Unraveling the thermal decomposition and chemical ionization of methionine using ion mobility spectrometry and computational chemistry

Abstract

Methionine, an essential amino acid, plays a crucial role in various biological processes and exhibits antioxidant properties, significantly impacting the health and well-being of humans, livestock, poultry, and fisheries. This study focuses on the thermal decomposition products of methionine utilizing ion mobility spectrometry (IMS) in conjunction with computational chemistry. This research focuses on analyzing the thermal decomposition products of methionine using ion mobility spectrometry (IMS) combined with computational chemistry. The IMS spectra of methionine were acquired under normal conditions and after subjecting the samples to elevated temperatures (280 °C) for different durations (5, 10, and 15 s) prior to analysis. By comparing the IMS spectra with those of pure compounds, analyzing changes in peak intensities over elapsed time, and employing a two-reference method to predict the masses of ionic species, the study aimed to identify and characterize the thermal degradation products of methionine at this temperature. Density functional theory (DFT) was employed to further interpret the IMS spectra to predict the fragments generated during decomposition. To achieve this, the decomposition pathways of methionine and protonated methionine from the N, O, and S centers are considered comprehensively. Consequently, four potential energy surfaces (PESs) are constructed with suitable details. The obtained PESs revealed that the saddle points and produced fragments in protonated molecules are more stable than neutral molecules compared to the respective reactant. This led us to conclude that the initial high impact between the hydronium ion and methionine plays a crucial role in the fragmentation process. The theoretical findings align well with the IMS spectra. Moreover, the methodology employed in this study can be applied to exactly interpret the IMS spectra of any similar molecular system.

Supplementary Information

The online version contains supplementary material available at 10.1038/s41598-025-18103-w.

Introduction

The chemistry of small biological molecules, such as amino acids, is essential across multiple disciplines. These molecules are studied for their behavior in gas and solution phases, their applications in biotechnology (e.g., biosensors), and their significance in astrochemistry and astrobiology^1–3. Recent advances in experimental and theoretical techniques have facilitated a comprehensive investigation into complex systems, ranging from individual amino acids to larger structures like peptides and proteins. These progresses enhance our ability to analyze and predict the detailed chemical and physical behaviors of living organisms^1,4,5.

Methionine (Met), a sulfur-containing essential amino acid (α-amino-γ-methylthiobutyric acid), is critical for protein synthesis, methyl group donation (e.g., choline, adrenaline), and antioxidant activity^6–8. Its applications span agriculture, corrosion inhibition, and therapeutics^9–12. Despite its importance, Met’s thermal stability and sublimation enthalpies remain debated^13,14. Met’s thioether group renders it susceptible to oxidation, forming methionine sulfoxide (MetSO) and sulfone (MetSO₂) under oxidative stress^15,16. These studies, along with others, integrate Met’s biochemical roles, decomposition pathways, and advances in analytical methodologies to elucidate its thermal and oxidative behavior^17–23.

Met decomposes via distinct pathways under varied conditions. Electron-impact studies reveal fragmentation at C–S and C–C bonds²⁴while hydrothermal decomposition in high-temperature/pressure water yields CO₂, NH₃, and H₂O²⁵. Thermogravimetric-mass spectrometric analyses (323–593 K) identify cyclic condensates and peptide bonds as key intermediates²⁶. Met’s oxidation, a major degradation route in biotherapeutics, alters hydrophobicity and hydrogen bonding, impacting protein stability^27–31. Theoretical delineation of aqueous oxidation by hydroxyl radicals highlights pH-dependent kinetics^17,32. Future work should prioritize real-time monitoring of oxidative intermediates and standardization of thermal decomposition protocols. Met’s dual role as a biochemical building block and a thermally labile compound underscores the need for advanced analytical techniques.

Ion mobility spectrometry (IMS) is a widely used ambient-pressure ion separation technique known for its low detection limits, rapid response, and suitability for online analysis. This technique offers advantages such as ease of sample preparation, high sensitivity, low cost, and the ability to analyze various sample matrices directly^33,34. IMS finds applications in diverse fields, including drug detection, environmental monitoring, food quality assessment, and the study of ion-molecule reaction kinetics^35–38. Furthermore, IMS has been utilized for the separation, identification, and quantification of amino acids for several decades^39–41. IMS, combined with computational models, bridges experimental and theoretical insights, offering a robust platform for studying decomposition pathways. For sulfur-rich Met, IMS detects volatile sulfur compounds (e.g., CH₃SH) and elucidates reaction kinetics under physiologically relevant conditions⁴².

Here, we employ a customized IMS system with delayed injection to investigate the thermal decomposition and ionization of L-Met. We elucidate the fragmentation mechanisms of both neutral and protonated L-Met by integrating the temporal evolution of peak intensities, mass-mobility correlations, and density functional theory (DFT) calculations at the M06-2X level. This dual approach not only deciphers complex IMS signatures but also validates decomposition pathways, offering a framework for interpreting thermal degradation processes in biomolecules. The synergy of experimental and computational insights advances the application of IMS in studying thermally labile compounds, with implications for fields ranging from food chemistry to pharmaceutical stability testing.

Materials and methods

Experimental method

L-Methionine (Merck, Product No. M9625) was dissolved in deionized water to prepare a 400 ppm solution. Aliquots (1 µL) were introduced into the ionization chamber of an IMS-300 ion mobility spectrometer (TOF Tech. Pars. Co., Isfahan, Iran) using a calibrated microsyringe. In this device, the sample, following evaporation in the injection chamber, is carried into the ionization region by a carrier gas, where ionization occurs. Subsequently, the generated ions enter the drift region through a shutter grid and are propelled toward the collector by a uniform electric field. Ion separation within the drift region occurs based on ion mobility, resulting in the arrival of different ion species at the collector at distinct drift times. The arrival of each ion group at the collector generates a signal, where the drift time corresponds to the ion species and the signal intensity reflects the ion concentration. The IMS-300 operates via a corona discharge ionization source (positive mode), where evaporated analytes are transported by nitrogen carrier gas into the ionization region. Reactant ions, predominantly hydrated hydronium ions (H₃O⁺·(H₂O)_n), initiate ionization. Ions are then pulsed into a 12-cm drift tube under a uniform electric field (500 V/cm), with separation governed by mobility differences. Drift times and signal intensities were recorded using PicoScope 6 software (Pico Technology Ltd), while VisIMS 2.0.2 software enabled advanced analyses, including mass-mobility correlations and time-dependent peak profiling. Key operational parameters are summarized in Table 1.

Table 1.

IMS operation parameters.

Parameter	Setting
Drift and carrier gas	N2
Flow rate of dri ft gas (N2)	500 (ml/min)
Flow rate of carrier gas (N2)	500 (ml/min)
cell temperature	200 °C
Normal injection port temperature	260 °C
Post-injection delay temperature	280 °C
Post-injection delay time	0, 5, 10, and 15 s
Drift field	500 (V/cm)
Shutter grid pulse width	30 (µs)

Thermal decomposition was probed using a pulsed carrier gas method adapted from Tabrizchi et al.⁴³. Unlike continuous flow, this approach introduces a programmable delay (1–60 s) between sample injection and vapor transport to the ionization region. During the delay, carrier gas flow is halted, allowing controlled thermal treatment of the sample at 280 °C, introduced by Pokorný et al.⁴⁴ within the injection chamber. This method enhances sensitivity for stable compounds by concentrating vapors before ionization, while for thermally labile species like methionine, it enables time-resolved decomposition studies. Delay times of 0, 5, 10, and 15 s were selected to monitor fragmentation dynamics. Scheme 1 compares carrier gas flow patterns in normal injection versus post-injection delay operation. In the delayed mode, sample vapors are held at 280 °C in the injection port for predefined intervals (1–60 s) without carrier gas flow. After the delay, carrier gas resumes, transporting thermally modified species to the ionization region.

Scheme 1.

Carrier gas flow patterns: (a) normal injection; (b) post-injection delay method.

