Tracking the disulfide rearrangement of heated lactoglobulin by matrix-assisted laser desorption/ionization-in-source decay top-down analysis.

Disulfide bond rearrangement is a common occurrence during protein analysis or treatment. A convenient and rapid method has been developed to investigate heat-induced disulfide rearrangement of lactoglobulin using matrix-assisted laser desorption/ionization-in-source decay (MALDI-ISD) technology. By analyzing heated lactoglobulin in reflectron and linear mode, we demonstrated that cysteines C66 and C160 exist as free residues other than linked ones in some protein isomers. This method provides a straightforward and expeditious way to assess the cysteine status and structural changes of proteins under heat stress.

Fully synthetic phosphorylated Tau181, Tau217, and Tau231 calibrators for Alzheimer's disease diagnosis.

Background: The calibrator in immunoassay plays an essential role in diagnosing Alzheimer's disease (AD). Presently, the most well-studied biomarkers for AD diagnosis are three phosphorylated Tau (p-Tau): p-Tau231, p-Tau217, and p-Tau181. Glycogen synthase-3beta (GSK3ß)-phosphorated Tau-441 is the most commonly used calibrator for p-Tau immunoassays. However, the batch-to-batch inconsistency issue of the commonly used GSK3ß-phosphorylated Tau-441 limits its clinical application. Methods: We have successfully generated and characterized 61 Tau monoclonal antibodies (mAbs) with distinct epitopes by using the hybridoma technique and employed them as capture or detection antibodies for p-Tau immunoassays. Through chemical synthesis, we synthesized calibrators, which are three peptides including capture and detection antibody epitopes, for application in immunoassays that detect p-Tau231, p-Tau217, and p-Tau181. The novel calibrators were applied to Enzyme-linked immunosorbent assay (ELISA) and Single-molecule array (Simoa) platforms to validate their applicability and establish a range of p-Tau immunoassays. Results: By employing the hybridoma technique, 49 mAbs recognizing Tau (1-22), nine mAbs targeting p-Tau231, one mAb targeting p-Tau217, and two mAbs targeting p-Tau181 were developed. Peptides, including recognition epitopes of capture and detection antibodies, were synthesized. These peptides were used as calibrators to develop 60 immunoassays on the ELISA platform, of which six highly sensitive immunoassays were selected and applied to the ultra-sensitive Simoa platform. Remarkably, the LODs were 2.5, 2.4, 31.1, 32.9, 46.9, and 52.1 pg/ml, respectively. Conclusion: Three novel p-Tau calibrators were successfully generated and validated, which solved the batch-to-batch inconsistency issue of GSK3ß-phosphorylated Tau-441. The novel calibrators exhibit the potential to promote the standardization of clinical AD diagnostic calibrators. Furthermore, we established a series of highly sensitive and specific immunoassays on the Simoa platform based on novel calibrators, which moved a steady step forward in p-Tau immunoassay application for AD diagnosis.

Evaluation of the reliability of MS1-based approach to profile naturally occurring peptides with clinical relevance in urine samples.

RATIONALE: The profiling of natural urinary peptides is a valuable indicator of kidney condition. While front-end separation limits the speed of peptidomic profiling, MS1-based results suffer from limited peptide coverage and specificity. Clinical studies on chronic kidney disease require an effective strategy to balance the trade-off between identification depth and throughput. METHODS: CKD273, a urinary proteome classifier associated with chronic kidney disease, in samples from diabetic nephropathy patients was profiled in parallel using capillary electrophoresis-mass spectrometry (CE-MS), liquid chromatography with mass spectrometry (LC-MS), and matrix-assisted laser desorption/ionization-mass spectrometry (MALDI-MS). Through cross-comparison of results from MS1 of unfractionated peptides and elution-time-resolved MS1 as well as MS/MS in LC- and CE-MS approaches, we evaluated the contribution of false-positive identification to MS1-based identification and quantitation, and analyzed the benefit of front-end separation in terms of accuracy and efficiency. RESULTS: In LC- and CE-MS, although MS1 data resulted in higher number of identifications than MS/MS, elution-time-dependent analysis revealed extensive interference by non-CKD273 peptides, which would contribute up to 50% to quantitation if they are not separated from genuine CKD273 peptides. In the absence of separation, MS1 data resulted in lower numbers of identifications and abundance pattern that significantly deviated from those by liquid chromatography with tandem mass spectrometry (LC-MS/MS) or capillary electrophoresis with tandem mass spectrometry (CE-MS/MS). CE showed higher identification efficiency even when less sample was used or achieved faster separation. CONCLUSIONS: To ensure the reliability of MS1-based urinary peptide profiling, front-end separation should not be omitted, and elution time should be used in addition to intact mass for identification. Including MS/MS in data acquisition does not compromise the speed or identification number, while benefiting data reliability by providing real-time sequence verification.

