Probe oscillation control in tapping-mode scanning probe electrospray ionization for stabilization of mass spectrometry imaging.

Mass spectrometry imaging (MSI) is used for visualizing the distribution of components in solid samples, such as biological tissues, and requires a technique to ionize the components from local areas of the sample. Tapping-mode scanning probe electrospray ionization (t-SPESI) uses an oscillating capillary probe to extract components from a local area of a sample with a small volume of solvent and to perform electrospray ionization of those components at high speed. MSI can be conducted by scanning the sample surface with a capillary probe. To ensure stable extraction and ionization for MSI, the probe oscillation during measurements must be understood. In this study, we examined the changes in oscillation amplitude and phase due to the interaction between the oscillating probe and the brain tissue section when the probe tip was dynamically brought close to the sample surface. The changes in the probe oscillation depended on the oscillation frequency and polarity of the bias voltage applied to the solvent because an electrostatic force shifted the frequency of the probe oscillation. These findings suggest that controlling the probe oscillation frequency is important for stabilizing MSI by t-SPESI.

A Method for High Throughput Free Fatty Acids Determination in a Small Section of Bovine Liver Tissue Using Supercritical Fluid Extraction Combined with Supercritical Fluid Chromatography-Medium Vacuum Chemical Ionization Mass Spectrometry.

A novel ionization technique named medium vacuum chemical ionization (MVCI) mass spectrometry (MS), which is a chemical ionization using oxonium (H3O+) and hydroxide (OH-) formed from water, has excellent compatibility with the supercritical fluid extraction (SFE)/supercritical fluid chromatography (SFC). We have studied a method to determine free fatty acids (FFAs) in a small section of bovine liver tissue using SFE/SFC-MVCI MS analysis without further sample preparation. A series of FFA molecules interact with the C18 stationary phase, exhibiting broad chromatographic peaks when using a non-modified CO2 as the mobile phase. It can be optimized by adding a small content of methanol to the mobile phase as a modifier; however, it may dampen the ionization efficiency of MVCI since the proton affinity of methanol is slightly higher than water. We have carefully evaluated the modifier content on the ion detection and column efficiencies. The obtained result showed that an optimized performance was in the range of 1 to 2% methanol-modified CO2 mobile phase for both column efficiency and peak intensity. Higher methanol content than 2% degrades both peak intensity and column efficiency. Using optimized SFC conditions, a section of bovine liver tissue sliced for 14 µm thickness by 1 mm square, which is roughly estimated as about 3300 hepatocytes, was applied to determine 18 FFAs amounts for carbon chains of C12-C24. An amount of each tested FFA was estimated as in the range of 0.07 to 2.6 fmol per cell.

Estimation of the Spatial Extent of the Transient Gain Drop in a Microchannel Plate.

The gain of the microchannel plate temporally drops after an ion initiates an electron avalanche. Electron multiplication was expected to deplete the charge from the microchannel wall and produce the depleted charge (wall charge). Moreover, it was reported that the gain drop occurred not only in the activated channels, where the electrons are multiplied, but also in the surrounding channels. One mechanism of the gain-drop spatial extension has been considered as that the wall charges in the activated channels change the electric field in the surrounding channels. Anacker et al. assumed that the wall charge is a uniform line charge; the gain-drop spatial extent should be proportional to the amount of the wall charges. We considered that the wall charges exponentially increased in the channel toward the exit. In this study, the electric field produced by the wall charges was calculated, considering the distribution of the wall charges. The transverse electric field generated by the wall charges was expected to disturb the electron trajectory near the channel exit and decrease the number of secondary electrons emitted per collision (gain per collision), resulting in a gain drop. The gain per collision was calculated to decrease by 22% for the position where the gain decreased significantly in the presence of the transverse electric field of 3×105 V/m. In our model, the gain-drop spatial extent extended proportionally to the square root of the wall charges when the distance from the activated channel exceeded 50 µm.

Aeromicelle-A new form of liquid aerosol for delivering aqueous samples into a single-particle mass spectrometer.

