Design of a dual Ir-Eu tag for fluorescent visualization and ICP-MS quantification of SIRPα and its host cells.

With the expansion of ICP-MS application into the field of bioanalysis, there is an urgent need for novel element tags today. Here, we report the design of a dual-element Ir-Eu tag, opening the door to simultaneous fluorescent imaging and ICP-MS quantification. The ratio of 153Eu/193Ir may serve as a precision control of the labeling process, allowing internal validation of the quantitative results obtained. As for SIRPα and its host cell analysis exemplified here, the Ir-Eu tag demonstrated superior figures of ICP-MS quantification with the LOD (3σ) down to 0.5 (153Eu) and 1.1 (193Ir) pM SIRPα and 220 (153Eu) and 830 (193Ir) RAW264.7 cells more than 130 times more sensitive compared with the LOD (3σ) of 65.2 pM SIRPα at 612 nm using fluorometry. Not limited to these demonstrations, we believe that the design ideas of the dual Ir-Eu tags should be applicable to various cases of bioanalysis when dual optical profiling and ICP-MS quantification are indispensable.

Two SOX11 variants cause Coffin-Siris syndrome with a new feature of sensorineural hearing loss.

Coffin-Siris syndrome (CSS, OMIM#135900) is a rare congenital disorder associated with neurodevelopmental and dysmorphic features. The primary cause of CSS is pathogenic variants in any of 9 BAF chromatin-remodeling complex encoding genes or the genes SOX11 and PHF6. Herein, we performed whole-exome sequencing (WES) and a series of analyses of growth-related, auditory, and radiological findings in two probands with syndromic sensorineural hearing loss and inner ear malformations who exhibited distinctive facial features, intellectual disability, growth retardation, and fifth finger malformation. Two de novo variants in the SOX11 gene (c.148A>C:p.Lys50Asn; c.811_814del:p.Asn271Serfs*10) were detected in these probands and were identified as pathogenic variants as per ACMG guidelines. These probands were diagnosed as having CSS based upon clinical and genetic findings. This is the first report of CSS caused by variants in SOX11 gene in Chinese individuals. Deleterious SOX11 variants can result in sensorineural hearing loss with inner ear malformation, potentially extending the array of phenotypes associated with these pathogenic variants. We suggest that both genetic and clinical findings be considered when diagnosing syndromic hearing loss.

Zero-Interfacing µHPLC to ICPMS.

The chromatography-mass spectrometry hyphenated technique is the most widely adopted tool for quantifying trace analytes in a complex biosample. One issue we frequently encountered, however, is that the separated analyte-containing chromatographic peaks broaden and even remix prior to mass spectrometric quantification due to the inevitable molecular diffusion within the dead-volume introduced by hyphenation. We developed a zero-interfacing approach for coupling microbore (µ) HPLC with inductively coupled plasma mass spectrometry (ICPMS). Zero-interfacing µHPLC to ICPMS has been achieved by a column-nebulizer assembly (COL-NEB) of a self-designed glass framework with a tapered nozzle, in which a capillary chromatographic column can be harbored while an Ar gas flow is blown through the nozzle mouth. The COL-NEB can be positioned just before the base of the Ar-ICP serving as the central sampling channel of a conventional Ar-ICP torch for online nebulization and transportation of the analytes separated on µHPLC into ICPMS, maintaining the molecular resolution obtained on µHPLC and the limit of detection (LOD) of ICPMS. For example, the full width at half-maximum of a SLUGT peptide chromatographic peak was reduced to 1.71 ± 0.07 s (n = 5) with a 0.72 fg LOD (3σ) of 80Se. Moreover, at least 32 Se-containing peptides were determined in the trypsin lysate of the water-soluble fraction (≥3000 MW) from Se-enriched yeast CRM SELM-1 within a 10 min run, the highest record to date. We believe such an approach paves the way to determining accurate information on a heteroatom and its binding biomolecules that play key roles during life processes.

Quantification of active selenols in cells: a selenol-specific recognition europium-switched signal-amplification ICP-MS approach.