Point-of-Care Test Paper for Exhaled Breath Aldehyde Analysis via Mass Spectrometry.

Volatile organic compounds (VOCs) from exhaled breath (EB) are considered to be promising biomarkers for lung diseases. A convenient and sensitive point-of-care (POC) testing method for EB VOCs is essential. Here, we developed a POC test paper for the analysis of EB aldehydes, which are potential biomarkers for lung cancer. A probe molecule, 4-aminothiophenol (4-ATP), was anchored on a paper substrate to specifically capture gas-phase aldehydes through the Schiff base reaction. Meanwhile, thin-film reaction acceleration was utilized to increase capture efficiency. By directly coupling the test paper to a mass spectrometer through paper spray, high sensitivity (0.1 ppt) and a wide quantification linear range (from 10 ppt to 1 ppm) were obtained. Analysis of EB from lung cancer patients with the test paper showed a significant increase in several reported aldehyde markers compared to EB from healthy volunteers, indicating the potential of this method for sensitive, low-cost, and convenient lung cancer screening and diagnosis.

MALDI-TOF/TOF tandem mass spectrometry imaging reveals non-uniform distribution of disaccharide isomers in plant tissues.

Mass spectrometry imaging (MSI) is a powerful technique for investigating the biomolecular locations within tissues. However, the isomeric compounds are rarely distinguished due to inability of MSI to differentiate isomers in the probing area. Coupling tandem mass spectrometry with MSI can facilitate differentiating isomeric compounds. Here MALDI-TOF/TOF tandem mass spectrometry imaging approach was applied to probing the spatial distributions of isomeric disaccharides in plant tissues. First, MS/MS imaging analysis of disaccharide-matrix droplet spots demonstrated the feasibility of distinguishing isomeric species in tissues, by measuring the relative intensity of specific fragments. Then, tandem MS imaging of disaccharides in onion bulb tissues indicated that sucrose and other unknown non-sucrose disaccharides exhibit heterogeneous locations throughout the tissues. This method enables us to image disaccharide isomers differentially in biological tissues, and to discover new saccharide species in plant. This work also emphasizes the necessity of considering isobaric compounds when interpreting MSI results.

MALDI-TOF mass spectrometry-based quantification of C-peptide in diabetes patients.

BACKGROUND: Serum C-peptide concentrations reflect insulin secretion and beta cell function and can be used to diagnose and distinguish type-1 and type-2 diabetes. C-peptide is a more accurate indicator of insulin status than direct insulin measurement for monitoring patients with diabetes. However, the current methods available for C-peptide quantification exhibit poor reproducibility, are costly, and require highly trained laboratory personnel. Here, we have developed and evaluated a matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS)-based assay to standardize C-peptide measurements, providing highly accurate and comparable results across testing systems and laboratories. METHODS: C-peptide from human serum was enriched using antibody-conjugated magnetic beads. The eluted isolates were further modified with 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate (AQC) to enhance the ionization of naturally acidic C-peptide. After desalting with ZipTips, the samples were subjected to MALDI-TOF MS analysis. Recombinant human C-peptide was used to develop the assay, and a heavy isotope labeled human C-peptide was used as an internal standard for quantification. RESULTS: The MALDI-TOF MS method was validated in accordance with the restrictions of the device, with a limit of quantitation of 25 pmol/L. A correlation between the MAL-DI-TOF MS assay and a reference method was conducted using patient samples. The resulting regression revealed good agreement. CONCLUSIONS: A simple, high-throughput, cost effective and quantitative MALDI-TOF MS C-peptide assay has been successfully developed and validated in clinical serum samples.

Mass, Size, and Density Measurements of Microparticles in a Quadrupole Ion Trap.

