Cell Differentiation and Development

ABSTRACTProteomics is nowadays increasingly becoming part of the routine clinical practice of diagnostic laboratories, especially due to the advent of advanced mass spectrometry techniques. This review focuses on the application of proteomic analysis in the identification of pathological conditions in a hospital setting, with a particular focus on the analysis of protein biomarkers. In particular, the main purpose of the review is to highlight the challenges associated with the identification of specific disease‐causing proteins, given their complex nature and the variety of posttranslational modifications (PTMs) they can undergo. PTMs, such as phosphorylation and glycosylation, play critical roles in protein function but can also lead to diseases if dysregulated. Proteomics plays an important role especially in various medical fields ranging from cardiology, internal medicine to hemato‐oncology emphasizing the interdisciplinary nature of this field. Traditional methods such as electrophoretic or immunochemical methods have been mainstay in protein detection; however, these techniques are limited in terms of specificity and sensitivity. Examples include the diagnosis of multiple myeloma and the detection of its specific protein or amyloidosis, which relies heavily on these conventional methods, which sometimes lead to false positives or inadequate disease monitoring. Mass spectrometry in this respect emerges as a superior alternative, providing high sensitivity and specificity in the detection and quantification of specific protein sequences. This technique is particularly beneficial for monitoring minimal residual disease (MRD) in the diagnosis of multiple myeloma where traditional methods fall short. Furthermore mass spectrometry can provide precise typing of amyloid proteins, which is crucial for the appropriate treatment of amyloidosis. This review summarizes the opportunities for proteomic determination using mass spectrometry between 2012 and 2024, highlighting the transformative potential of mass spectrometry in clinical proteomics and encouraging its wider use in diagnostic laboratories.

ABSTRACTMatrix‐assisted laser desorption electrospray ionization (MALDESI) conventionally utilizes a mid‐infrared (IR) laser for the desorption of neutrals, allowing for detection of hundreds to thousands of analytes simultaneously. This platform enables mass spectrometry imaging (MSI) capabilities to not only detect specific molecules but also reveal the distribution and localization of a wide range of biomolecules across an organism. However, an IR laser comes with its disadvantages when imaging plants. At a mid‐IR wavelength (2970 nm), the compartmentalized endogenous water within the leaf structure acts as an internal matrix, causing rapid heating, and, in turn, degrades the spatial resolution and signal quality. An ultraviolet (UV) laser operates at wavelengths that overlap with the absorption bands of secondary metabolites allowing them to serve as sacrificial matrix molecules. With the integration and optimization of a 355 nm UV laser into the MALDESI‐MSI NextGen source for the analysis of plants, we were able to detect diverse molecular classes including flavonoids, fatty acid derivatives, galactolipids, and glucosinolates, at higher ion abundances when compared to the mid‐IR laser. These results show that re‐visiting UV‐MALDESI‐MSI, without the need for an exogenous matrix, provides a promising approach for the detection and imaging of important analytes in plants.

ABSTRACTThe development of LA‐ICPMS and LA‐MC‐ICPMS resulted in analytical methods for zircon geochronology using a 20‐ to 60‐μm laser spot size. A high amount of complexly zoned zircons promotes the requirement of U–Pb dating at smaller spot sizes. When spot size reduces, downhole fractionation (DHF) increases, increasing the DHF discrepancy between zircon grains and the primary reference zircon standard and resulting in inaccurate results. With MC‐ICPMS's high sensitivity and multi‐collector capabilities, this work attempts to accurately determine age with spatial resolutions below 20 μm. Three well‐characterized zircon standards (91500, GJ‐1, and Plešovice) were tested at spot sizes of 35, 20, 15, and 10 μm. Laser and mass spectrometry tuning, laser shot count, and ablation time have been optimized to reduce the DHF effect on measurement accuracy. Static ablation of 35‐ to 15‐μm spots with 150 laser shot counts (30 s) yielded precision of less than 1.5% and age offset of less than 2%. The DHF differs significantly from the reference standard and two test zircon samples used for validation, with an age offset of 4% at 10‐μm spot sizes. Masking shot counts from the end enhanced accuracy, notably for lower laser shot counts and shorter ablation times. At 75 laser shot counts, precision reached 1.4% and age offsets reduced to 1.6% for 206Pb/238U age. This method minimizes laser shot counts to avoid sampling two age groups. Higher zircon DHF variability at smaller spot sizes may influence a well‐calibrated, sensitive LA‐MC‐ICP‐MS analytical figure of merit.

