Mass Spectrometry for Paper-Based Immunoassays: Toward On‑Demand Diagnosis

Current analytical methods, either point-of-care or centralized detection, are not able to meet recent demands of patient-friendly testing and increased reliability of results. Here, we describe a two-point separation on-demand diagnostic strategy based on a paper-based mass spectrometry immuno­assay platform that adopts stable and cleavable ionic probes as mass reporter; these probes make possible sensitive, interruptible, storable, and restorable on-demand detection. In addition, a new touch paper spray method was developed for on-chip, sensitive, and cost-effective analyte detection. This concept is successfully demonstrated via (i) the detection of <i>Plasmodium falciparum</i> histidine-rich protein 2 antigen and (ii) multiplexed and simultaneous detection of cancer antigen 125 and carcinoembryonic antigen

Stable-Isotope N‑Me Aziridination Enables Accurate Quantitative CC Isomeric Lipidomics

Accurate lipid quantification is essential to revealing their roles in physiological and pathological processes. However, difficulties in the structural resolution of lipid isomers hinder their further accurate quantification. To address this challenge, we developed a novel stable-isotope N-Me aziridination strategy that enables simultaneous qualification and quantification of unsaturated lipid isomers. The one-step introduction of the 1-methylaziridine structure not only serves as an activating group for the CC bond to facilitate positional identification but also as an isotopic inserter to achieve accurate relative quantification. The high performance of this reaction for the identification of unsaturated lipids was verified by large-scale resolution of the CC positions of 468 lipids in serum. More importantly, by using this bifunctional duplex labeling method, various unsaturated lipids such as fatty acids, phospholipids, glycerides, and cholesterol ester were accurately and individually quantified at the CC bond isomeric level during the mouse brain ischemia. This study provides a new approach to quantitative structural lipidomics

Stable-Isotope N‑Me Aziridination Enables Accurate Quantitative CC Isomeric Lipidomics

Accurate lipid quantification is essential to revealing their roles in physiological and pathological processes. However, difficulties in the structural resolution of lipid isomers hinder their further accurate quantification. To address this challenge, we developed a novel stable-isotope N-Me aziridination strategy that enables simultaneous qualification and quantification of unsaturated lipid isomers. The one-step introduction of the 1-methylaziridine structure not only serves as an activating group for the CC bond to facilitate positional identification but also as an isotopic inserter to achieve accurate relative quantification. The high performance of this reaction for the identification of unsaturated lipids was verified by large-scale resolution of the CC positions of 468 lipids in serum. More importantly, by using this bifunctional duplex labeling method, various unsaturated lipids such as fatty acids, phospholipids, glycerides, and cholesterol ester were accurately and individually quantified at the CC bond isomeric level during the mouse brain ischemia. This study provides a new approach to quantitative structural lipidomics

2,3,4,5-Tetrakis(3′,4′-dihydroxylphenyl)thiophene: A New Matrix for the Selective Analysis of Low Molecular Weight Amines and Direct Determination of Creatinine in Urine by MALDI-TOF MS

Small organic matrixes are still the most commonly used ones in matrix-assisted laser desorption/ionization mass spectrometry (MALDI MS) because of their advantages of high sensitivity, convenience, and cost-effectiveness. However, due to the matrix interference in the low mass region, the direct analysis of low molecular weight amines in complex surroundings with conventional organic matrixes remains a challenge. Here, a new Brønsted–Lowry acid compound 2,3,4,5-tetrakis­(3′,4′-dihydroxylphenyl)­thiophene (DHPT) was designed, synthesized, and applied as a matrix for analysis of low molecular weight amines by MALDI-TOF MS. DHPT displays good selectivity in the analysis of amines without matrix-related interference and the low picomole/femtomole limit-of-detection was obtained in positive ion mode. With DHPT, the metabolites including creatinine, glycine, alloxan, allantoin, and 3-hydroxyhippuric acid in human urine were directly analyzed by MALDI-TOF MS. The identity of these metabolites was confirmed by tandem mass spectrometry. Furthermore, the urine creatinine was quantitatively determined using isotope-labeled internal standard. This DHPT-assisted LDI MS method provides a general approach for both qualitative and quantitative analysis of low molecular weight amines

Carbon Nanodots As a Matrix for the Analysis of Low-Molecular-Weight Molecules in Both Positive- and Negative-Ion Matrix-Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry and Quantification of Glucose and Uric Acid in Real Samples

Carbon nanodots were applied for the first time as a new matrix for the analysis of low-molecular-weight compounds by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) in both positive- and negative-ion modes. A wide range of small molecules including amino acids, peptides, fatty acids, as well as β-agonists and neutral oligosaccharides were analyzed by MALDI MS with carbon nanodots as the matrix, and the lowest 0.2 fmol limits-of-detection were obtained for octadecanoic acid. Clear sodium and potassium adducts and deprotonated signals were produced in positive- and negative-ion modes. Furthermore, the glucose and uric acid in real samples were quantitatively determined by the internal standard method with the linear range of 0.5–9 mM and 0.1–1.8 mM (<i>R</i>² > 0.999), respectively. This work gives new insight into the application of carbon nanodots and provides a general approach for rapid analysis of low-molecular-weight compounds

Quantitative Assessment of Protein Adsorption on Microparticles with Particle Mass Spectrometry

