissociation was explored to reduce P _i adduct formation, but it appeared to disrupt the RNase A+dC ₅ and RNase A+dC ₂AC ₂ complexes, and so this approach was abandoned (data not shown). Although it was not attempted, centrifugal desalting of the RNase A stock solution might have eliminated P _i adducts and improved the quality of the mass spectra in Figure 1. As an added benefit, in the context of an undergraduate lab, desalting would also introduce students to a common sample preparation technique. It is unclear why the decrease in the total ion abundance for the ligand-bound forms of RNase A was observed at higher deoxyoligonucleotide concentrations ( Figure 2). Previously, the ion intensity ratio of free RNase A to the RNase A+cytidine 2′-monophosphate (2′-CMP) complex was observed to vary with charge state as follows: +8 (0.65), +7 (0.73), +6 (1.1) ¹² . This led Zhang et al. to suggest that either the binding of ligand, or the presence of ligand in the analyzed RNase A samples, created a change of the charge state distribution for the protein-ligand complex ¹² . In the present work, the binding of deoxyoligonucleotide, or the presence of deoxyoligonucleotide in samples, could have shifted some of the total ion abundance of free and/or ligand-bound RNase A from the +8 charge state to lower charge states, which were beyond the mass range of our ion-trap mass analyzer. This highlights an inherent limitation of this work, which was the inability to gather data for all free and ligand-bound RNase A charge states. Kitova et al. stated the importance of including all ligand-bound and free protein ions in the calculation of R, and emphasized that the “sometimes-used practice” of employing a particular charge state to determine K _a should be avoided ¹³ . Thus, the lack of data for the +7 and +6 charge states of RNase A hindered accurate collection of total ion abundance data, which may have affected calculations of R and led to the negative K _d values at low ligand concentrations ( Table 6). Other factors to consider, that could have affected measurements, include non-ideal ionization conditions and non-specific ligand binding. Benkestock et al. showed that instrument-derived parameters (e.g. capillary-to-cone distances) could affect the protein-ligand complex to free protein ratio ¹⁹ . They also demonstrated that compared to pneumatically assisted ESI, which was used in this work, nESI better reflects the equilibrium between free protein and protein–ligand complexes in solution. Furthermore, Kitova et al. noted that changes in the magnitude of Ka, with changes in ligand concentration, might indicate nonspecific ligand binding ¹³ . As seen in Table 6, Kd values varied with the deoxyoligonucleotide concentration. Therefore, it is reasonable to suspect that non-specific binding may have contributed to the decreased total ion abundance of the ligand-bound forms of RNase A at higher ligand concentrations ( Figure 2). Notwithstanding these possibilities, the positive K _d values in Table 6 are of similar magnitude to those determined by Zhang et al. for 2′-CMP and CTP, via a nESI-Q-TOF-MS titration experiment, which were 1.7 ± 0.3 μM and 0.8 ± 0.2 μM, respectively ¹² . They are also in the neighborhood of in solution K _d measurements (3-24 μM) observed for the binding of short fluorescein-labeled deoxyoligonucleotides to RNase A ²⁰ . In conclusion, while RNase A is an excellent model for many experiments, instructors wishing to use a LCQ ESI-IT-MS instrument to investigate protein-ligand interactions are encouraged to consider other protein-ligand systems that would enable all charges states (of the free and ligand-bound protein) to be observed.

The data referenced by this article are under copyright with the following copyright statement: Copyright: � 2018 Clark DD

Data associated with the article are available under the terms of the Creative Commons Zero "No rights reserved" data waiver (CC0 1.0 Public domain dedication).

Dataset 1. LCQ *.raw data files for all samples. 10.5256/f1000research.14268.d198373 ²¹

Data files 1–6 are for samples that contained free RNase A (6 replicates), Data files 7–18 are for samples that contained RNase A and dC ₅ (3 replicates per [dC ₅]), Data files 19–30 are for samples that contained RNase A and dC ₂AC ₂ (3 replicates per [dC ₂AC ₂]).