ABSTRACTMS analysis of cyclic peptides in negative ion mode has been a challenge, in particular if the peptide does not contain acidic functional groups. In this paper, we present a way to easily produce negative ions from anionic peptide adducts, utilising collision‐induced dissociation (CID)‐mediated elimination. Using two different mass spectrometers, we have generated series of adducts of three cyclic and one linear peptide with various anions. They were then isolated and subjected to CID with a range of collision energies. The deprotonation percentage was then calculated from the resultant spectrum, and compared between the spectrometers, as well as with an external reference—proton affinity values. The susceptibility to deprotonate by detaching a HX moiety is proportional to the proton affinity of the X− species. Also, the linear peptide deprotonated more readily than the cyclic ones. On the other hand, lack of amino or acidic groups resulted in higher collision voltage (CV) necessary for the formation of deprotonated species. Moreover, the exact propensity for neutral loss depends on the ion temperature, which differs between mass spectrometers. We have developed a facile method for generating peptide anions for MS analysis of cyclic peptides, which works even if the peptide in question does not have easily ionisable groups. The deprotonated species generated in this way can be fragmented again in order to identify the peptide.

ABSTRACTEthyl propionate (C5H10O2, EP) has been extensively studied in the fields of biofuels and atmospheric chemistry. However, its vacuum ultraviolet (VUV) photoionization has not been investigated. This study examines the photoionization process of EP using tunable VUV synchrotron radiation, coupled with a reflectron time‐of‐flight mass spectrometer. This method yielded the photoionization mass spectrum of EP and photoionization efficiency (PIE) spectra of 10 identified fragment ions (i.e., C4H7O2+., C3H7O2+., C3H6O2+., C3H5O2+., C3H6O+., C3H5O+., C3H4O+., C2H5O+., C2H5+., and C2H4+.). The results, interpreted with the aid of high‐accuracy theoretical calculations, conclude possible formation mechanisms for each fragment ion. In the dissociation pathway of EP's cation, intramolecular hydrogen shifts and bond cleavage are the predominant processes. The C3H7O2+. and C2H4+. reaction channels do not arise from one‐step bond cleavage, but their reaction energy barriers are influenced by product energy, making them comparable to direct reaction channels. The active reaction sites within the molecules are elucidated using Laplacian bond order (LBO). Rate constants are calculated using RRKM theory, which confirms the kinetic factors governing the EP reaction process. This study provides a detailed understanding of the photoionization and dissociation of the main ions of EP within the 9.35–15.50 eV photon energy range.

ABSTRACTThis study focuses on investigating the conformational structure and zinc(II) affinity of a zinc finger‐like motif (ZFM) peptide with the sequence acetyl‐His1‐Cys2‐Gly3‐Pro4‐Gly5‐His6‐Cys7, where bold highlights the potential zinc(II) binding sites. Zinc fingers are crucial protein motifs known for their high specificity and affinity for zinc ions. The ZFM peptide's sequence contains the 2His‐2Cys zinc‐binding sites similar to those in natural zinc finger proteins but without the hydrophobic core, making it a valuable model for studying zinc(II)–peptide interactions. Previous research on related peptides showed that collision cross sections and B3LYP modeling predicted that the His‐2Cys‐carboxyl terminus coordination of zinc(II) was more stable than the 2His‐2Cys. Employing a comprehensive approach integrating ion mobility–mass spectrometry and theoretical modeling techniques, various zinc(II) binding modes of the ZFM have been thoroughly compared to ascertain their influence on the competitive threshold collision‐induced dissociation method for measuring the relative gas‐phase Zn(II) affinity of the ZFM peptide. The measured Zn(II) affinity of ZFM is greater than those measured recently for two peptides with similar primary structures, acetyl‐His1‐Cys2‐Gly3‐Pro4‐Gly5‐Gly6‐Cys7 and acetyl‐Asp1‐His2‐Gly3‐Pro4‐Gly5‐Gly6‐Cys7, indicating the preference for the His1‐Cys2‐His6‐Cys7 side groups for coordinating zinc(II) over the His‐2Cys‐carboxyl terminus or Asp‐His‐Cys‐carboxyl terminus in these related heptapeptides.