Computational method

The electronic structure of the involved species, such as methionine (Rs), saddle points (TSs), post-reactive complexes (CPs), intermediates (INs), and products (Ps), is considered by the validated⁴⁵ M06-2X method. This method is based on the meta-hybrid density functional theory (DFT) formalism that has precise results for the mechanistic study of organic molecules. Therefore, geometries of R, TSs, CPs, INs, and Ps are optimized by the M06-2 ×⁴⁶ method to design target reaction routes. A suitable double zeta basis set, 6–31 + G(d, p)^47,48, is used to optimize the mentioned stationary points. The structures involved in the reaction routes are characterized by the harmonic vibrational calculations at the M06-2X/6–31 + G(d, p) level. The results indicate that minimum structures such as R, CPs, IN, and Ps exhibit no imaginary frequency. In contrast, the TS structures are characterized by the presence of a single imaginary frequency. The intrinsic reaction coordinate^49,50 (IRC) Calculations performed at the M06-2X/6–31 + G(d, p) level validate the proposed reaction routes from a computational perspective⁴⁵. It is important to highlight that the M06-2X/6–31 + G(d, p) level was chosen for studying protonated methionine reactions due to its superior accuracy in accounting for noncovalent interactions compared to many other DFT methods⁵¹. All DFT computations were performed in the gas phase using Gaussian 09⁵².

This combined approach bridges experimental IMS data with theoretical models, enabling mechanistic interpretation of methionine’s thermal decomposition and ionization behavior.

Results and discussion

Experimental results

Normal ion mobility spectrum of methionine

In IMS, utilizing a corona discharge ionization source, a steady flow of reactant ions is generated by ionizing the gas present in the ionization region. Ionization of analytes occurs through proton transfer reactions from reactant ions, primarily hydrated hydronium ions, to the analyte molecules. These analyte molecules are first vaporized at the sample injection port and then carried into the ionization region by the carrier gas. Figure 1 displays the ion mobility spectrum resulting from the injection of 1 µL of a 400 ppm methionine solution, along with its corresponding background spectrum. The background ion mobility spectrum (Fig. 1a) reveals several peaks in the drift time range of 4.2 to 5.2 ms, with the most intense signal attributed to hydrated hydronium ions⁵³.

Fig. 1.

(a) Background ion mobility spectrum, and (b) methionine ion mobility spectrum.

The ion mobility spectrum of methionine, as depicted in Fig. 1b, exhibits five well-defined product ion peaks (PIPs) superimposed on background signals. The most intense peak, PIP1, corresponds to protonated methionine (MH⁺) and exhibits the longest drift time of 6.81 ms, along with a calculated K₀ value of 1.61 cm²/V·s⁵⁴. PIP5, with a drift time of 5.45 ms, was attributed to protonated methyl mercaptan (CH₃SH⁺), as confirmed by reduced mobility comparison with authentic standard measurements (Supporting Information S1). The three intermediate PIPs (drift times: 6.63 ms, 6.11 ms, and 5.95 ms) presumably correspond to ionic fragments with masses between methyl mercaptan and methionine.

Product ion formation in IMS involves complex processes governed by multiple factors: analyte chemical structure, temperature conditions, ionization source characteristics, and reactant ion chemistry. Fragmentation pathways in IMS typically occur via two distinct mechanisms: (a) Thermal Decomposition, in which analyte pyrolysis prior to ionization generates neutral fragments that subsequently undergo protonation; and (b) Ionization-Induced Decomposition, in which protonated molecular ions undergo dissociation due to energy transfer during ionization. Notably, implementation of a post-injection delay period typically enhances the intensity of peaks originating from thermally-derived decomposed species.

Ion mobility spectrum of methionine with post-injection delay

As described in the Materials and Methods, our delayed injection system temporarily retains the sample at the injection port for controlled time intervals. The sample is maintained at 280 °C during this holding period before being transferred to the ionization region. We systematically evaluated holding times of 5, 10, and 15 s to investigate their effects on sample analysis. The ion mobility spectra demonstrated significant time-dependent peak pattern variations (Fig. 2), with the 15-second delay producing the most pronounced spectral modifications.

Fig. 2.

Ion mobility spectra of methionine under varying delayed sample injection conditions: (a) no delay, (b) 5-second delay, (c) 10-second delay, and (d) 15-second delay.

The delayed injection system significantly altered the spectral profile, with two key observations. First, the maximum intensity of PIP1 decreased progressively with longer delay times (5–15 s), consistent with thermal decomposition of methionine at 280 °C. Second, a new product ion peak emerged at 5.6 ms drift time, whose intensity showed a strong positive correlation with delay duration. Additionally, most other peaks show intensity enhancement following thermal exposure, suggesting temperature-dependent fragmentation or ionization efficiency changes. These collective results demonstrate that both decomposition kinetics and reaction product formation are time- and temperature-dependent under the experimental conditions.

It is important to note that the spectra presented in Figs. 1 and 2 represent the maximum intensity recorded at each drift time, representing peak values rather than integrated signal intensities. In ion mobility spectrometry, peak intensity does not directly correlate with species abundance due to differing formation kinetics. A minor component forming continuously may yield a low-intensity peak, while an abundant but short-lived species can produce a sharp, intense signal. This temporal dependence means that maximum-intensity spectra alone cannot reliably quantify relative abundances. Therefore, a comprehensive analysis of thermal decomposition effects requires examination of both peak intensities and their temporal evolution profiles.

Temporal variation in peak intensities

To determine the origin of the ionic species corresponding to the observed ion mobility peaks, one useful strategy is to analyze how these peaks evolve over time. After sample introduction into the ionization source, the peak intensities typically follow a distinct pattern: an initial increase, a maximum intensity, and a gradual decline as the sample is consumed. In this study, the detector records spectra at a rate of 10 spectra per second. Plotting the intensity of each peak over time reveals the origin and relative stability of the corresponding ionic species. Accordingly, the temporal evolution of the peaks detected after a 10-second delay (Fig. 2c) was analyzed and is displayed in Fig. 3.

Fig. 3.

Time evolution of detected peaks in the methionine ion mobility spectrum with a delay time of 10 s.

Following a 10-second delay, when the sample enters the ionization region, the reactant ion peak (hydronium) shows a sharp drop, temporarily disappearing, followed by a gradual increase towards its initial intensity (Fig. 3a). In contrast, PIP1’s signal rises quickly, reaching a maximum after approximately 5 s, and subsequently declines as the sample is depleted in the ionization region. Unlike PIP1 (Fig. 3b), PIP2 exhibited a markedly different temporal profile. Upon the 10 s delay, PIP2 emerged, rapidly reached a peak maximum intensity, and subsequently experienced a significant decline. A similar, though less pronounced, trend was observed for PIP3 (Fig. 3c), while PIP6 (Fig. 3f) exhibited an even more dramatic intensity drop. This temporal behavior strongly suggests that these peaks originate primarily from fragments generated at the injection port during the thermal decomposition of methionine. Due to their smaller size, these fragments are carried into the ionization region slightly faster than intact methionine molecules by the flow of the carrier gas. Their early arrival allows for preferential ionization. Subsequently, as the methionine molecules enter the ionization region, their higher proton affinity outcompetes the fragments for available protons, leading to a suppression in the ionization and detection of the fragments.

Although the intensity of PIP4 exhibits some dependence on delay time (Fig. 2), its temporal behavior offers a distinct perspective. Unlike PIP2, PIP3, and PIP6, which exhibit a rapid decline-PIP4 persists until the departure of the injected sample. Moreover, its temporal profile closely resembles that of the parent ion (protonated methionine) (Fig. 3d). This similarity suggests that PIP4 formation involves a distinct mechanism from the thermal decomposition at the injection port. Specifically, it points primarily to the fragmentation of the protonated methionine molecule within the ionization region as a significant contributor to the formation of PIP4.