For applying highly sensitive mass spectrometry to chemical analysis of aqueous samples, we have developed a novel technique using a new form of liquid droplets, which we call "aeromicelle" (AM), to deliver aqueous sample solutions directly into the vacuum region of a single-particle mass spectrometer in liquid form and conduct immediate mass analysis. AMs are generated by spraying an aqueous solution containing a surfactant at a concentration significantly lower than its critical micelle concentration (CMC). When the solution is sprayed, liquid droplets containing the surfactant are formed, which gradually dry in an air flow. Upon drying, the surfactant concentration in the droplet exceeds its CMC, and consequently, the surfactant molecules begin to cover the droplet surface. Finally, the surface is expected to be fully covered with surfactant molecules such as reverse micelles. The surface coverage helps suppress the evaporation of water, thereby enhancing the residence time of the liquid droplet. Our experimental results show that the AMs retained a liquid form for at least 100 s in air and survived even under vacuum conditions for further mass analysis: each AM delivered in the vacuum region of a single-particle mass spectrometer is ablated with an intense laser pulse and then, mass analyzed. Individual AMs generated from an aqueous solution containing CsCl were analyzed using a single-particle mass spectrometer. The Cs+ ion peak was observed even in AMs generated from the 10 nM solution. The number of Cs atoms in each AM was estimated to be approximately 7 × 103, which corresponds to 1.2 × 10-20 mol (12 zmol). Meanwhile, in the mass analysis of tyrosine, both positive and negative fragmentation ions from tyrosine in AMs were observed in the mass spectrum and 4.6 × 105 (760 zmol) tyrosine molecules were detected.

Solvent effects of N,N-dimethylformamide and methanol on mass spectrometry imaging by tapping-mode scanning probe electrospray ionization.

Mass spectrometry imaging (MSI) is an effective technique for visualizing the distribution of lipids in tissues. The direct extraction-ionization methods using minute volumes of solvent for local components have the advantage of rapid measurement without any sample pretreatment. For effective MSI of tissues, it is necessary to understand the effect of solvent physicochemical properties on ion images. In this study, we report solvent effects on the lipid imaging of mouse brain tissue by tapping-mode scanning probe electrospray ionization (t-SPESI) which is capable of extraction-ionization using sub-pL solvents. To precisely measure lipid ions, we developed a measurement system incorporating a quadrupole-time-of-flight mass spectrometer. The differences in signal intensity and spatial resolution of lipid ion images were investigated using N,N-dimethylformamide (non-protic polar solvent), methanol (protic polar solvent) and their mixture. The mixed solvent was suitable for the protonation of lipids, and it provided high spatial resolution MSI. Results indicate that the mixed solvent improves the extractant transfer efficiency and minimizes charged droplets from an electrospray. The solvent selectivity study revealed the importance of solvent selection based on physicochemical properties for the advancement of MSI by t-SPESI.

Distinct diurnal chemical compositions and formation processes of individual organic-containing particles in Beijing winter.

Organic aerosols (OA) are major components of fine particulate matter, yet their formation mechanism remains unclear, especially in polluted environments. In this study, we investigated the diurnal chemical compositions and formation processes of OA in carbonaceous particles during winter in Beijing using aerosol time-of-flight mass spectrometry. We found that 84.5% of the measured carbonaceous particles underwent aging processes, characterized by larger diameter and more secondary species compared to fresh carbonaceous particles, and presented different chemical compositions of OA in the daytime and nighttime. During the day, under high O3 concentrations, organosulfates and oligomers existed in the aged carbonaceous particles, which were mixed with a higher signal of nitrate compared with sulfate. At night, under high relative humidity, distinct spectral signatures of hydroxymethanesulfonate and organic nitrogen compounds, and minor signals of other hydroxyalkylsulfonates and high molecular weight organic compounds were present in the aged carbonaceous particles, which were mixed with a higher signal of sulfate compared with nitrate. Our results indicated that photochemistry contributed to OA formation in the daytime, while aqueous chemistry played an important role in OA formation in the nighttime. The findings can help improve the performance of air quality and climate models on OA simulation.

Space and Time Coherent Mapping for Subcellular Resolution of Imaging Mass Spectrometry.