ABSTRACTThe development of a real‐time system for characterizing individual biomolecule‐containing aerosol particles presents a transformative opportunity to monitor respiratory conditions, including infections and lung diseases. Existing molecular assay technologies, although effective, rely on costly reagents, are relatively slow, and face challenges in multiplexing, limiting their use for real‐time applications. To overcome these challenges, we developed digitalMALDI, a laser‐based mass spectrometry system designed for single‐particle characterization. digitalMALDI operates as a near real‐time platform that directly samples aerosols, bypassing the need for complex and time‐consuming sample preparation. To demonstrate the feasibility of this approach, intact insulin protein was used as a representative target. Results showed that digitalMALDI is capable of detecting 1 pg of insulin protein in single aerosol particles, suggesting that the system has a broad application for disease diagnosis, environmental monitoring, and biosecurity management.

ABSTRACTPerfluoroalkyl and polyfluoroalkyl substances (PFAS) are a class of emerging contaminants that have been in use industrially since the 1940s. Their long‐term and extensive commercial use has led to their ubiquitous presence in the environment. The ability to measure the bioconcentration and distribution of PFAS in the tissue of aquatic organisms helps elucidate the persistence of PFAS as well as environmental impacts. Traditional analysis by LC–MS/MS can measure total PFAS concentrations within an organism but cannot provide comprehensive spatial information regarding PFAS concentrations within the organism. In the current study, we used infrared matrix‐assisted laser desorption electrospray ionization (IR‐MALDESI) to determine the limit of detection (LOD) of several PFAS utilizing a commercial standard mix spotted on mouse liver tissue. The traditional ice matrix and an alternative matrix, 1,8‐bis (tetramethylguanidino)naphthalene (TMGN), were explored when determining the limits of detection for various PFAS by IR‐MALDESI. The ice matrix alone resulted in a higher response than the combination of TMGN and ice. The resulting LOD for perfluorooctane sulfonic acid (PFOS) on a per voxel basis was 0.16 fmol/voxel. For comparison, zebrafish that were exposed to perfluorooctanoic acid (PFOA), PFOS, and perfluorohexanesulfonic acid (PFHxS) at different concentrations were homogenized, and PFAS were extracted by solid–liquid extraction, purified by solid phase extraction, and analyzed by LC–MS/MS to determine the level of bioaccumulation in the zebrafish. PFOS resulted in the highest level of bioaccumulation (731.9 μg/kg, or 234.2 fg/voxel). A zebrafish that had been exposed to a PFAS mixture of PFOA (250 ng/L), PFOS (250 ng/L), and PFHxS (125 ng/L) was cryosectioned and analyzed by IR‐MALDESI. Images could not be generated as the accumulation of PFAS in the sectioned tissue was below detection limit of the technique.

ABSTRACTIn our previous work, the sodiation of melittin, cytochrome c, and ubiquitin in a 1 mM NaOH water/methanol solution was studied by electrospray mass spectrometry. It was suggested that the α‐helix is more resistant to sodiation than the β‐sheet. In this study, sodiation of enhanced green fluorescent protein (EGFP) composed of a β‐barrel was studied in 1% CH3COOH (AcOH) or 1 mM NaOH water/methanol solution by electrospray mass spectrometry. Although EGFP was denatured in an acidic solution, it maintains a near‐native structure in a basic solution. For the 1% AcOH solution, the protonated EGFP, [EGFP + nH − mH + mNa]n+, with n = 14 − 36 and m = 0 was detected. For 1 mM NaOH, the number n for [EGFP + nH − mH + mNa]n+ was found to increase with the sodiation number m and vice versa for [EGFP + nH − mH + mNa]n−. Namely, Na+ adducts counteract the negative charges of deprotonated acidic residues. The protonated EGFP detected as major ions for basic 1 mM NaOH was ascribed to the more surface‐active H3O+(aq) than OH−(aq).

ABSTRACTThis study presents a comprehensive evaluation of the application of online multi‐internal standard calibration (M.ISC) in determining iodine concentrations through inductively coupled plasma mass spectrometry (ICP‐MS). Notably, M.ISC streamlines the calibration process by requiring only a single standard solution, thereby enhancing sample throughput and minimizing liquid waste. In addition, unlike conventional internal standard (IS) methods, M.ISC omits the need for time‐consuming species identification by utilizing multiple IS species simultaneously to minimize signal biases. The effectiveness of M.ISC was validated through the analysis of six standard reference samples, with the results of LOD and LOQ also being calculated by the error propagation approach. The traditional chemical analytical methods (TCAM), external standard calibration (EC) and single IS methods were also evaluated as comparative purpose. Nonetheless, M.ISC emerges as a straightforward matrix‐correction strategy, offering a simple and efficient alternative to traditional calibration methods for iodine detection by ICP‐MS.