The temporal evolution of PIP5, derived from protonated methyl mercaptan, is depicted in Fig. 3e. Notably, PIP5 exhibits a delayed appearance, with minimal intensity observed immediately after injection. Subsequently, its signal intensity increases as the intensity of other peaks diminishes. This behavior is consistent with the lower proton affinity of methyl mercaptan compared to methionine and other amine-containing species present in the environment. Consequently, methyl mercaptan formation likely proceeds through two pathways: thermal decomposition of neutral methionine and fragmentation of protonated methionine. Computational analysis confirms this dual-origin mechanism for methyl mercaptan (CH₃SH) generation (Sect. “Results and discussion”). The temporal persistence of its precursor ion PIP5 (CH₃SH⁺) across thermal delay conditions stems from two complementary routes: (1) thermal decomposition of neutral methionine at 280 °C, yielding CH₃SH through kinetically accessible pathways like R10 (ΔE^≠ = 71.14 kcal/mol) and leading to thermodynamically stable products P8/P10; and (2) fragmentation of protonated methionine (M-XH⁺), where site-specific protonation (notably at S/N centers) enables near-barrierless CH₃SH release (ΔE^≠ ≤ 10.94 kcal/mol). This dual pathway explains PIP5’s sustained intensity despite depletion of intact methionine (PIP1).

Following sample injection, the peak intensity of ammonium ions initially decreased before rising steadily, eventually reaching ~ 4 nA, substantially higher than its initial intensity. This trend strongly suggests in situ ammonia generation. Although ammonia could originate from either thermal decomposition of methionine or fragmentation of protonated methionine, this observation unequivocally demonstrates that [M-NH₃]H⁺, resulting from the decomposition and ionization of methionine, constitutes a significant product in the system. Computational thermodynamics corroborates dual NH₃-release pathways. For neutral methionine, NH₃ elimination (R9: Met → D + NH₃) is thermodynamically viable at the temperature of 280 °C used in this study (ΔG = 10.51 kcal/mol, Table 2. Kinetically, the most accessible fragmentation pathway for neutral methionine involves ammonia (NH₃) elimination. These align with PIP2’s significant delay-dependent intensity (Fig. 2b-d), confirming thermal decomposition as its primary source. For protonated methionine (M-NH⁺), NH₃ release from J-NH⁺ (R18-N, P11b-N) is highly favorable (ΔG = − 12.00 kcal/mol, Table 3). However, PIP2’s temporal profile (Fig. 3b) shows rapid initial intensity followed by suppression when methionine enters the ionization region. This occurs because fragment D (132 amu, PIP2 precursor) lacks the primary amine group, reducing its proton affinity relative to intact methionine (PA(D) < PA(Met)), allowing methionine to outcompete D for protons. Thus, while both pathways are accessible, thermal decomposition dominates PIP2 formation under experimental conditions.

Table 2.

Relative energy, internal energy, enthalpy, Gibbs free energy, and entropy of R, tss, cps, and Ps involved in the thermal decomposition of methionine.

Species	∆E	∆U0	∆H0	∆G0	T∆S0
Methionine	0.00	0.00	0.00	0.00	0.00
CP1	51.80	52.90	52.90	50.71	2.19
CP2	25.89	28.40	28.40	20.81	7.59
CP3	37.42	38.90	38.89	35.58	3.31
CP5	20.65	21.09	21.09	19.38	1.72
CP6	21.31	22.33	22.33	18.80	3.53
CP8	37.41	38.81	38.81	35.67	3.14
CP9	9.86	10.95	10.95	7.73	3.22
CP10	17.87	18.71	18.71	16.17	2.54
CP11	69.70	69.01	69.01	71.38	−2.37
CP13	71.11	71.78	71.78	70.03	1.75
CP14	69.84	69.07	69.07	71.84	−2.77
CP15	54.95	54.08	54.08	57.41	−3.34
CP16	19.54	19.92	19.92	19.16	0.76
CP17	48.50	47.99	47.99	50.23	−2.24
IN1	61.95	61.73	61.73	62.82	−1.09
TS1	93.64	93.71	93.71	94.21	−0.49
TS2	69.83	70.28	70.28	69.20	1.08
TS3	77.20	77.33	77.33	77.43	−0.10
TS4	50.71	50.49	50.49	50.83	−0.35
TS5	121.19	120.92	120.92	122.23	−1.30
TS6	62.97	62.69	62.69	63.40	−0.72
TS7	72.69	72.63	72.62	72.38	0.25
TS8	71.55	71.70	71.70	71.58	0.12
TS9	61.20	60.80	60.80	62.16	−1.37
TS10	71.14	71.20	71.20	71.52	−0.32
TS11	74.91	74.23	74.23	76.77	−2.54
TS12	89.84	89.81	89.81	90.29	−0.48
TS13	124.76	124.38	124.38	126.14	−1.76
TS14	88.22	87.57	87.57	90.35	−2.78
TS15	63.29	62.06	62.06	65.76	−3.70
TS16	130.59	130.63	130.63	130.76	−0.13
TS17	62.96	62.38	62.38	64.46	−2.09
P1	58.89	59.89	60.49	48.66	11.83
P2	34.67	35.92	37.10	14.55	22.55
P3	42.51	43.67	44.27	32.04	12.23
P4	18.69	18.63	18.63	19.15	−0.52
P5	25.87	25.83	26.42	14.12	12.30
P6	24.64	25.05	25.05	24.61	0.44
P7	21.49	22.28	22.87	10.51	12.37
P8	23.23	22.75	23.34	12.72	10.62
P9	86.32	87.00	87.59	76.04	11.55
P10	22.48	21.73	22.91	2.48	20.43

Table 3.

Relative energy, internal energy, enthalpy, Gibbs free energy, and entropy of R, tss, cps, and Ps involved in the decomposition pathways of the N-site protonated methionine (M-NH⁺ molecule).

Species	∆E	∆U0	∆H0	∆G0	T∆S0
R	0.00	0.00	0.00	0.00	0.00
CR-N	−69.55	−69.78	−70.37	−60.19	−10.18
M-NH+ + H2O	−47.55	−47.43	−47.44	−48.77	1.34
CP1-N	−35.52	−34.66	−34.66	−36.42	1.76
CP3-N	−5.28	−3.97	−3.97	−6.82	2.86
CP5-N	−38.07	−36.87	−36.87	−42.35	5.48
CP6-N	−41.41	−40.67	−40.67	−42.96	2.29
CP8-N	−14.78	−13.37	−13.37	−17.35	3.98
CP10-N	−39.28	−38.42	−38.42	−41.29	2.87
CP12-14-N	−32.72	−32.75	−32.75	−32.78	0.03
CP16-N	−32.44	−31.26	−31.26	−35.94	4.67
CP18-N	−59.08	−58.11	−58.11	−62.86	4.75
IN1-N	7.08	7.18	7.18	7.03	0.15
TS1-N	25.09	25.77	25.77	23.82	1.95
TS3-N	28.10	28.36	28.36	26.67	1.69
TS4-N	−2.68	−2.72	−2.72	−2.88	0.16
TS5-N	58.33	58.25	58.25	58.57	−0.32
TS6-N	39.25	39.44	39.44	38.51	0.93
TS7-N	20.53	20.56	20.56	20.04	0.52
TS8-N	26.62	26.76	26.76	26.23	0.53
TS10-N	10.94	10.82	10.82	10.94	−0.12
TS12-14a-N	10.89	10.58	10.58	11.27	−0.69
TS12-14b-N	26.98	26.83	26.83	27.10	−0.27
TS12-17-N	24.58	24.23	24.23	25.23	−1.00
TS12-N	35.78	35.77	35.77	35.63	0.14
TS13-N	40.47	40.11	40.11	41.23	−1.12
TS16-N	51.50	51.64	51.64	51.25	0.39
TS18-N	2.57	2.55	2.55	2.68	−0.13
P1a-N	−17.27	−16.02	−15.43	−28.38	12.95
P1b-N	8.87	10.58	11.77	−11.78	23.55
P3a-N	5.61	6.93	7.52	−5.76	13.28
P3b-N	13.11	14.60	15.79	−7.21	22.99
P4-N	−36.21	−36.08	−36.08	−36.46	0.37
P5-N	−26.04	−26.00	−25.41	−38.46	13.05
P6-N	−28.77	−28.31	−28.31	−30.60	2.29
P8a-N	−25.60	−25.85	−25.26	−37.21	11.95
P8b-N	36.49	37.15	38.34	14.02	24.32
P9a-N	0.88	1.46	2.05	−12.00	14.05
P9b-N	9.47	9.29	10.47	−12.24	22.71
P10-N	21.87	22.12	22.71	11.29	11.42
P11a-N	−51.92	−51.94	−51.35	−62.43	11.08
P11b-N	8.66	9.55	10.73	−12.00	22.73
P11c-N	16.28	17.31	19.08	−14.63	33.71
P-TS13-N	−4.84	−4.79	−4.79	−4.98	0.19