Selenium (Se) is a mysterious thus tempting element playing a dual bio-chemical function, mainly through selenol, during life processes. Quantification of the selenols is thus of great significance for understanding the biological roles of Se, but remains a big challenge. Herein we report a selenol-specific recognition-mediated and europium (Eu) signal-switched amplification inductively coupled plasma mass spectrometry (ICP-MS) approach for quantifying the free active selenols (act-SeH) in cells. A bifunctional molecule, 2,4-dinitrobenzenesulfonyl-piperidin-4-yl-1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic europium (DNBS-DOTA-Eu), was designed and synthesized for the specific recognition and highly sensitive quantification of act-SeH via switching Se to more sensitive Eu ICP-MS signals. The limit of detection (LOD, 3σ) of 3.41 pg/mL (22.43 pmol/L), corresponding to the absolute mass LOD of 6.82 ag act-SeH per cell, is almost 25 times lower than 83.76 pg/mL (1.06 nmol/L), 167.52 ag, when monitoring 80Se. The results indicate that act-SeH in the selenite-precultured cancerous HepG2 and paracancerous HL7702 cells are 0.090 ± 0.002 pg/cell (n = 7) and 0.021 ± 0.006 pg/cell (n = 7), more than 4.28 times higher in HepG2 than in HL7702. Preliminary application of this approach to the cells from real hepatic tissue samples suggested that act-SeH has a positive relationship with the degree of hepatic disease. act-SeH in cells appears to be a very promising relevant index for understanding the biochemical functions of Se, besides the total Se in cells and blood serum and/or plasma.

Multivalent Duplexed-Aptamer Networks Regulated a CRISPR-Cas12a System for Circulating Tumor Cell Detection.

Although circulating tumor cells (CTCs) have great potential to act as the mini-invasive liquid biopsy cancer biomarker, a rapid and sensitive CTC detection method remains lacking. CRISPR-Cas12a has recently emerged as a promising tool in biosensing applications with the characteristic of fast detection, easy operation, and high sensitivity. Herein, we reported a CRISPR-Cas12a-based CTC detection sensor that is regulated by the multivalent duplexed-aptamer networks (MDANs). MDANs were synthesized on a magnetic bead surface by rolling circle amplification (RCA), which contain multiple duplexed-aptamer units that allow structure switching induced by cell-binding events. The presence of target cells can trigger the release of free "activator DNA" from the MDANs structure to activate the downstream CRISPR-Cas12a for signal amplification. Furthermore, the 3D DNA network formed by RCA products also provided significantly higher sensitivity than the monovalent aptamer. As a proof-of-concept study, we chose the most widely used sgc8 aptamer that specifically recognizes CCRF-CEM cells to validate the proposed approach. The MDANs-Cas12a system could afford a simple and fast CTC detection workflow with a detection limit of 26 cells mL-1. We also demonstrated that the MDANs-Cas12a could directly detect the CTCs in human blood samples, indicating a great potential of the MDANs-Cas12a in clinical CTC-based liquid biopsy.

DNAzyme-Au nanoprobe coupled with graphene-oxide-loaded hybridization chain reaction signal amplification for fluorometric determination of alkaline phosphatase.

A sensing platform is presented for the determination of alkaline phosphatase (ALP) activity based on the cooperation of DNAzyme-Au spherical nucleic acid nanoprobe with the graphene-oxide-loaded hybridization chain reaction (HCR/GO) system to achieve good detection sensitivity and specificity. This assay takes advantage of the strong affinity of pyrophosphate (PPi) to Cu2+ ions and the fact that ALP can hydrolyze pyrophosphate (PPi) to release free Cu2+ ions. In the presence of ALP, the released Cu2+ can promote the Cu2+-dependent DNAzyme to cleave the substrate that generates a shorter DNA fragment, which is responsible for further triggering the HCR/GO system to form a long fluorescence dsDNA and thereby giving an amplified fluorescence signal. Linear calibration range was obtained from 0.2 to 10 U L-1, and the limit of detection (LOD) is about 0.14 U L-1. The feasibility of the proposed method was validated by spiking ALP standards in bovine serum. The recovery ranged from 97.2 to 104.6%, and a coefficient of variation (CV) of less than 8% (n = 3) was obtained. This assay strategy was also applied to evaluate the ALP inhibitor efficiency, which indicates that the assay has potential for drug screening.