Space and time coherent mapping (STCM) is a technology developed in our laboratory for improved matrix-assisted laser desorption ionization (MALDI) time of flight (TOF) imaging mass spectrometry (IMS). STCM excels in high spatial resolutions, which probe-based scanning methods cannot attain in conventional MALDI IMS. By replacing a scanning probe with a large field laser beam, focusing ion optics, and position-sensitive detectors, STCM tracks the entire flight trajectories of individual ions throughout the ionization process and visualizes the ionization site on the sample surface with a subcellular scale of precision and a substantially short acquisition time. Results obtained in thinly sectioned leech segmental ganglia and epididymis demonstrate that STCM IMS is highly suited for (1) imaging bioactive lipid messengers such as endocannabinoids and the mediators of neuronal activities in situ with spatial resolution sufficient to detail subcellular localization, (2) integrating resultant images in mass spectrometry to optically defined cell anatomy, and (3) assembling a stack of ion maps derived from mass spectra for cluster analysis. We propose that STCM IMS is the choice over a probe-based scanning mass spectrometer for high-resolution single-cell molecular imaging.

Microscale supercritical fluid extraction combined with supercritical fluid chromatography and proton-transfer-reaction ionization time-of-flight mass spectrometry for a magnitude lower limit of quantitation of lipophilic compounds.

The application of proton transfer ionization reaction mass spectrometry (PTR MS) combined with microscale supercritical fluid extraction (SFE) and supercritical fluid chromatography (SFC) aiming to quantitate single-cell fatty acid analysis levels was investigated. Using a microscale extraction vessel, the obtained low limits of quantitation (LLOQs) of arachidonic acid and arachidic acid were 1.2 and 2.7 fmol, respectively, by using less than 1 µL of sample on stainless steel frit. A series of phthalate, vitamin K1, and α-tocopherol were also tested, and the LLOQ was less than one femtomole for phthalate and 35 and 13 fmol for vitamin K1 and α-tocopherol, respectively. A microliter portion of SFE extracts was introduced into the SFC column by split injection, improving the reproducibility of the chromatography and separation efficiency. The method in the present study has great potential to quantitate lipophilic molecules on the nanogram scale of a sample without complex preparation procedures.

Matrix-Assisted Laser Desorption/Ionization (MALDI) Mass Spectrometry Imaging of L-4-Phenylalanineboronic Acid (BPA) in a Brain Tumor Model Rat for Boron Neutron Capture Therapy (BNCT).

Boron neutron capture therapy (BNCT) is a cell-selective particle therapy for cancer using boron containing drugs. Boron compounds are accumulated in high concentration of tens ppm level of boron in target tumors to cause lethal damage to tumor tissue. The examination of boron distribution in target tumor and normal tissue is important to evaluate the efficiency of therapy. The matrix-assisted laser desorption/ionization (MALDI) mass spectrometry imaging (MSI) is a powerful tool to visualize the distribution of target analyte in biological samples. In this manuscript, we report a trial to visualize the distribution of a typical BNCT drug, L-4-phenylalanine boronic acid (BPA) in a brain tumor model rat using MALDI-MSI technique. We performed a MALDI-MSI with high mass resolution targeting to [BPA+H]+ at m/z 210 in a BPA-treated rat brain section using a spiral orbit-type time of flight (SpiralTOF) mass spectrometer. Several BPA ion species, [BPA+H]+, [BPA-H2O+Na]+, [BPA+DHB-2H2O+Na]+ and [BPA+DHB-2H2O+K]+ were detected separate from peaks originated from biomolecules or matrix reagent by achieving the mass resolving power of approximately 20,000 (full width at half maximum; FWHM) at m/z 210. The mass images with 60 µm spatial resolution obtained from these BPA ion species in a mass window of 0.02 Da revealed their localization in the tumor region. Additionally, the mass image obtained from [BPA+H]+ also likely showed the distribution of BPA inside the tumor. MALDI-MSI with high mass resolution targeting to [BPA+H]+ has a great potential to visualize the distribution of BPA in brain tissue with tumor.

Attempts to Detect Lipid Metabolites from a Single Cell Using Proton-Transfer-Reaction Mass Spectrometry Coupled with Micro-Scale Supercritical Fluid Extraction: A Preliminary Study.

Proton-transfer-reaction (PTR) mass spectrometry (MS), a widely used method for detecting trace-levels of volatile organic compounds in gaseous samples, can also be used for the analysis of small non-volatile molecules by using supercritical fluid as a transporter for the molecules. Supercritical fluid extraction (SFE) is a method that permits lipophilic compounds to be rapidly and selectively extracted from complex matrices. The combination of the high sensitivity of PTR MS with the SFE is a potentially novel method for analyzing small molecules in a single cell, particularly for the analysis of lipophilic compounds. We preliminarily evaluated this method for analyzing the components of a single HeLa cell that is fixed on a stainless steel frit and is then directly introduces the SFE extracts into the PTR MS. A total of 200/91 ions were observed in positive/negative ion mode time-of-flight mass spectra, and the masses of 11/10 ions could be matched to chemical formulae obtained from the LipidMaps lipids structure database. Using various authentic lipophilic samples, the method could be used to detect free fatty acids in the sub-femtomole to femtomole order in the negative ion mode, the femtomole to sub-picomole order for fat-soluble vitamins, and the picomole order for poly aromatic hydrocarbons in both the positive and negative ion mode.