Mass-drift time correlation using two standard masses

Ion mobility spectrometry separates ions based on their drift time (t_d) through a buffer gas under an electric field. The reduced mobility (K₀) is inversely proportional to t_d:

1

Empirically, K₀ correlates logarithmically with ion mass (m) for structurally similar ions:

2

​Traditional mass-mobility relationships (e.g., Eq. 2) require explicit knowledge of instrumental parameters (drift length, temperature, and pressure) to compute K₀. Equation 3, described in our previous work⁵⁵circumvents this by using two calibration ions of known mass (m₁, m₂) and measured drift times (t₁, t₂). This directly relates an unknown ion’s drift time (t) to its mass (m) without instrument-specific constants, significantly simplifying mass estimation.

3

This method utilizes two well-characterized reference peaks as reference points. By using the known mass and drift time of these two peaks, the observed drift time in the ion mobility spectrum can be converted to a corresponding mass value. While Eq. 3 enables drift-time-based mass estimation, its accuracy inherently depends on structural similarity between analyte ions and reference standards. As demonstrated in IM-MS studies^37,54the logarithmic mass-mobility relationship holds rigorously only for ions of comparable collision cross-sections (CCS) and charge distributions. For structurally divergent species (e.g., linear vs. branched ions), CCS variations introduce systematic errors in mass predictions⁵⁶. In this work, PIP mass assignments should thus be interpreted as approximations, with larger uncertainties expected for fragments exhibiting distinct geometries (e.g., PIP3/PIP4). We recently validated this approach for asymmetric dimer detection⁵⁷but emphasize that definitive identification requires complementary techniques (e.g., IM-MS). A key limitation of this approach lies in its decreasing accuracy of the predicted mass diminishes as the target peak moves further away from the two reference peaks. However, for peaks falling within the drift time range bracketed by the two reference points - and particularly for those proximate to either reference - the method demonstrates good mass prediction accuracy with minimal error.

As shown in Fig. 4, a correlation plot between drift time and predicted mass was constructed for the various methionine fragments. This plot reveals a logarithmic relationship between drift time and mass. The correlation is particularly strong for PIP2 and PIP6, indicating high predictive accuracy for these fragments. However, as is evident from the plot, PIP3 and PIP4 show significant deviations from this overall trend, lying below the trendline.

Fig. 4.

Correlation plot of drift time versus predicted mass using two reference masses for fragmentation products of methionine.

PIP1 (150 amu, corresponding to protonated methionine) and PIP5 (49 amu, corresponding to protonated methyl mercaptan) were selected as reference peaks to convert the ion mobility spectrum of Fig. 2b to a mass spectrum. Using this conversion, the predicted mass for PIP2 was determined to be 133 amu, precisely matching the expected mass of the MH⁺-NH₃ species. Similarly, a predicted mass of 57 amu was obtained for PIP6, exhibiting excellent agreement (1% error) with the mass of protonated allylamine. A fragment with a mass of 57 amu is a known degradation product of methionine, formed through the sequential loss of one carbon dioxide and methyl mercaptan molecule upon heating (M-CO₂-CH₃SH)⁵⁸.

The ion mobility PIP3 (drift time: 6.11 ms) was assigned a predicted mass of 92 amu using the two-reference calibration method. Computational analysis (Sect. “Results and discussion”) resolves this apparent discrepancy by identifying three co-eluting fragment sources with near-identical CCS: MH⁺-CO₂ (106 amu) generated via direct decarboxylation of protonated methionine (R18-N, TS18-N: ΔE^≠ = 2.57 kcal/mol); B-NH⁺ (104 amu) formed through formic acid loss from N-protonated methionine (R5-N/R6-N, ΔG = − 38.46 kcal/mol); and the [B-NH⁺ + CO + H₂O] ensemble produced by concerted elimination from O-protonated species (R8-O, P5b-O). These fragments exhibit indistinguishable ion mobility due to their shared linear aliphatic backbone geometries. While MH⁺-CO₂ dominates thermodynamically (ΔG = − 62.43 kcal/mol), B-NH⁺ formation is kinetically favored (ΔE^≠ = 23.72 kcal/mol versus 62.97 kcal/mol for its neutral analog), making PIP3 a composite peak arising from competitive protonation-site-dependent pathways.

Assignment of PIP4 (drift time: 5.95 ms) has been established as the MH⁺-CH₃SH fragment (102 amu) based on a convergence of both experimental and computational evidence. Experimentally, PIP4’s intensity profile closely mirrors that of protonated methionine (PIP1), strongly suggesting that it originates from the in-source fragmentation of protonated species. Computationally, the loss of methyl mercaptan can be explained by two distinct mechanisms. The first is the thermal decomposition of neutral methionine, yielding a neutral fragment of 101 amu, which then protonates to form E-H⁺ (102 amu). The second pathway involves the direct fragmentation of protonated methionine itself. This can occur either through N-protonation, leading to E-NH⁺ (102 amu), or S-protonation, which produces Q⁺ (102 amu). The structural similarities among E-H⁺, E-NH⁺, and Q⁺ account for their co-elution at a single drift time. Further experimental evidence confirms this dual-origin mechanism: PIP4’s intensity varies with delay time, confirming a thermal decomposition component, while its consistent presence during ionization points to the barrierless fragmentation of the protonated species. This combined evidence resolves previous mass discrepancies and is consistent with the detection of methyl mercaptan (PIP5).

The significant mass discrepancies observed for PIP3 and PIP4 arise from fundamental limitations in mass-mobility correlations when structural heterogeneity exists. As demonstrated in several studies^59–62significant deviations occur when ions exhibit divergent CCS due to differences in charge localization, molecular branching, and functional group density.

Theoretical results

Total energy, internal energy, enthalpy, Gibbs free energy, and entropy of all species are in Tables S1-S4. Graphs of the IRC of all saddle points are in Tables S5-S8. Cartesian coordinates of all species are in Tables S9-S12.

Thermal decomposition of methionine

The thermal decomposition reaction routes of methionine are proposed to provide a deeper understanding of the experimental results. For this, the following reaction routes are investigated mechanistically.