Sequential Bilateral Cochlear Implantation in a Child with Severe External, Middle, and Inner Ear Malformations: Surgical Considerations and Practical Aspects.

Cochlear implantation (CI) is a safe and beneficial surgery for children with congenital inner ear malformations, with the exception of cochlear nerve aplasia. The combination of microtia with middle and inner ear abnormalities is extremely uncommon and sufficiently severe to make a surgical approach to the cochlea difficult. We report herein the case of a 2-year-old girl who presented with profound bilateral sensorineural hearing loss, congenital aural atresia, microtia, and inner ear malformations. High-resolution computed tomography revealed poor development of the bilateral middle ear spaces, absence of the incus and stapes, aberrant courses of facial nerves, aplastic lateral semicircular canals, and covered round windows. With intraoperative imaging assistance, sequential bilateral CI was performed using a transmastoid approach with no complication. We propose that CI is feasible in patients with severe external and middle ear malformations. However, major malformations increase the risk of complications. As the facial nerve and cochlea are difficult to locate due to the lack of important anatomical landmarks, detailed planning and adequate preparation, including review of the preoperative imaging data, and the use of facial nerve monitoring and intraoperative imaging are very important. In addition, experienced surgeons should perform CI to ensure the success of the operation.

A Biochemical Lanthanide-Encoding Approach Enables Quantitative Monitoring of the Bacterial Response to Vancomycin Treatment.

A pathogenic bacterium has its own mechanisms for not only pathogenic attack but also exogenous invasion defense, in which the bacterial cell wall is the front line of attack and defense. We developed a biochemical lanthanide-encoding approach to quantify the uncanonical d-amino acid (d-X) that was edited in a small proportion into the terminal acyl-d-Ala-d-X of nascent peptidoglycan UDP-MurNAc-pentapeptides in the bacterial cell wall. This approach overcomes the difficulties regarding quantification and accuracy issues encountered by the popular optical imaging and traditional high-performance liquid chromatography-based methods. Newly synthesized azide-d-Leu and ketone-d-Met were used together with alkynyl-d-Ala for their metabolic assembly and then bioorthogonally encoded by the correspondingly fabricated DBCO-DOTA-Gd, H2NO-DOTA-Eu, and azide-DOTA-Sm tags. This approach allows direct quantification of the d-X in situ in the cell wall using 158Gd, 153Eu, and 154Sm species-unspecific isotope dilution inductively coupled plasma mass spectrometry, avoiding any tedious and complex "cell-broken" pretreatment procedures that might induce racemization of the d-X. The obtained site-specific and accurate in situ information about the d-X enables quantitative monitoring of the bacterial response when Staphylococcus aureus meets vancomycin, showing that the amounts of azide-d-Leu and ketone-d-Met assembled are more important after determining the structure- and composition-dependent bacterial antibiotic resistance mechanisms. In addition, we found that the combined use of vancomycin and d-Ala restores the efficacy of vancomycin and might be a wise and simple way to combat vancomycin intermediate-resistant S. aureus.

Orderly MOF-Assembled Hybrid Monolithic Stationary Phases for Nano-Flow HPLC.