Rapid Analysis of α-Tocopherol and Its Oxidation Products Using Supercritical Carbon Dioxide and Proton Transfer Reaction Ionization Mass Spectrometry.

We have developed a rapid and sensitive analytical method for α-tocopherol and its oxidative products by combining online hyphenation of supercritical fluid extraction-supercritical fluid chromatography (SFC) with proton transfer reaction (PTR) ionization mass spectrometry (MS). α-Tocopherol is a well-known antioxidant that plays a vital role in the antioxidant defense system in plant cells. However, studies on the cellular mechanisms of α-tocopherol have been limited owing to the lack of a rapid analytical method, which limits the comparison of plant cells incubated in various conditions. Additionally, complex sample preparation and long chromatography separation times are required. Moreover, the majority of the involved molecules are a combination of isomers, which must be separated before applying tandem MS. α-Tocopherol produces the α-tocopheroxyl radical in the first step of its antioxidant function; another ion with the same mass may also be generated from the source. SFC separation effectively distinguished the observed ions from their oxidative products in the sample and those produced during the ionization reaction process. This method enabled the measurement of α-tocopherol and its oxidative products such as α-tocopheroxyl radical and α-tocopheryl quinone in approximately 3 min per sample, including the time required for sample preparation.

A Method for Expanding Mass Range on a Multi-Turn Time-of-Flight Mass Spectrometer by a Lap Superimposed Spectrum.

A time-of-flight mass spectrometer that uses a closed-orbit flight path can achieve a high mass resolving power and a high mass accuracy with a small instrument footprint. It has long been known that a drawback to a closed flight path is an obtained spectrum may contain peaks by ions at a different number of laps. A lower m/z ion may overtake higher m/z ions, resulting in the peak being superimposed on an acquired mass spectrum; therefore, such a mass bandwidth of the analyzer is limited to a narrow range given the current situation. However, recent research has documented a solution to the problem based on careful study of the equation of motion of an ion in a closed-path analyzer. All of the ions in the analyzer remain in motion in orbit by the nature of the closed flight path, thus resulting in a superimposed spectrum with the width of the orbital period of the highest mass in the sample matrix, which contains several different lap numbers. When target ions for the sample are known in advance, the time-of-flight for a given m/z can be determined regardless of the lap number under given analyzer conditions, and peak assignment can be self-validated by comparison to a mass spectrum acquired at a different lap condition. Furthermore, the m/z value for an unknown ion can also be determined by comparing time-of-flight values on spectra acquired at different lap conditions.

Analysis of Nonvolatile Molecules in Supercritical Carbon Dioxide Using Proton-Transfer-Reaction Ionization Time-of-Flight Mass Spectrometry.

Proton-transfer-reaction (PTR) mass spectrometry (MS) is capable of detecting trace-level volatile organic compounds (VOCs) in gaseous samples in real time. Therefore, PTR-MS has become a popular method in many different study areas. Most of the currently reported PTR-MS applications are designed to determine volatile compounds. However, the method might be applicable for nonvolatile organic compound detection. Supercritical fluid chromatography (SFC) has been studied in the last 5 decades. This approach has high separation efficiency and predictable retention behavior, making separation optimization easy. Atmospheric ionization techniques, such as atmospheric chemical ionization (APCI) and electrospray ionization (ESI), are the most studied SFC-MS interfaces. These processes require the addition of makeup solvents to prevent precipitation or crystallization of the solute while depressurizing the mobile phase. In contrast, the PTR process is carried out in a vacuum; supercritical carbon dioxide may release solute into the PTR flow tube without a phase transition as long as it is maintained above a critical temperature. Therefore, this might constitute yet another use for the SFC-MS interface. Caffeine and a few other nonpolar compounds in supercritical carbon dioxide were successfully detected with time-of-flight MS without adding solvent by using preliminarily assembled supercritical flow injection and supercritical fluid extraction (SFE)-PTR interfaces.