The physical properties of microparticles, such as mass, size, and density, are critical for their functions. The comprehensive characterization of these physical parameters, however, remains a fundamental challenge. Here, we developed a particle mass spectrometry (PMS) methodology for determining the mass, size, and density of microparticles simultaneously. The collisional cross-section (CCS) and mass spectrometry (MS) measurements were performed in a single quadrupole ion trap (QIT), and the two modes can be switched easily by tuning the electric and gas hydrodynamic fields of the QIT. The feasibility of the method was demonstrated through a series of monodispersed polystyrene (PS) and silica (SiO2) particle standards. The SiO2/polypyrrole core-shell particles were also successfully characterized, and the measured results were verified by using conventional methods.

A Miniature Particle Mass Spectrometer.

Microparticles play important roles in our life. Besides chemical compositions and morphology, the size of microparticles will also decide their behavior in the environment or in organisms. Weighing the mass of microparticles by mass spectrometry is a useful method to characterize their size. In this technical note, a miniature particle mass spectrometer with an aerodynamic desorption/ionization ion source has been developed. We used a compact main control board to produce an ac voltage for trapping and ejecting the particles. The sampling process and data acquisition were also controlled by this board. We utilized this instrument to measure polystyrene spheres, silica particles, and mice red blood cells. Mass distributions of these particles were obtained rapidly with good accuracy.

Linkage and sequence analysis of neutral oligosaccharides by negative-ion MALDI tandem mass spectrometry with laser-induced dissociation.

Mass spectrometry (MS) has become the primary method for high-sensitivity structural determination of oligosaccharides. Fragmentation in the negative-ion MS can provide a wealth of structural information and these can be used for sequence determination. However, although negative-ion MS of neutral oligosaccharide using the deprotonated molecule [M-H]- as the precursor has been very successful for electrospray ionization (ESI), it has only limited success for matrix-assisted laser desorption/ionization (MALDI). In the present study, the features of negative-ion MALDI primary spectra were investigated in detail and the product-ion spectra using [M-H]- and [M+Cl]- as the precursors were carefully compared. The formation of [M-H]- was the main difficulty for MALDI while [M+Cl]- was proved to be useful as alternative precursor anion for MALDI-MS/MS to produce similar fragmentation for sequencing of neutral oligosaccharides. N-(1-naphthyl)ethylenediamine dihydrochloride was then used as both the matrix and the Cl- dopant to evaluate the extent of structural information that can be obtained by negative-ion fragmentation from [M+Cl]- using laser-induced dissociation (LID)-MS/MS for linkage assignment of gluco-oligosaccharides and for typing of blood-group ABO(H) and Lewis antigens on either type 1 or type 2 backbone-chains.

Enhancing surface-assisted laser desorption ionization mass spectrometry performance by integrating plasmonic hot-electron transfer effect through surface modification.

Surface-assisted laser desorption ionization mass spectrometry (SALDI MS) performances were enhanced by modifying surfaces to include a plasmonic hot-electron transfer effect. After modification, the core material's specificity was not only maintained but also enhanced. The MS imaging of ischemic tissues with the modified material indicated some sulfatide changes, which have been barely reported.

Mass spectrometry imaging of the in situ drug release from nanocarriers.

It is crucial but of a great challenge to study in vivo and in situ drug release of nanocarriers when developing a nanomaterial-based drug delivery platform. We developed a new label-free laser desorption/ionization mass spectrometry (MS) imaging strategy that enabled visualization and quantification of the in situ drug release in tissues by monitoring intrinsic MS signal intensity ratio of loaded drug over the nanocarriers. The proof of concept was demonstrated by investigating the doxorubicin (DOX)/polyethylene glycol-MoS2 nanosheets drug delivery system in tumor mouse models. The results revealed a tissue-dependent release behavior of DOX during circulation with the highest dissociation in tumor and lowest dissociation in liver tissues. The drug-loaded MoS2 nanocarriers are predominantly distributed in lung, spleen, and liver tissues, whereas the accumulation in the tumor was unexpectedly lower than in normal tissues. This new strategy could also be extended to other drug-carrier systems, such as carbon nanotubes and black phosphorus nanosheets, and opened a new path to evaluate the drug release of nanocarriers in the suborgan level.

Hot electron transfer promotes ion production in plasmonic metal nanostructure assisted laser desorption ionization mass spectrometry.