We report an approach that polymerizable handle-modified nanosized metal organic frameworks (MOFs) are used as independent monomers to be covalently organized by crosslinking molecules (CLMs) into an orderly MOF-assembled hybrid monolithic stationary phase, overcoming the respective problems of previously reported MOF-mixed or embedded stationary phases so far. It has a hierarchical micro-, meso-, and macropore structure throughout the monolithic matrix that is donated from MOF themselves, formed via CLM crosslinking in-between MOFs and expended by porogenic solvents, and a tunable surface chemistry derived inherently from MOFs, regulated by CLMs and initiated by the mobile phases as well. Such a pore structure and surface chemistry display multiplex interactions of sieving and electrostatic repulsion in addition to the polarity-based interactions that synergistically govern the partitioning way and degree of target molecules between the stationary and mobile phases, thus offering the ability to simultaneously separate small and large molecules during one chromatographic run on a nano-flow capillary high-performance liquid chromatography platform. A baseline mutual separation with the HETP and Rs of, for example, 9.2 µm butylbenzene and 4.56 (butylbenzene and pentylbenzene), 7.9 µm (phenylalanine) and 3.50 (tryptophan and phenylalanine), and 7.0 µm (myoglobin) and 1.91 (bovine serum albumin and myoglobin) was achieved when UiO-66/NH-methacrylate was exemplified as a model of MOFs and 1,6-hexanediol dimethacrylate and stearyl methacrylate together as CLMs. Not limited to the MOFs and CLMs demonstrated here, other available MOFs and CLMs or newly designed and synthesized ones are expected to be used for constructing one's own desired monolithic stationary phases toward her/his particular purposes.

Direct Infusion ICP-qMS of Lined-up Single-Cell Using an Oil-Free Passive Microfluidic System.

When coupled online with mass spectrometry (MS), widely applied water-in-oil droplet-based microfluidics for single cell analysis met problems. For example, the oil phase rumpled the stability, efficiency, and accuracy of MS, the conventional interface between MS and the microfluidic chip suffered the low sample introduction efficiency, and the transportation rates sometimes unmatched the readout dwell times for transient signal acquisition. Considering cells are already "droplets" with hydrophilic surface and elastic hydrophobic membrane, we developed an oil-free passive microfluidic system (OFPMS) that consists of alternating straight-curved-straight microchannels and a direct infusion (dI) micronebulizer for inductively coupled plasma quadrupole-based mass spectrometry (ICP-qMS) of lined-up single-cell. OFPMS guarantees exact single cell isolation one by one just using a thermo-decomposable NH4HCO3 buffer, eliminating the use of any oil and incompatible polymer carriers. It is more flexible and facile to adapt to the dwell time of ICP-qMS owing to the adjustable throughput of 400 to 25000 cells/min and the controllable interval time of at least 20 ms between the lined-up adjacent single cells. Quantitative single-cell transportation and high detection efficiency of more than 70% was realized using OFPMS-dI-ICP-qMS exemplified here. Thus, cell-to-cell heterogeneity can be simply uncovered via the determination of metals in the individual cells.

Viruslike Element-Tagged Nanoparticle Inductively Coupled Plasma Mass Spectrometry Signal Multiplier: Membrane Biomarker Mediated Cell Counting.

Although rare cancerous cells are considered as more objective indications for a precise early diagnosis of cancers, accurate counting of them still is a spirited challenge. We reported a signal multiplication strategy by constructing element-tagged viruslike nanoparticles (VLNPs) with a precise number of atoms for a membrane biomarker mediated higher sensitive cell counting using inductively coupled plasma mass spectrometry (ICPMS). Typical bacteriophage MS2 was exemplified to demonstrate the effectiveness of the element-tagged VLNPs as signal multipliers. Dibenzylcyclooctyne-poly(ethylene glycol)-folate (DBCO-PEG-FA) and DOTA-Eu complex tag modified (FA-PEG)69-MS2-(DOTA-Eu)965 targeted the folate receptor (FR) on KB cells as low as subzeptomole FRs could be quantified by 153Eu-species unspecific isotope dilution ICPMS, allowing us to be able to count at least 5 KB cells. While more than 2197 KB cells were needed to give a significant ICPMS signal using FA-PEG-DOTA-Eu, demonstrating more than 2 orders of magnitude signal multiplication and resulting in total 4.0 × 108 times signal amplification relative to one KB cell. We believe that such a signal multiplication strategy can be expanded to quantify and count other membrane biomarkers and their host cells using various VLNPs modified with different kinds and precise numbers of elements and guiding groups. In this way, prescribed multiples of signal amplification can be realized for a more accurate ICPMS-based quantitative bioanalysis because targeted molecules/cells in a complicated biological system might exist in orders of magnitude wide concentration range.