Evaluation of microchannel plate gain drops caused by high ion fluxes in time-of-flight mass spectrometry: A novel evaluation method using a multi-turn time-of-flight mass spectrometer.

Use of a two-stage microchannel plate (MCP) detector is common in time-of-flight (TOF) mass spectrometry because it shows excellent time resolution with sufficient gains. However, the gain drops significantly when the detector detects intense ion fluxes, such as matrix ions, by matrix-assisted laser desorption/ionization mass spectrometry. As a result, significant ion signals corresponding to analytes, such as proteins, are hidden, thereby hampering the mass spectral interpretation. However, details of this phenomenon have not previously been investigated using ions because of the lack of suitable measurement methods and apparatus. Thus, we herein report a novel method for controlling the TOF of two selected ions, as a function of time differences between each other using a multi-turn TOF mass spectrometer. This method involves the use of an isotope cluster of ions that fly in a figure-of-eight orbit and the extraction of an ion at a given lap number. A series of time differences (∆t) between two ions in a TOF spectrum can be generated using this method. We evaluated the time constants of gain recovery after high ion-flux detection for two sets of two-stage MCP detectors to obtain values of 1,600 and 180 µs for channel plate resistances of 500 and 71 MΩ, respectively. The obtained time constants from the gains determined at various values of ∆t were 0.48 and 0.38 fold (for 500 and 71 MΩ, respectively) of the values suggested from the channel plate resistance and capacitance.

Selective Extraction of a Monoisotopic Ion While Keeping the Other Ions in Flight on a Multi-Turn Time-of-Flight Mass Spectrometer.

Using a multi-turn time-of-flight (TOF) mass spectrometer, we have extracted a single xenon isotope ion, 129Xe+, from its orbit at given a lap number without disturbing the rest of isotopes. After detecting the 129Xe+ at 20 laps, the rest of the xenon isotope spectrum was obtained at 30 laps, which generated a TOF spectrum where the TOF difference between 129Xe+ and 130Xe+ was 87.4 µs while 130Xe+ and 131Xe+ were 1.03 µs. The time distance between 129Xe+ and other isotopes can be set by any lap difference that is a factor of 8.7 µs, which depends on the acceleration voltage and the mass of the ion. Method accuracy was verified by comparing the isotopic abundance ratio of the xenon sample after withdrawing one of the ions from the isotope cluster to the abundance ratio obtained from the conventional method. The TOF stability was also evaluated at various lap numbers between 10 to 230.

New Method for Improving LC/Time-of-Flight Mass Spectrometry Detection Limits Using Simultaneous Ion Counting and Waveform Averaging.

Simultaneous ion counting and waveform averaging implemented on a field-programmable gate array compiled with a high-speed digitizer was applied to ultraperformance liquid chromatography-time-of-flight mass spectrometric analysis of sulfa drugs. Ion counting was carried out by a "Peak Detection" (PKD) function that works together with signal averaging (AVG). Sulfadimidine (SDD) and sulfadimethoxine (SDMX) were measured in human serum (HS) model sample matrix. By using simultaneous PKD and AVG acquisition, we observed a unified calibration curve for more than 3 orders of magnitude of sample amounts (0.010-100.0 pmol). The ion count rate for the "practical" sample amounts, such as less than 1 pmol, was below 30%, which is suitable for PKD-based ion counting for quantitative accuracy and excellent peak identification performance. Samples containing 200 fmol or less could not be identified from the AVG waveform. Adding HS treated with acetonitrile severely suppressed the SDMX ion to less than one-half (58.1%). However, a linear response was observed for chromatographic peak area for analytes calculated from PKD waveforms. Also, the mass-resolving power calculated from the peak on the PKD waveform was 24% better than the corresponding AVG waveform, which also improves performance for analyte identification.

Improved Quantitative Dynamic Range of Time-of-Flight Mass Spectrometry by Simultaneously Waveform-Averaging and Ion-Counting Data Acquisition.