Plasmonic metal nanostructures have been used as effective materials for enhancing the laser desorption ionization (LDI) efficiency in mass spectrometry analysis. But the ionization mechanism and the origin of initial charges have not been fully understood yet. Here we provided experimental evidence showing that the hot electron transfer in localized surface plasmon resonance (LSPR) plays a key role in ionizing molecules during the LDI process.

A novel mass spectrometry method based on competitive non-covalent interaction for the detection of biomarkers.

We report a novel biosensor platform based on competitive non-covalent interaction between ssDNA and a mass tag towards AuNPs, which detects PSA biomarkers sensitively, observed using MALDI MS. A detection limit of 57 pg mL-1 has been achieved, showing an improvement of two orders of magnitude compared to the traditional spectroscopic method.

Heat-Induced Rearrangement of the Disulfide Bond of Lactoglobulin Characterized by Multiply Charged MALDI-TOF/TOF Mass Spectrometry.

Disulfide bonds are an important post-translational modification of proteins and play a significant role in stabilizing protein structure. While both mass spectrometry-based bottom-up and top-down proteomics are widely used in the identification of disulfide linkages, the top-down approach can avoid potential information loss of disulfide linkage occurring in the bottom-up analysis. In the present work, we applied matrix-assisted laser desorption/ionization tandem Time-of-Flight (MALDI-TOF/TOF) mass spectrometry to investigate the heat-induced disulfide rearrangement of ß-lactoglobulin (ß-LG). Since ß-LG (18 kDa) is too large for common TOF/TOF analysis, we use 2-nitrophloroglucinol (2-NPG) as a matrix to generate multiply charged proteins by MALDI. Fragmentation of doubly charged protein ions yields characteristic triplet peaks of disulfide bonds. We found that the characteristic fragments derived from the heterolytic cleavage of disulfide bonds decreased sharply when the incubation temperature of ß-LG solution reached the critical point of 75 °C. These results indicate that the disulfide linkage between C160 and C66 has been broken during the heating process and, probably, new disulfide formed. In conclusion, our work highlights the analytical value of the multiply charged MALDI-TOF/TOF method in the identification of larger proteins (>12 kDa) and disulfide-containing proteins.

Utilizing a Mini-Humidifier To Deposit Matrix for MALDI Imaging.

MALDI mass spectrometry imaging (MALDI-MSI) is a powerful tool to study endogenous metabolites. The process of matrix deposition is crucial for a high-quality imaging result. Commercial instruments for matrix deposition are expensive. Low-cost methods like airbrushing will generate matrix crystals that are too large for high-spatial-resolution imaging. Sublimation may cause some compounds to go undetected because of the lack of solvent. Herein, we utilized a mini-humidifier, costing less than 5 dollars, to deposit matrix for MALDI-MSI. Compared with Imageprep, a commercialized instrument, our device based on the humidifier provided higher sensitivity and much smaller matrix crystals with diameters of less than 10 µm. High-quality ion images with 10 µm spatial resolution were obtained using our method. The enhancement of sensitivity by the humidifier could provide a sufficient amount of ions to perform tandem mass imaging. We also performed MALDI-MS/MS imaging to separate two lipids in mouse brain.

Laser Cleavable Probes-Based Cell Surface Engineering for in Situ Sialoglycoconjugates Profiling by Laser Desorption/Ionization Mass Spectrometry.

Cell-surface sialoglycoconjugates (sialoglycoproteins and sialoglycolipids) play important roles in cell-cell interactions and related tumor metastasis process. Although there have been some analytical methods to evaluate the sialoglycoconjugates, an effective method providing both qualitative and quantitative information is still deficient. Here we establish an extraction-free, sensitive, and high-throughput platform to realize in situ detection of the cell-surface sialoglycoconjugates on various cell lines, e.g., cancer and normal cells by laser desorption/ionization mass spectrometry (LDI MS). In this proposal, azide groups were introduced into the ends of cell-surface sialoglycoconjugates by the biorthogonal method, and then the sialoglycoconjugates were armed with a laser-cleavable probe (Tphsene) through click chemistry. We can easily get the probes signal under laser irradiation, which reflected the presence of cell-surface sialoglycoconjugates. Different cell lines were discriminated simultaneously, and the LDI relative quantification agreed with fluorescent results. Besides, a linear quantitation relationship in the range of 100 fmol to 100 pmol was obtained with a designed and synthesized internal standard (phTsane) added. A detection limit of 5 fmol was obtained with good reproducibility. Based on the quantitative and high-throughput ability, we conducted pharmacodynamics study of drug (tunicamycin) on cancer cells. In addition, we found the tag was safe from sweet-spot effect of matrix adding. The simultaneous detection of sialoglycoconjugates and metabolites was therefore achieved. We believe that this laser cleavable probes-based cell-surface engineering for sialoglycoconjugates platform means great significance to diagnosis, prognosis, and therapeutic purposes. Besides, this strategy can be applied to other glycoconjugates which is hard to detect and the related disease processes when more corresponding chemically modified sugar substrates and exact biorthogonal reactions are developed.