Methionine→ TS1→ CP1→ P1 (A + H₂O) (R1)

Methionine→ TS2→ CP2→ P2 (B + H₂O + CO) (R2)

Methionine→ TS3→ CP3→ P3 (C + H₂O) (R3)

Methionine→ TS4→ P4 (R4)

Methionine→ TS4→ P4→ TS5→ CP5→ P5 (B + HCOOH) (R5)

Methionine→ TS6→ CP6→ P5(B + HCOOH) (R6)

Methionine→ TS7→ P6 (R7)

Methionine→ TS7→ P6 → TS8→ CP8→ P3(C + H₂O) (R8)

Methionine→ TS9→ CP9→ P7 (D + NH₃) (R9)

Methionine→ TS10→ CP10→ P8 (E + CH₃SH) (R10)

Methionine→ TS11→ CP11→ P9 (F + CH₃) (R11)

Methionine→ TS12→ IN1→ TS13→ CP13→ P5(B + HC(OH)₂) (R12)

Methionine→ TS12→ IN1→ TS14→ P9 (F + CH₃) (R13)

Methionine→ TS12→ IN1→ TS14→ P9 → TS15→ CP15→ P10 (G + CO₂ + CH₃SH) (R14)

Methionine→ TS12→ IN1→ TS16→ CP16→ P8 (E + CH₃SH) (R15)

Methionine→ TS12→ IN1→ TS16→ CP16→ TS17→ CP17→ P10 (G + CO₂ + CH₃SH) (R16)

Table 2; Fig. 5 show the energies and structures of all stationary points in the thermal decomposition of neutral methionine. Routes R1 and R3 show the separation of a water molecule from methionine by the saddle points TS1 and TS3, respectively. The residual molecules, A and C, are structural isomers with the same molecular mass (131.040 amu). The second route simulates the separation of water and carbon monoxide. The saddle point of R2 is TS2 with a relative energy of 68.93 kcal mol⁻¹. The molecular mass of residual structure B is 103.046 amu. The fourth route (R4) includes an H atom transfer from the C2 atom to the carbon atom of the carboxylic functional group via TS4. The final product P4 shows that another hydrogen atom is transferred from the NH₂ group to the O atom of the carboxylic group. The shift of this hydrogen occurs spontaneously, without encountering a saddle point. The rationale behind this transfer lies in the stability of the P4 adduct. In R4, there is no change in the physical mass of the initial molecule; however, a significant chemical transformation is seen. Route R5 shows the separation of formic acid from the P4 adduct by TS5 with a relative energy of 121.19 kcal mol⁻¹, leading to the formation of the final product P5 (B + HCOOH). The molecular mass of B and formic acid are 103.046 and 46.005 amu, respectively. In R6, the HOCOH group is separated from methionine by TS6. It is better to say that the HOCOH fragment easily converts to formic acid by a hydrogen shift from the oxygen atom to the carbon atom. The corresponding saddle point relative energy is 62.97 kcal mol⁻¹. Thus, the final products of R6 have the same mass as R5. In path R7, an intramolecular hydrogen shift from the carbon C2 to the O1 atom of the carboxylic group occurs, leading to the formation of the P6 adduct. Thus, there are no differences between the mass of the reactant and the product in R7. The pathway R8 shows a water molecule separation from P6 by TS8 with a relative energy of 71.55 kcal mol⁻¹. The separation of ammonia happens by route R9 by TS9 with an energy barrier of 61.20 kcal mol⁻¹. The structure D is generated by this process with a mass of 132 amu. In the R10 and R11 routes, the separation of the methyl mercaptan molecule takes place by the saddle points TS10 and TS11, respectively, leading to the formation of E and F isometrical structures with a mass of 101 and 134 amu, respectively. The computed energy barriers for TS10 and TS11 are 71.14 and 74.91 kcal mol⁻¹, respectively. The first step of the routes R12-R16 is the formation of IN1 by the saddle point TS12 with an energy barrier of 89.84 kcal mol⁻¹. This saddle point involves an H atom shift from the atom C4 to the atom S. In the second step of the routes R12-R16, the intermediate IN1 can convert to the CP3, P9, P15a, CP16, and CP17 after surmounting the saddle points TS13-TS17, respectively. The calculated energy barriers for TS13 -TS17 are 124.76, 88.22, 63.29, 130.59, and 62.96 kcal mol⁻¹, respectively. In the exit channels of routes R12-R16, the products B, E, F, G, CH₃, HCOOH, CO₂, and CH₃SH are yielded. The mass of the G structure is 57.059 amu. All pathways for suggested products obtained by theoretical investigations, as well as experimental methods, are schematized in Fig. 6.

Fig. 5.

The structures involved in the thermal decomposition of neutral methionine.

Fig. 6.

The PES of the fragmentation of neutral methionine computed at the M06-2X/6–31 + G(d, p) level of theory.

Kinetically, the most accessible fragmentation pathway for neutral methionine involves ammonia (NH₃) elimination via Route R9 (ΔE^≠ = 61.20 kcal/mol at TS9), yielding product P7 (D + NH₃). Species D corresponds to the precursor of PIP2 observed in experimental IMS spectra. Competing pathways include methyl mercaptan (CH₃SH) loss (R10, ΔE^≠ = 71.14 kcal/mol at TS10), which generates P8 (E + CH₃SH). Species E contributes to PIP4 in IMS spectra, while this pathway further corroborates experimental detection of methyl mercaptan - manifested as PIP5 - among methionine’s thermal decomposition products. A third significant route involves formic acid (HCOOH) formation (R6, ΔE^≠ = 62.97 kcal/mol at TS6), producing P5 (B + HCOOH). As noted experimentally, protonated M-HCOOH⁺ is a key candidate for PIP3 formation.

Thermodynamically, the most stable product is P10 (G + CO₂ + CH₃SH, ΔG = 2.48 kcal/mol), arising from concerted decarboxylation and CH₃SH elimination. This species - experimentally assigned to PIP6 - emerges as the dominant thermodynamic product of neutral methionine decomposition. Subsequent stability is observed for P5 (B + HCOOH, ΔG = 14.12 kcal/mol) formed via C–C cleavage, and P8 (E + CH₃SH, ΔG = 12.72 kcal/mol), aligning with their respective contributions to PIP3 and PIP5 in experimental spectra.

Critically, no direct CO₂ elimination pathway was computationally identified for neutral methionine. CO₂ formation occurs exclusively through secondary fragmentation of intermediates (e.g., P10 in R14/R16) or necessitates prior protonation. This stands in sharp contrast to protonated methionine (next section), where CO₂ loss proceeds barrierlessly in pathways like R18-N (ΔE^≠ = 2.57 kcal/mol).

Fragmentation of protonated methionine

First, we highlight that the reaction routes for protonated molecules have been simulated, similar to the neutral molecule paths. For this, we skip discussing the mechanism of protonated routes in more detail. Therefore, we will focus on the more important routes of the fragmentation mechanism of protonated molecules. Additionally, several new paths are proposed. Figure 7 shows three sites, namely N, O, and S, for the protonation of methionine. The S1, N1, and O1 centers are the most probable sites for protonation. It should be noted that the N site is the most probable center among all energetically. This does not mean that other sites are less important. Thus, we consider all sites’ protonation and reactions similar to the suggested routes for neutral methionine.

Fig. 7.

Methionine structure highlighting protonation sites N1, O1, and S1.

Protonation of methionine from the N site

The structures and PES of protonated methionine from the N site are shown in Figs. 8 and 9, respectively. Also, the relative energies and thermodynamic parameters of the involved species are in Table 3. The probable reaction routes are as follows.

Fig. 8.

The structures involved in the N-site protonated methionine decomposition pathways.

Fig. 9.

The PES of the fragmentation of the N-Site protonated methionine (M-NH⁺) computed at the M06-2X/6–31 + G(d, p) level of theory.

Methionine + H₃O⁺→ CR-N→ M-NH⁺+ H₂O→TS1-N→ CP1-N→

P1a-N (A-NH⁺ + H₂O) → P1b-N (A⁺ +NH₃ + H₂O) (R1-N)

Methionine + H₃O⁺→ CR-N→M-NH⁺+ H₂O→ TS3-N→ CP3-N→

P3a-N (C-NH⁺+H₂O) → P3b-N (C⁺ + NH₃ + H₂O) (R3-N)

Methionine + H₃O⁺→ CR-N→M-NH⁺+ H₂O→ TS4-N→ P4-N (R4-N)

Methionine + H₃O⁺→ CR-N→M-NH⁺+ H₂O→ TS4-N→

P4-N → TS5-N→ CP5-N→ P5-N (B-NH⁺+HCOOH) (R5-N)

Methionine + H₃O⁺→ CR-N→M-NH⁺+ H₂O→ TS6-N→

CP6-N→ P5-N (B-NH⁺+HCOOH) (R6-N)

Methionine + H₃O⁺→ CR-N→M-NH⁺+ H₂O→ TS7-N→ P6 (R7-N)