Two different types of data acquisition methods, "averaging mode" and "ion-counting mode", have been used in a time-of-flight (TOF) mass spectrometry. The most common method is an averaging mode that sums waveform signals obtained from each flight cycle. While it is possible to process many ions arriving at the same TOF in one flight cycle, low-abundance ions are difficult to measure because ion signals are overwhelmed by noises from the detection system. An ion-counting mode is suitable for the detection of such low-concentration ions, but counting loss occurs when two or more ions arrive at the detector within the dead time of the acquisition system. In this study, we introduce a technique that combines two methods to measure target ions with a high concentration difference, i.e., averaging mode and ion-counting mode are used simultaneously for high abundant and trace ions, respectively. By processing waveforms concurrently during data acquisition, one can choose to analyze either or both types of data to achieve a highly quantitative mass spectrum over a wide range of sample concentrations. The result of the argon isotope analysis shows that this method provides a more accurate determination of the isotope ratio compared to averaging mode alone at one-twentieth of the analysis time required by ion-counting alone. Graphical Abstract ᅟ.

A new approach for accurate mass assignment on a multi-turn time-of-flight mass spectrometer.

A simple, effective accurate mass assignment procedure for a time-of-flight mass spectrometer is desirable. External mass calibration using a mass calibration standard together with an internal mass reference (lock mass) is a common technique for mass assignment, however, using polynomial fitting can result in mass-dependent errors. By using the multi-turn time-of-flight mass spectrometer infiTOF-UHV, we were able to obtain multiple time-of-flight data from an ion monitored under several different numbers of laps that was then used to calculate a mass calibration equation. We have developed a data acquisition system that simultaneously monitors spectra at several different lap conditions with on-the-fly centroid determination and scan law estimation, which is a function of acceleration voltage, flight path, and instrumental time delay. Less than 0.9 mDa mass errors were observed for assigned mass to charge ratios ( m/z) ranging between 4 and 134 using only 40Ar+ as a reference. It was also observed that estimating the scan law on-the-fly provides excellent mass drift compensation.

Instrumentation and Method Development for On-Site Analysis of Helium Isotopes.

Helium isotope determination may be useful in measuring volcanic activity and issuing earlier warnings of possible eruptions. A method is presented for measuring the 3He/4He ratio in a gas sample using a multiturn time-of-flight mass spectrometer "infiTOF". In contrast to conventional waveform averaging, peaks are determined by counting ion pulses from each time-of-flight trigger. Samples were also measured by conventional magnetic-sector mass spectrometry for comparison. Magnetic sector results were used to designate a standard for infiTOF measurement and to calculate a ratio for each sample measured by infiTOF. Mass assignment error for ultrapure 3He+ standard was 4.30 × 10-5 Da. Mass assignment error of 4He2+ and 3He+ for sample cylinders was 3.00 × 10-8 Da and 2.25 × 10-4 Da, respectively. Abundance ratios determined by infiTOF were found to be within 2% of the abundance ratios determined by magnetic-sector mass spectrometry. Mass drift was <50 × 10-6 Da over 10 h. Sample flow rate was not found to affect the results as long as the reference sample was analyzed under the same conditions. Results indicate that the infiTOF system may be a viable tool for measuring helium isotopes, which may eventually lead to earlier warnings of volcanic activity.

N-Terminal Derivatization with Structures Having High Proton Affinity for Discrimination between Leu and Ile Residues in Peptides by High-Energy Collision-Induced Dissociation.

De novo sequencing is still essential in the identification of peptides and proteins from unexplored organisms whose sequence information is not available. One of the remaining problems in de novo sequencing is discrimination between Leu and Ile residues. The discrimination is possible based on differences in side chain fragmentation between Leu and Ile under high-energy collision-induced dissociation (HE-CID) conditions. However, this is observed only when basic residues, such as Arg and Lys, are present near the N- or C-terminal end. It has been shown that the charge derivatization at the N-terminal end by a quarternary ammonium or phosphonium moiety facilitates the side chain fragmentation by HE-CID. However, the effective backbone fragmentation by low-energy CID (LE-CID) is often hampered in those derivatives with a fixed charge. Previously, we demonstrated that the N-terminal charge derivatization with the structures having high proton affinity induced the preferential formation of b-ions under LE-CID conditions, allowing straightforward interpretation of product ion spectra. In the present study, we further investigated whether the same derivatization approach is also effective for discrimination between Leu and Ile under HE-CID conditions. Consequently, the side chain fragmentation of Leu and Ile residues was most effectively enhanced by the N-terminal derivatization with 4-(guanidinomethyl)benzoic acid among the tested structures. This derivatization approach, which is compatible with both HE- and LE-CID analysis, offers a straightforward and unambiguous de novo peptide sequencing method.