Inhibitory Covalent Labeling and Clickable-Eu-Tagging-Based ICPMS: Measurement of pH-Dependent Absolute Activities of the Cathepsins in Hepatocyte Lysosomes.

We report an inhibitory covalent labeling and clickable-element-tagging strategy for measuring the absolute activity of a protease in cells using inductively coupled plasma mass spectrometry (ICPMS). Epoxysuccinyl-leucine-tyrosine-6-aminocaproic-lysine-amino-Boc-alkyne (epoxysuccinyl-LYK-alkyne) was designed and synthesized to achieve irreversibly labeling of the cysteine cathepsins, recording their momentary activities. L and Y assisted epoxysuccinyl-LYK-alkyne in accessing the deprotonated -S- of Cys25, located at the bottom of the long cathepsin active domain. Quantitative Eu-tagging was followed using azido-DOTA-Eu through a bioorthogonal 1:1 copper-catalyzed azide-alkyne-cycloaddition click reaction. The Eu tag could be absolutely quantified using 153Eu-species-nonspecific-isotope-dilution ICPMS coupled with HPLC, serving as a Eu ruler and allowing us to simultaneously measure the pH-dependent activities of cathepsins B, L, and S as well as the pH in the lysosomal microenvironment of liver cancerous C7721 and paracancerous C7701 cells. As long as suitable labeling molecules and elemental tags are designed and synthesized, we believe that such a tandem labeling and tagging ICPMS approach can be applied to the measurement of the activities of other proteases in cells, providing more accurate information on the proteases' biofunctions and thus implementing precise clinical diagnoses.

Counting and Recognizing Single Bacterial Cells by a Lanthanide-Encoding Inductively Coupled Plasma Mass Spectrometric Approach.

Counting and recognizing single bacterial cells are crucial to the diagnosis of bacterium-induced disease and study of cell-to-cell variability as well as the related antibiotic resistance mechanism. A higher sensitive and selective method has always been desired for a more accurate single bacterial cell analysis. We report a lanthanide-encoding inductively coupled plasma (ICP) mass spectrometric approach for counting and recognizing single bacterial cells for the first time. When noncanonical alkyne-d-alanine ( aDA) was added to five typical bacterial strains of Escherichia coli, Staphylococcus aureus, Listeria monocytogenes, Shigella dysenteriae, and Vibrio parahemolyticus, aDA was metabolically assembled into the peptidoglycan layer-supported bacterial cell wall followed by post-clickable europium-tagging with 1,4,7,10-tetraazacyclododecane-1,4,7-tris-acetic acid-10-azidopropyl ethylacetamide-europium complex ( azide-DOTA-Eu). Such Eu-tagged bacterial cells can be deemed as Eu-engineered particles, delivering more than 5 orders of magnitude self-signal-amplification outcome relative to the single bacterial cells themselves when 151/153Eu is determined by single particle ICP mass spectrometry ( spICPMS). This metabolic assembly of aDA mediated Eu-encoding signal amplification strategy breaks through the detection limit of spICPMS and ensures that we directly count a single bacterial cell. The individual bacterial strains we counted can be simultaneously recognized through their corresponding lanthanide (Ln)-coded polyclonal antibody (Ln = 139La, 141Pr, 142Nd, 152Sm, and 160Gd, respectively), serving as a specific bacterial identification (Ln- pAb-ID). Moreover, the developed approach was applied to show the different behavior between genetically identical Staphylococcus aureus under the treatments of vancomycin and Ag nanoparticles, demonstrating that such a lanthanide-encoding spICPMS approach provided a new way to discover still ambiguous cell-to-cell variability.

Fluorescent and mass spectrometric evaluation of the phagocytic internalization of a CD47-peptide modified drug-nanocarrier.