Methionine + H₃O⁺→ CR-N→M-NH⁺+ H₂O→ TS7-N→ P6 →

TS8-N→ CP8-N→ P3a-N (C-NH⁺+H₂O) → P3b-N (C⁺ + NH₃ + H₂O) (R8-N)

Methionine + H₃O⁺→ CR-N→M-NH⁺+ H₂O→ TS10-N→ CP10-N→

P8a-N (E-NH⁺ + CH₃SH)→ P8b-N (E⁺ + NH₃ + CH₃SH) (R10-N)

Methionine + H₃O⁺→ CR-N→M-NH⁺+ H₂O→ TS12-N→ IN1-N→

TS13→ P13-N (R12-N)

Methionine + H₃O⁺→ CR-N→M-NH⁺+ H₂O→ TS12-14a-N→ P9a-N (CPA + CH₃SH)→

P9b-N (H₂NCHCOOH⁺+C₂H₄ + CH₃SH) (R13a-N)

Methionine + H₃O⁺→ CR-N→M-NH⁺+ H₂O→ TS12-14b-N→ P9a-N (CPA + CH₃SH)→

P9b-N (H₂NCHCOOH⁺+C₂H₄ + CH₃SH) (R13b-N)

Methionine + H₃O⁺→ CR-N→M-NH⁺+ H₂O→ TS12-N→ IN1-N→ TS16-N→ CP16-N→

P8a-N (E-NH⁺ + CH₃SH)→ P8b-N (E⁺ + NH₃ + CH₃SH) (R15-N)

Methionine + H₃O⁺→ CR-N→M-NH⁺+ H₂O→ TS12-17-N → P10-N (I⁺ + CO₂) (R16-N)

Methionine + H₃O⁺→ CR-N→M-NH⁺+ H₂O→ TS18-N→ CP18-N→ P11a-N (J-NH⁺+CO₂)→

P11b-N (CPB-N + NH₃ + CO₂)→ P11c-N (C₂H₄ + CH₃SCH₂⁺+NH₃ + CO₂) (R18-N)

An impact between a hydronium ion and the N site of methionine led to the formation of the CR-N complex (intermediate) with a relative energy of −69.55 kcal mol⁻¹. Through this impact, the proton of H₃O⁺ transfers to the N site of methionine, leading to the formation M-NH⁺ molecule. After that, the M-NH⁺ unimolecular dissociation reactions are investigated by the abovementioned routes. In routes R1-N -R13-N and R15-N, the saddle points TS1-N-TS8-N, TS10-N, TS12-N, and TS16-N exhibit behavior similar to the transition states in neutral methionine. The results show that the saddle points of protonated molecules are more stable compared to the corresponding TSs in neutral methionine dissociation reactions. The forecast stability for TS1-N-TS8-N, TS10-N, TS12-N, and TS16-N is 68.55, 49.10, 53.39, 62.87, 23.72, 52.16, 44.93, 60.19, 11.20, 84.30, and 79.09 kcal mol⁻¹, respectively, compared to the corresponding saddle points in neutral methionine. It should be noted that similar post-reactive complexes and products are more stable than the respective species in a neutral system. The fragment A-NH⁺ (132.048 amu) is the isometrical structure of the A molecule produced from the neutral molecule. Also, A-NH⁺ converts to A⁺ by losing an NH₃ molecule. The geometries of B-NH⁺ (104.053 amu) and C-NH⁺ (132.048 amu) fragments are the same as B and C structures, respectively. The fragments B-NH⁺, C-NH⁺, and E-NH⁺ can lose an NH₃ molecule and produce C⁺ (115.022 amu) and E⁺ (85.029 amu). The E-NH⁺ (102.056) fragment has a mass near to B-NH⁺. The saddle points TS12-14a-N, TS12-14b-N, and TS12-17-N show the transferring of two hydrogen atoms simultaneously, where in neutral molecules, respective hydrogens are shifted by two individual saddle points. The relative energies of TS12-14a-N, TS12-14b-N, and TS12-17-N are 10.89, 26.98, and 24.581 kcal mol⁻¹, respectively. The P9a product generates distinct fragments in contrast to the P9 product. First, it produces a complex (CPA) and methyl mercaptan. Then, CPA dissociates into H₂NCHCOOH⁺ (74.024 amu) and C₂H₄ (28.031 amu) fragments. R18-N is a new route that has not been seen for neutral methionine. The saddle point TS18-N has a relative energy of 2.57 kcal mol⁻¹. The fragments CO₂ (43.990 amu) and J-NH⁺ (106.069 amu) are generated by R18-N. Finally, an NH₃ molecule leaves the J-NH⁺ fragment, and I⁺ (106.069 amu) is yielded. Also, the CPB-N fragment can further dissociate with C₂H₄ and CH₃SCH₂⁺ (61.011 amu) molecules.

Protonation of methionine from the O site

The involved structures and PES in protonated methionine from the O site are displayed in Figs. 10 and 11, respectively. Also, the important energetics of the involved species are tabulated in Table 4. The possible products are generated by the following routes.

Fig. 10.

The structures involved in the O-site protonated methionine decomposition pathways.

Fig. 11.

The PES of the fragmentation of the O-site protonated methionine (M-OH⁺) computed at the M06-2X/6 − 3 + G(d, p) level of theory.

Table 4.

Relative energy, internal energy, enthalpy, Gibbs free energy, and entropy of R, tss, cps, and Ps involved in the decomposition pathways of the O-site protonated methionine (M-OH⁺ molecule).

Species	∆E	∆U0	∆H0	∆G0	T∆S0
R	0.00	0.00	0.00	0.00	0.00
CR-O	−45.69	−45.69	−46.28	−36.95	−9.33
M-OH+	−18.81	−18.61	−18.61	−19.62	1.01
CP4B-O	6.30	6.86	6.86	5.50	1.36
CP8a-O	−12.78	−11.38	−11.38	−14.77	3.39
CP8b-O	−19.95	−17.56	−16.97	−35.27	18.30
CP10-O	−9.41	−9.66	−9.66	−8.39	−1.27
CP18-O	−58.89	−58.01	−58.01	−61.68	3.67
IN1-O	31.26	31.70	31.70	30.85	0.85
TS4a-O	−4.68	−4.63	−4.63	−4.74	0.10
TS4b-O	32.49	32.73	32.73	32.08	0.65
TS8-O1	23.19	23.40	23.40	22.58	0.83
TS10-O	22.61	22.44	22.44	23.83	−1.40
TS12-O	62.40	62.65	62.64	61.88	0.77
TS13-O	50.25	50.33	50.33	50.43	−0.09
TS16-O	58.29	58.12	58.12	59.57	−1.46
TS17-O	−41.91	−42.64	−42.64	−40.57	−2.07
TS18-O	15.94	15.81	15.81	16.00	−0.19
P4a-O	−36.21	−36.08	−36.08	−36.46	0.37
P4b-O	33.05	34.27	34.87	21.72	13.15
P5b-O	−17.20	−15.82	−14.63	−38.11	23.48
P8a-O	3.40	3.17	3.76	−8.10	11.87
P8b-O	−18.99	−20.07	−19.48	−29.05	9.57
P12-O	24.51	24.66	25.25	12.48	12.77
P13-O	1.58	1.84	1.84	1.69	0.15
P16a-O	−25.67	−26.25	−26.25	−23.98	−2.27
P16b-O	−11.36	−10.53	−9.93	−22.18	12.25
P-TS17-O	−40.83	−41.14	−41.14	−40.61	−0.53
P-TS18-O	−51.92	−51.94	−51.35	−62.43	11.08

Methionine + H₃O⁺→ CR-O→ M-OH⁺+ H₂O→ TS4-O→ P4a-O (R4a-O)

Methionine + H₃O⁺→ CR-O→ M-OH⁺+ H₂O→ TS4a-O→ P4-O→

TS4b-O→ CP4b-O→ P4b-O (D⁺+H₂O) (R4b-O)

Methionine + H₃O⁺→ CR-O→M-OH⁺+ H₂O→ TS8-O→ CP8a-O→

CP8b-O + H₂O → P5b-O (B-NH⁺+CO + H₂O) (R8-O)