Mixing O-Containing and N-Containing Directing Groups for C-H Activation: A Strategy for the Synthesis of Highly Functionalized 2,2'-Biaryls.

A strategy combining O- and N-containing directing groups has been developed for the synthesis of 2,2'-biaryl via Pd-mediated C-H bond activation and oxidative coupling. This new transformation may proceed through a mechanism involving Pd(II) and Pd(IV) intermediates. We found the use of PTSA and HFIP to be critical for the reaction and suggest that these reagents could serve as efficient ligands for this C-C bond formation. This methodology provides broad functional group tolerance, excellent reactivity, and high yields.

Differentiation and Relative Quantitation of Disaccharide Isomers by MALDI-TOF/TOF Mass Spectrometry.

Saccharide isomer differentiation has been a challenge in glycomics, as the lack of technology to decipher fully the diverse structures of compositions, linkages, and anomeric configurations. Several mass spectrometry-based methods have been applied to the discrimination of disaccharide isomers, but limited quantitative analyses have been reported. In the present study, MALDI-LIFT-TOF/TOF has been investigated to differentiate and relatively quantify underivatized glucose-containing disaccharide isomers that differ in composition, connectivity or configuration. N-(1-naphthyl)ethylenediamine dihydrochloride (NEDC) was used as a highly sensitive matrix without matrix interferences in low mass range, thus yielding intense chloride-attached disaccharide ions [M + Cl]-, which could be fragmented to give diagnostic characteristic fragment patterns for distinguishing these isomers. Three different types of disaccharide isomers were successfully relatively quantified in a binary mixture using the specific product ion pairs. Finally, this method was utilized to identify and relatively quantify two disaccharide isomers in Medicago leaf (maltose and sucrose) without numerous preparation steps. In general, this method is a fast, effective, and robust method for rapid differentiation and quantitation of disaccharide isomers in complex medium.

Improving the Performance of the Mini 2000 Mass Spectrometer with a Triboelectric Nanogenerator Electrospray Ionization Source.

Balancing the contradiction between portability and analytical performances of a miniaturized mass spectrometer is vital to extend its on-site applications. In this study, triboelectric nanogenerator (TENG)-driven ion sources were coupled with our home-built Mini 2000 system and applied to the analyses of different samples. Compared with the conventional direct current (DC) nanoelectrospray ionization (nanoESI) source, the ion intensity of the TENG-nanoESI miniature mass spectrometer was improved by ∼3 times. Moreover, maybe due to the different pathways of ion formation in comparison with DC electrospray, TENG electrospray is shown to reduce the salt suppression effect during ionization. With these figures of merit, the direct detection of reserpine in saliva was demonstrated using the TENG-Mini 2000 system.

N-Phenyl-2-naphthylamine as a Novel MALDI Matrix for Analysis and in Situ Imaging of Small Molecules.

Due to its strong ultraviolet absorption, low background interference in the small molecular range, and salt tolerance capacity, N-phenyl-2-naphthylamine (PNA) was developed as a novel matrix in the present study for analysis and imaging of small molecules by matrix-assisted laser desorption/ionization mass spectrometry time-of-fight (MALDI-TOF MS). The newly developed matrix displayed good performance in analysis of a wide range of small-molecule metabolites including free fatty acids, amino acids, peptides, antioxidants, and phospholipids. In addition, PNA-assisted LDI MS imaging of small molecules in brain tissue of rats subjected to middle cerebral artery occlusion (MCAO) revealed unique distributions and changes of 89 small-molecule metabolites including amino acids, antioxidants, free fatty acids, phospholipids, and sphingolipids in brain tissue 24 h postsurgery. Fifty-nine of the altered metabolites were identified, and all the changed metabolites were subject to relative quantitation and statistical analysis. The newly developed matrix has great potential application in the field of biomedical research.