Ru(bpy)3@SiO2-COOH and Ru(bpy)3@SiO2@CD47-peptide nanoparticles (NPs) with fluorescent and mass spectrometric properties were designed and synthesized as the models of drug-nanocarriers. Their phagocytic internalization could be quantitatively measured using more sensitive inductively coupled plasma mass spectrometry (ICPMS) (102Ru) versus traditional laser confocal scanning microscope (λex/em = 458/600 nm) for the first time. Modification of a self-signal trigging CD47-peptide on the NPs' surface decreased internalization by 10 times, (2.79 ± 0.21) × 104 Ru(bpy)3@SiO2-COOH and (0.28 ± 0.04) × 104 Ru(bpy)3@SiO2@CD47-peptide NPs per RAW264.7 macrophage (n = 5). The alkynyl-linked CD47-peptide allowed us to quantify the number (2412 ± 250) of CD47-peptide modified on the NP and the total content (5.14 ± 0.25 amol) of signal regulatory protein α (SIRPα) on the macrophage by measuring the clickable tagged Eu using ICPMS. Furthermore, the interaction between CD47-peptide and SIRPα as well as the changes of the remaining free SIRPα during the internalization process of Ru(bpy)3@SiO2@CD47-peptide NPs were quantitatively evaluated, providing direct experimental evidence of the longspeculated crucial CD47-SIRPα interaction for drug-nanocarriers to escape internalization by phagocytic cells. Remarkable difference in the internalization ratio of 12.3 ± 4.8 of Ru(bpy)3@SiO2-COOH NPs and 4.3 ± 0.5 Ru(bpy)3@SiO2@CD47-peptide NPs with and without the protein corona indicated that CD47-peptide still worked when the protein corona formed. Not limited to the evaluation of the NPs studied here, such a fluorescent and mass spectrometric approach is very much expected to apply to the assessment of other drug-nanocarriers designed by chemists and before their medical applications. Graphical abstract.

Selective determination of nitrite/nitrate based on photo-induced redox activity of titanium dioxide.

Given its unique photocatalytic and structural properties, titanium dioxide was used as a sensitizer for the quantification of nitrite and nitrate contents by high-performance liquid chromatography with ultraviolet/nano-titanium dioxide photo-induced chemiluminescence detection. The photo-induced chemiluminescence signal was enhanced after the introduction of titanium dioxide. Ethylenediaminetetraacetic acid, as a positive hole scavenger, considerably improved the signal. The peak area of the chemiluminescence signal was enhanced 85 times after ethylenediaminetetraacetic acid was added to 1 × 10-6  mol/L of nitrite. The detection limits of nitrite and nitrate were 9.0 × 10-9 and 1.4 × 10-7  mol/L, respectively. Our method was applied for the determination of nitrite and nitrate contents in water samples. In contrast to other methods, our method is simple and environmentally friendly and enables the simultaneous determination of nitrite and nitrate.

Near-Infrared Neodymium Tag for Quantifying Targeted Biomarker and Counting Its Host Circulating Tumor Cells.

Quantitative information on a targeted analyte in a complex biological system is the most basic premise for understanding its involved mechanisms, and thus precise diagnosis of a disease if it is a so-called biomarker. Here, we designed and synthesized a neodymium (Nd)-cored tag [1,4,7,10-tetraazacyclododecane-1,4,7-trisacetic acid (DOTA)-Nd complex together with a light-harvesting antenna aminofluorescein (AMF, λex/em = 494/520 nm), AMF-DOTA-Nd] with duplex signals, second near-infrared (NIR) window luminescence (λem = 1065 nm, 2.5 µs), and stable isotopic mass (142Nd). AMF-DOTA-Nd covalently linked with a urea-based peptidomimetic targeting group, 2-[3-(1,3-dicarboxypropyl)-ureido]pentanedioic acid (DUPA)-8-Aoc-Phe-Phe-Cys (DUPAaFFC) (DUPAaFFC-AMF-DOTA-Nd), allowing us to detect and quantify prostate-specific membrane antigen (PSMA) and its splice variants (total PSMA, tPSMA), which was set as an example of targeted biomarkers in this study, using NIR and inductively coupled plasma mass spectrometry (ICPMS) with the limit of detection (LOD) (3σ) of 0.3 ng/mL. When it was applied to the analysis of 80 blood samples from prostate cancer (PCa) and benign prostatic hyperplasia (BPH) patients as well as healthy volunteers, we found that 320 and 600 ng/mL tPSMA could be recommended as the threshold values to differentiate BPH from PCa and for the diagnosis of PCa. Moreover, PSMA-positive circulating tumor cells (CTCs) were counted using ICPMS being from 134 to 773 CTCs in the PCa blood samples of the Gleason score from 6 to 9 when the cell membrane-spanning mPSMA was tagged. Such a methodology developed could be expected to be applicable to other clinic-meaningful biomolecules and their host CTCs in liquid biopsy, when other specific targeting groups are modified to the NIR Nd tag.