Methionine + H₃O⁺→ CR-O→ M-OH⁺+ H₂O→ TS10-O→ CP10-O→

P8a-O (E-OH⁺+CH₃SH) (R10-O)

Methionine + H₃O⁺→ CR-O→ M-OH⁺+ H₂O→ TS12-O→ IN1-O→

P12-O (NH₂CHCOOH + L⁺) (R12a-O)

Methionine + H₃O⁺→ CR-O→ M-OH⁺+ H₂O→ TS12-O→ IN1-O→

TS13→ P13-O (R12b-O)

Methionine + H₃O⁺→ CR-O→ M-OH⁺+ H₂O→ TS12-O→ IN1-O→

TS16-O→ P16a-O→P16b-O (K⁺+NH₃) (R15-O)

Methionine + H₃O⁺→ CR-O→ M-OH⁺+ H₂O→ TS17-O→

P17-O (M-SH⁺)→ P8b-O (E-CH⁺+CH₃SH) (R16-O)

Methionine + H₃O⁺→ CR-O→ M-OH⁺+ H₂O→ TS18-O→

CP18-O→ P18-O (J-NH⁺ + CO₂) (R17-O)

When hydronium ion collides with methionine from the O site of the -COOH group, the CR-O complex is yielded with a relative energy of −45.69 kcal mol⁻¹. The result of this collision is the transfer of a proton from H₃O⁺ to the O site of methionine. Thus, an M-OH⁺ molecule is generated. Routes R4a-O, R4b-O, R8-O, R10-O, R12a-O, R12b-O, R15-O, R16-O, and R17-O are unimolecular dissociation routes of M-OH⁺ to different fragments. The stability of TS4-O, TS8-O, TS10-O, TS13-O, TS16-O, and TS17a-O is 55.38, 48.37, 48.53, 74.51, 72.30, and 104.87 kcal mol⁻¹ larger in comparison with the saddle points TS4, TS8, TS10, TS13, TS16, and TS17a, respectively. Also, the product complexes CP8a-O and CP10-O are 50.19 and 27.28 kcal mol⁻¹ more stable than CP8 and CP10. The new routes, R4b-O and R15-O, show the separation of water and NH₃ molecules from the P4 and P16a adducts, leading to the formation of D⁺ (132.048 amu) and K⁺ (133.032 amu) fragments, respectively. The saddle point TS4b-O has a relative energy of 32.49 kcal mol⁻¹. The geometry of the fragment E-OH⁺ is the same as E. The only difference is the protonation of the O center in the -COOH group. Route R12a-O shows that IN1-O can produce the fragments NH₂CHCOOH (75.032 amu) and L⁺ (75.027 amu). The product of P16a-O loses an NH₃ molecule and generates K⁺.

Protonation of methionine from the S site

The structures and energetics of all species are in Fig. 12; Table 5, respectively. The PES of protonated methionine from the S site is sketched in Fig. 13. The reaction routes of this PES are as follows.

Fig. 12.

The structures involved in the S-site protonated methionine (M-SH⁺) decomposition pathways.

Table 5.

Relative energy, internal energy, enthalpy, Gibbs free energy, and entropy of R, tss, cps, and Ps involved in the decomposition pathways of the S-site protonated methionine (M-SH⁺ molecule).

Species	∆E	∆U0	∆H0	∆G0	T∆S0
R	0.00	0.00	0.00	0.00	0.00
CR-S	−47.43	−46.91	−47.51	−39.93	−7.58
M-SH+	−32.76	−32.75	−32.75	−32.92	0.17
CP2-S	−0.45	1.99	1.99	−4.64	6.62
CP3-S	−4.60	−3.03	−3.03	−7.17	4.14
CP5-S	−18.82	−18.04	−18.04	−19.84	1.80
CP6-S	−13.80	−12.94	−12.94	−15.53	2.59
CP7-S	1.94	3.48	3.48	−0.28	3.75
CP8b-S	1.94	3.48	3.48	−0.28	3.75
CS9a	−32.32	−31.97	−31.97	−33.08	1.11
CS9b	−35.14	−34.52	−34.52	−37.26	2.74
CP9c	2.84	5.36	5.36	−1.99	7.35
TS2-S	43.59	43.92	43.92	42.92	1.01
TS3-S	42.20	42.35	42.35	42.14	0.21
TS4-S	23.59	23.19	23.19	24.49	−1.30
TS5-S	97.97	97.93	97.93	98.10	−0.17
TS6-S	76.38	76.70	76.70	75.53	1.17
TS7-S	34.88	34.79	34.79	34.96	−0.16
TS8-S	32.08	32.24	32.23	31.82	0.42
TS8b-S	32.09	32.24	32.24	31.82	0.41
TS8B-S	32.09	32.24	32.24	31.82	0.41
TS9a-S	19.56	19.90	19.90	18.51	1.39
TS9b-S	6.52	7.11	7.11	5.31	1.80
TS9c-S	14.25	14.96	14.96	12.84	2.11
P2a-S	9.29	10.67	11.85	−11.42	23.28
P2b-S	11.63	12.10	13.87	−19.03	32.91
P3a-S	10.06	11.18	11.77	−0.46	12.23
P3b-S	8.93	9.19	10.37	−12.13	22.50
P4a-S	−9.53	−9.48	−9.48	−9.55	0.07
P4b-S	−18.18	−19.25	−18.66	−28.47	9.81
P5a-S	0.49	0.59	1.18	−11.85	13.03
P5b-S	2.83	2.01	3.20	−19.46	22.66
P6a-S	−12.94	−12.68	−12.68	−13.11	0.43
P6b-S	−12.01	−12.83	−12.24	−22.26	10.02
P7a-S	−21.82	−22.51	−21.91	−32.61	10.69
P7b-S	19.53	19.96	21.14	−1.44	22.59

Fig. 13.

The PES of the fragmentation of the S-site protonated methionine (M-SH⁺) computed at the M06-2X/6–31 + G(d, p) level of theory.

Methionine + H₃O⁺→ CR-S→ M-SH⁺+H₂O→ TS2-S→ CP2-S→

P2a-S (B-SH⁺+H₂O + CO) → P2b-S (O⁺+CH₃SH + H₂O + CO) (R2-S)

Methionine + H₃O⁺→ CR-S→ M-SH⁺+H2O→TS3-S→ CP3-S→

P3a-S (C-SH⁺+H₂O)→ P3b-S (N⁺+CH₃SH + H₂O) (R3-S)

Methionine + H₃O⁺→ CR-S→ M-SH⁺+H₂O→TS4-S→ P4a-S→ P4b-S(T⁺+ CH₃SH) (R4-S)

Methionine + H₃O⁺→ CR-S→ M-SH⁺+H₂O→TS4-S→ P4-S→

TS5-S→ CP5-S→ P5a-S (B-SH⁺+HCOOH) → P5b-S (O⁺+CH₃SH + HCOOH) (R5-S)

Methionine + H₃O⁺→ CR-S→ M-SH⁺+H₂O→TS6-S→ CP6-S→

P5a-S (B-SH⁺+HCOOH) → P5b-S(O⁺+CH₃SH + HCOOH) (R6-S)

Methionine + H₃O⁺→ CR-S→ M-SH⁺+H₂O→TS7-S→ P6a-S → P6b-S (U⁺ + CH₃SH) (R7-S)

Methionine + H₃O⁺→ CR-S→ M-SH⁺+H₂O→TS7-S→ P6 → TS8-S→ CP8-S→

P3a-S (C-SH⁺+H₂O)→ P3b-S (N⁺+CH₃SH + H₂O) (R8-S)

Methionine + H₃O⁺→ CR-S→ M-SH⁺+H₂O→TS9a-S→ CP9a-S→ P7a-S (Q⁺+CH₃SH) (R9a-S)

Methionine + H₃O⁺→ CR-S→ M-SH⁺+H₂O→TS9b-S →CP9b-S→ P7a-S (Q⁺+CH₃SH) (R9b-S)