ABSTRACTA possible TOF‐SIMS analysis of surface phase transitions has recently been proposed for limited cases such as polymers and ionic liquids. In the present study, we have extended this analysis to quench‐condensed noble gas films. The newly developed cryogenic TOF‐SIMS allowed both measurements of TOF‐SIMS below 4 K, and low‐energy ion scattering spectroscopy that is used to prepare a clean surface. It was found that the TOF‐SIMS intensity variation by increasing the temperature at a constant ramp rate (temperature‐programmed TOF‐SIMS) shows steep changes due to sublimation. Thus, the possibility of analyzing the surface phase transition at the local region defined by the incident ion beam of (cryogenic) TOF‐SIMS was demonstrated in the present study.

ABSTRACTA large geometry high resolution secondary ion mass spectrometer (HR‐SIMS) has been established as a part of the National Geochronology Facility (NGF) at Inter‐University Accelerator Centre (IUAC), New Delhi. The performance of the instrument related to high spatial resolution, high mass resolving power (MRP), sensitivity of the instrument to measure low abundant isotopes, and sensitivity of the instrument for 206Pb signal under different conditions are optimized and presented in this paper. We report the precision of the order of ~ 0.2‰ for oxygen isotopes measurement in 91500 reference material zircon and measured δ18O value of 10.08‰ ± 0.18‰ (2SD), which is in agreement with the recommended values. For U–Pb age measurement in zircon, Plešovice and FCT reference materials are measured as unknown and their 206Pb/238U ages agree with the reported values within the uncertainties. A long‐term evaluation of 206Pb/238UO and 206Pb/238U isotopic measurement ratio is also presented.

ABSTRACTOne critical issue in hydrogen/deuterium exchange mass spectrometry (HDX MS) analysis is the deleterious back exchange. Herein, we report that when matrix‐assisted laser desorption/ionization (MALDI) is used, the MALDI process itself can also cause significant back exchange. The back exchange occurred inside the reactive MALDI plume was investigated by depositing a fully deuterated sample prepared in D2O on top of a preloaded dried layer of matrix. A benzene‐1,3,5‐tricarboxamide (BTA) compound that can form supramolecular polymer in water and five peptides of angiotensin II (AT), pentaglycine (5G), pentaalanine (5A), cyclohexaglycine (C6G), and cyclohexaalanine (C6A) were selected as the testing compounds. Just like the situation in solution, the back exchange for the side chains and end groups is fast in the MALDI plume, while for the backbone amides, it is slow and dependent on the primary structure of the peptide. For the peptides tested, 5%–15% of D‐labels in the backbone amides can be lost during the MALDI process. This degree of back exchange, although not an unbearable problem for most HDX MS applications as 85%–95% of the informative labels would still survive, could seriously limit the use of MALDI in the HDX MS analysis of supramolecular assemblies. For these assemblies, the EX1‐like mechanism with two distinct distributions is common, and the back exchange could gravely distort or even merge the distinct isotopic distributions, which are the characteristic symbols of EX1.

ABSTRACTLC‐ESI‐MS/MS is a preferred method for detecting and identifying metabolites, including those that are unpredictable from the genome, especially in basal metazoans like Cnidaria, which diverged earlier than bilaterians and whose metabolism is poorly understood. However, the unexpected appearance of a “ghost peak” for dopamine, which exhibited the same m/z value and MS/MS product ion spectrum during an analysis of Nematostella vectensis, a model cnidarian, complicated its accurate identification. Understanding the mechanism by which “ghost peaks” appear is crucial to accurately identify the monoamine repertoire in early animals so as to avoid misassignments. Verification experiments showed that in‐source oxidation of tyramine, which produced an intense signal, was responsible for this “ghost peak.” This artifact commonly occurs among aromatic compounds with high signal intensities and appears at the same m/z as their respective in vivo oxidized metabolites. In metabolomics, spectra contain diverse signals from complex biological mixtures, making it difficult to recognize artifact peaks. To prevent misassignments, despite +16 Da differences, adequate chromatographic separation of metabolites from their respective in vivo oxidation precursors is necessary. Whereas both electrolysis and gas‐phase corona discharge can cause in‐source oxidation in ESI, corona discharge proved to be the dominant factor. Additionally, the presence of multiple oxygen atom sources was suggested by the voltage‐dependent mass shift of +16 Da to +18 Da of the “ghost peak” when using 18O‐labeled water as a solvent. Accurate metabolite identification using LC‐ESI‐MS/MS requires accounting for in‐source products that can mimic in vivo products.