Metabolism-Based Click-Mediated Platform for Specific Imaging and Quantification of Cell Surface Sialic Acids.

Although we believe that the cell surface sialic acids (Sias) are playing an important role in cell-cell interactions and related tumor metastasis processes, acquisition of their quantitative information has yet been a challenge to date. Here, we reported the construction of a new analytical platform for Sias-specific imaging and quantification. We used N-azidoacetyl-mannosamine tetraacylated as a metabolic sugar substrate to bioassemble azido-Sias on the surface of cells via the metabolic pathway of Sias de novo synthesis. These azido-Sias allow us to perform a duplex Sias-specific analysis with various fluorescent and elemental reporters such as DIBO-Alexa Fluor 647, DBCO-DOTA-Eu, and DBCO-PEG4-BODIPY, which can be easily labeled and/or tagged through an effective copper-free bioorthogonal click reaction. Compared to the previous reported strategies, we quantified the cell surface Sias with the LODs (3σ) down to 8.9 fmol and 0.24 pmol using 153Eu- and 10B-species unspecific isotope dilution ICPMS, in addition to their red- and green-CLSM profiling. Such a platform enables us to evaluate Sias regulation under the administration of paclitaxel, finding that 1 µM paclitaxel induced a significant Sias decrease of 67% on the surface of hepatic tumor cell SMMC-7721, while had no obvious adverse effect to that of para-carcinomatous liver cell LO2. Besides Sias, we believe that this metabolism-based click-mediated platform will provide opportunities to study other monosaccharides and their corresponding biological roles when more corresponding chemically modified sugar substrates and specific bioorthogonal reactions are developed.

ICPMS-Based Specific Quantification of Phosphotyrosine: A Gallium-Tagging and Tyrosine-Phosphatase Mediated Strategy.

Low-abundance tyrosine phosphorylation is crucial to not only normal but also aberrant life processes. We designed and synthesized a photocleavable magnetic nanoparticle-based gallium tag for tagging and enrichment as well as UV-release of the phosphate-bearing molecules/ions in cells. HPLC/71Ga species-unspecific isotope dilution (71Ga-SUID) ICPMS was subsequently developed for specific and absolute quantification of phosphotyrosine (pY) under the assistance of a protein tyrosine phosphatase-1B (PTP-1B). pY quantification was thus achieved via determination of Ga in the Ga-phosphate complexes that come exclusively from the Ga-tagged pY. In this way, the method detection limit of pY reached down to 30 amol with the RSD lower than 5.70% (n = 5 at pmol level). Feasibility of this proposed method was validated using VNQIGTLSEpYIK, VNQIGTLpSEpYIK, and extracellular regulated protein kinase 1 peptide (-pTEpY-) standards with the recovery of more than 96% (n = 5). It was applied to the absolute quantification of pY in human breast cancer MCF-7 cells, indicating that pY increased by 1.60 nmol (61.1%) in 3.0 × 106 MCF-7 cells after 100 nM insulin stimulation. We believe that, not limited to pY quantification, this element-tagging and protease-specific reaction mediated ICPMS methodology will pave a simple path for ever more applications of ICPMS to the studies of quantitative protein post-translational modifications (PTMs) when suitable element-tags are designed and specific proteases are available toward targeted PTMs.