Methionine + H₃O⁺→ CR-S→ M-SH⁺+H₂O→TS9b-S→ CP9b-S→ TS9c-S→

CP9c-S→ P7b-S (X⁺+CH₃SH + H₂O) (R9c-S)

Hydronium ion can impact the S center of methionine and form a complex, CR-S, with a relative energy of −47.43 kcal mol⁻¹. This impact leads to the migration of a proton from H₃O⁺ to the S site of methionine. The result of this process is the formation of a protonated M-SH⁺ molecule. M-SH⁺ can undergo several unimolecular reactions denoted by routes R2-S - R9c-S. The saddle points TS2-S - TS9-S in routes R2-S - R9a-S are similar to the saddle points of the R2-R9 pathways. The relative energies of TS2-S - TS9-S are 26.25, 35.00, 27.12, 23.23, 30.89, 40.61, 52.00, and 54.68 kcal mol⁻¹ more stable than the respective transition state in neutral methionine decomposition thermal pathways. Also, the post-reactive complexes CP2-S, CP3-S, Cp5-S, CP6-S, CP8-S, and CP9a-S exhibit greater stability compared to the corresponding complexes generated in neutral methionine decomposition, with the stabilities of 26.34, 42.02, 39.46, 35.11, 35.47, and 42.19 kcal mol⁻¹, respectively. The product P2a includes B-SH⁺, H₂O, and CO fragments. The B-SH⁺ fragment can dissociate into O⁺ and CH₃SH moieties. The product P3a contains C-SH⁺ and H₂O fragments. The C-SH⁺ molecule can further dissociate into the N⁺ (84.054 amu) and CH₃SH (48.003 amu) fragments. Also, the CH₃SH fragment can separate from the P4a, P6a, P7a, P7b, and P7c products and produce T⁺ (102.056 amu), U⁺(102.056 amu), Q⁺(102.056 amu), O⁺(56.050 amu), and X⁺ (84.045 amu) fragments, respectively.

DFT calculations establish the thermodynamic hierarchy of protonation sites: N1 (− 69.55 kcal/mol) > S1 (− 47.43 kcal/mol) > O1 (− 45.69 kcal/mol) (Table 3). While N-protonation dominates, all sites contribute to fragmentation kinetics, explaining observed PIP signatures (e.g., barrierless CO₂ loss at N-site vs. CH₃SH release at S-site).

The fragmentation of protonated methionine (M-XH⁺, where X = N, O, S) exhibits significantly enhanced kinetics and thermodynamics compared to the neutral system, driven by initial protonation via H₃O⁺. Site-specific protonation dictates distinct reaction pathways.

For N-protonated methionine (M-NH⁺), the loss of CO₂ through R18-N has an exceptionally low energy barrier (ΔE^≠ = 2.57 kcal/mol at TS18-N), yielding J-NH⁺ + CO₂. This product, along with [M - COOH] H⁺ from the decomposition of neutral methionine, contributes to the formation of PIP3. Methyl mercaptan loss (R10-N, ΔE^≠ = 10.94 kcal/mol at TS10-N) forms E-NH⁺ + CH₃SH. The low energy barrier for this pathway supports the claim that E-NH⁺ participates in the formation of PIP4, and it confirms the existence of two channels for methyl mercaptan production: decomposition of neutral methionine and fragmentation of its protonated form.

In O-protonated systems (M-OH⁺), a spontaneous rearrangement occurs via R4a-O (ΔE^≠ = −4.68 kcal/mol at TS4a-O) before water elimination. While dehydration pathways (e.g., R4b-O yielding D⁺ + H₂O) are kinetically feasible in protonated methionine (ΔE^≠ = 32.49 kcal/mol), their role is computationally subordinate to CO-release mechanisms. Pathway R8-O, which generates B-NH⁺ + CO + H₂O (P5b-O), exhibits superior kinetics (ΔE^≠ = 23.19 kcal/mol at TS8-O) and thermodynamics (ΔG = −38.11 kcal/mol). This concerted elimination of H₂O and CO effectively mimics formic acid (HCOOH) loss, producing the B-NH⁺ fragment that directly contributes to PIP3 in experimental IMS spectra. The 29% lower barrier and 51.26 kcal/mol enhanced stability of this pathway versus pure dehydration establish CO-coupled water elimination as the dominant fragmentation process, with simple water loss representing only a minor channel despite its formal accessibility.

S-protonated methionine (M-SH⁺) preferentially undergoes methyl mercaptan release through R9b-S (ΔE^≠ = 6.52 kcal/mol at TS9b-S) to produce Q⁺ + CH₃SH. This result confirms that the fragmentation of protonated methionine contributes to the formation of PIP4 and PIP5.

Thermodynamically, the most stable products across protonation sites are: J-NH⁺ + CO₂ (P11a-N, ΔG = − 62.43 kcal/mol) for N-protonation; J-NH⁺ + CO₂ (P-TS18-O, ΔG = − 62.43 kcal/mol) for O-protonation; and Q⁺ + CH₃SH (P7a-S, ΔG = − 32.61 kcal/mol) for S-protonation. These results demonstrate that protonation not only dramatically reduces fragmentation barriers but also stabilizes small-molecule elimination products, with CO₂ and CH₃SH formation being particularly favored both kinetically and thermodynamically across multiple protonation sites.

While neutral methionine fragmentation favors NH₃ elimination, as discussed in a previous section (Sect. “Thermal decomposition of methionine”), protonation drastically reshapes the decomposition landscape. As computed, protonated methionine exhibits barrierless CO₂ loss (N/O-protonation) and kinetically dominant CH₃SH release (S-protonation). The thermodynamics favor ions like J-NH⁺ (m/z 106) and Q⁺ (m/z 102). This aligns with IMS peaks PIP5 (CH₃SH⁺, 49 amu) and PIP6 (57 amu), confirming that initial H₃O⁺ protonation governs both fragment identities and formation rates.

Conclusion

In conclusion, this study investigated the ion mobility spectrum of methionine and its thermal decomposition products using a corona discharge ionization source coupled with a delayed sample injection system. The results revealed that methionine undergoes thermal decomposition at 280 °C, as evidenced by the decrease in the protonated methionine peak and the emergence of new peaks with increasing delay times. Temporal analysis of peak intensities revealed distinct fragmentation pathways, with some fragments formed at the injection port and others within the ionization region. Additionally, a mass-drift time correlation method provided valuable insights into the masses of observed fragment ions. Theoretical investigations using computational chemistry confirmed the fragmentation patterns of both neutral and protonated methionine molecules. The decomposition pathways for neutral (M) and protonated methionine M-XH⁺ (where X is N, O, and S sites) were systematically examined, and the probable decomposition products were proposed. Thus, the PES of all M and M-XH⁺ systems was constructed using the DFT-M06-2X method. Our results demonstrated that the protonated system fragmented into smaller components more easily than the neutral system due to the high impact of the H₃O⁺ species with methionine. The theoretical predictions aligned closely with the experimental observations, validating the proposed mechanisms.

These findings contribute to a deeper understanding of methionine fragmentation in ion mobility spectrometry and highlight the utility of combining delayed injection with temporal analysis for elucidating complex decomposition mechanisms. This integrated approach provides a powerful tool for studying thermal degradation mechanisms and can be extended to other molecular systems in future research. Also, future studies will incorporate collision cross-section (CCS) calculations via advanced tools (e.g., MobCal) to further validate fragment assignments.

Supplementary Information

Below is the link to the electronic supplementary material.

Author contributions

M.V. conceived and supervised the theoretical framework and corrected the manuscript. H.B. designed and supervised the experimental work. M.A. and H.D. carried out experiments, optimized methodologies, collected and analyzed data, and wrote both the experimental and theoretical sections of the manuscript. All authors contributed to the interpretation of results, manuscript discussions, and final approval of the content.

Data availability

All data analyzed during this study are included in this published article and its supplementary information files.

Declarations

Competing interests

The authors declare no competing interests.