In this paper, particle mass spectrometry (PMS), which consists of an aerodynamic desorption/ionization (AD) source, a quadrupole ion trap (QIT) mass analyzer, and a charge detector, was exploited to characterize the protein adsorption on microparticles based on the mass variations of microparticles before and after protein adsorption. This method is simple and has low sample cost. Importantly, its mass resolution is good enough to distinguish the microparticles with and without protein. For the adsorption of bovine serum albumin (BSA) on 3 μm porous poly styrene-divinylbenzene (poly S-DVB), the minimum mass increase that can be resolved by PMS corresponds to 128 fg (1.8 ng/cm²) or 1.17 × 10⁶ BSA molecules on each poly S-DVB particle. With PMS, the adsorption process of BSA on poly S-DVB spheres was successfully characterized, and the obtained maximum adsorption capacity <i>q</i>_m and dissociation constant <i>K</i>_d were consistent with that determined by the conventional depletion method. In addition, the influence of surface modification of silica particles on the enzyme immobilization was evaluated. Compared with C₄ (propyldimethylsilane), C₈ (octyldimethylsilane), and Ph (phenyldimethylchlorosilane), the CN (cyanoethyldimethylchlorosilane) functionalized silica particles were screened to be most beneficial for the immobilization of both lysozyme and trypsin

1,5-Diaminonaphthalene Hydrochloride Assisted Laser Desorption/Ionization Mass Spectrometry Imaging of Small Molecules in Tissues Following Focal Cerebral Ischemia

A sensitive analytical technique for visualizing small endogenous molecules simultaneously is of great significance for clearly elucidating metabolic mechanisms during pathological progression. In the present study, 1,5-naphthalenediamine (1,5-DAN) hydrochloride was prepared for matrix-assisted laser desorption/ionization (MALDI) mass spectrometry imaging (MSI) of small molecules in liver, brain, and kidneys from mice. Furthermore, 1,5-DAN hydrochloride assisted LDI MSI of small molecules in brain tissue of rats subjected to middle cerebral artery occlusion (MCAO) was carried out to investigate the altered metabolic pathways and mechanisms underlying the development of ischemic brain damage. Our results suggested that the newly prepared matrix possessed brilliant features including low cost, strong ultraviolet absorption, high salt tolerance capacity, and fewer background signals especially in the low mass range (typically <i>m</i>/<i>z</i> < 500), which permitted us to visualize the spatial distribution of a broad range of small molecule metabolites including metal ions, amino acids, carboxylic acids, nucleotide derivatives, peptide, and lipids simultaneously. Nineteen endogenous metabolites involved in metabolic networks such as ATP metabolism, tricarboxylic acid (TCA) cycle, glutamate-glutamine cycle, and malate-aspartate shuttle, together with metal ions and phospholipids as well as antioxidants underwent relatively obvious changes after 24 h of MCAO. The results were highly consistent with the data obtained by MRM MS analysis. These findings highlighted the promising potential of the organic salt matrix for application in the field of biomedical research

MALDI-TOF MS Imaging of Metabolites with a <i>N</i>‑(1-Naphthyl) Ethylenediamine Dihydrochloride Matrix and Its Application to Colorectal Cancer Liver Metastasis

Matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI MSI) is a label-free technique for identifying multiplex metabolites and determining both their distribution and relative abundance in situ. Our previous study showed that <i>N</i>-(1-naphthyl) ethylenediamine dihydrochloride (NEDC) could act as a matrix for laser desorption/ionization time-of-flight mass spectrometry (LDI-TOF MS) detection of oligosaccharides in solution. In the present study, NEDC-assisted LDI-TOF MSI yielded many more endogenous compound peaks between <i>m</i>/<i>z</i> 60 and <i>m</i>/<i>z</i> 1600 than 9-aminoacridine (9-AA). Our results show that NEDC-assisted LDI-TOF MSI is especially well-suited for examining distributions of glycerophospholipids (GPs) in addition to low molecular weight metabolites below <i>m</i>/<i>z</i> 400. Particularly, NEDC matrix allowed the LDI-TOF MSI of glucose in animal tissue. Furthermore, NEDC-assisted LDI-TOF MSI was applied to a mouse model of colorectal cancer liver metastasis. We revealed the distinct spatio-molecular signatures of many detected compounds in tumor or tumor-bearing liver, and we found that taurine, glucose, and some GPs decreased in tumor-bearing liver as the tumor developed in liver. Importantly, we also found a glucose gradient in metastatic tumor foci for the first time, which further confirms the energy competition between tumors and liver remnant due to the Warburg effect. Our results suggest that NEDC-assisted LDI MSI provides an in situ label-free analysis of multiple glycerophospholipids and low molecular weight metabolites (including glucose) with abundant peaks and high spatial resolution. This will allow future application to in situ definition of biomarkers, signaling pathways, and disease mechanisms

In Situ Bioconjugation and Ambient Surface Modification Using Reactive Charged Droplets

Molecular ions are generated in induced electrospray ionization, and they can be transported to grounded ambient surfaces in the form of charged microdroplets. Efficient amide bonds formation between amines and carboxylic acids were observed inside charged droplets during transfer to the surface. Biomolecules derivatized using this method were self-assembled on a bare gold surface via Au–S bonds under the charged microdroplet environment. Cyclic voltammetric analysis of the self-assembled molecular film showed accelerated protein derivatization with cysteine, which allowed the covalent immobilization of the protein to the gold surface. Cytochrome C-functionalized electrodes prepared using the induced dual nanoelectrospray process showed bioactivity toward aqueous solutions of hydrogen peroxide below 50 μM. In effect, we have developed a method that allows derivatization of biomolecules and their immobilization at ambient surfaces in a single experimental step