One-Pot Two-Nanoprobe Assay Uncovers Targeted Glycoprotein Biosignature.

We report a one-pot two-nanoprobe approach coupled to mass spectrometry for simultaneous quantification and post-translational modification (PTM) profiling of targeted protein in biofluid. Using N-glycoprotein as model, the assay employs two nanoprobes, antibody-conjugated SiO2 nanoparticles and lectin-conjugated magnetic Fe3O4 nanoparticles, to achieve target glycoprotein isolation from biofluid and subsequent glycopeptide enrichment in a single tube. As demonstrated on α-fetoprotein (AFP), a serum biomarker for hepatocellular carcinoma (HCC), the assay has high purification specificity (20 glycopeptides) with 2-fold and 10-fold superior total glycopeptide intensity compared to non-one-pot method (9 glycopeptides) or without enrichment (6 glycopeptides), respectively. By multiple reaction monitoring mass spectrometry (MRM-MS) analysis of the nonglycopeptides, the assay can quantify low abundant AFP expression (0.5 ng) with good correlation with conventional ELISA method (Pearson's r = 0.987). Furthermore, we present the first study revealing AFP glycopeptide signatures of individual HCC patients, comprised of 23 heterogeneous glycoforms of bi- and triantennary, core and terminal fucosylation, and mono- to trisialylation. In addition to 12 novel AFP glycoforms, our quantification result uncovers five abundant glycoforms in HCC, including 3 core-fucosylated (CF) forms. These identified CF forms may be evaluated in future studies as potential targets in a glycopeptide biomarker panel to further improve accuracy of conventional AFP-L3 tests. Through this one-pot assay, a comprehensive target protein profile comprised of protein expression and glycosylation pattern was achieved in simple protocol with high sensitivity, reduced analysis time, and minute starting material. This assay can be extended to other PTM biosignatures by conjugation of other affinity ligands on the nanoprobe.

Functionalized HgTe nanoparticles promote laser-induced solid phase ionization/dissociation for comprehensive glycan sequencing.

Glycoconjugates are ubiquitously present and play a critical role in various biological processes. Due to their low stability and incredibly high degree of structural diversity, the structural characterization of glycan generally requires chemical derivatization and sophisticated instrumentation. Herein, we report a method for complicated glycan characterization in a single assay by employing 2,5-dihydroxybenzoic acid functionalized mercury telluride nanoparticles (HgTe@DHB NPs) as a dual ionization-dissociation element in matrix-assisted laser desorption/ionization mass spectrometry. Using a linear glycan, HgTe@DHB NPs promote laser-induced extensive and intense dissociation of the glycan, superior to HgTe microparticles and other inorganic nanoparticles (TiO2, ZnO, and Mn2O3 NPs). Abundant generation of diagnostic glycosidic (Y-, and B-type ions) and cross-ring cleavage (A-type ions) ions permits unambiguous determination of the composition, sequence, branching, and linkage of labile sialylated glycans. The general utility of this approach was demonstrated by the characterization of labile sialylated glycans and two sets of complicated isomeric glycans. This phenomenon was delineated further by investigating the NP's physico-chemical characteristics, revealing that their nanoscale-dependent thermodynamic properties, including UV absorption, photoelectron release dynamics and thermal energy, were the key to levitate temperature synergistically, thus inducing spontaneous glycan decomposition during the nanoparticle-assisted laser desorption-ionization process. Our results show that this "pseudo-MS/MS" obtained by HgTe@DHB can be beneficial for the analysis of biologically relevant and more complicated carbohydrates, without the need for chemical pre-derivatization and conventional tandem mass spectrometry.

Imaging Endogenous Bilirubins with Two-Photon Fluorescence of Bilirubin Dimers.

On the basis of an infrared femtosecond Cr:forsterite laser, we developed a semiquantitative method to analyze the microscopic distribution of bilirubins. Using 1230 nm femtosecond pulses, we selectively excited the two-photon red fluorescence of bilirubin dimers around 660 nm. Autofluorescences from other endogenous fluorophores were greatly suppressed. Using this distinct fluorescence measure, we found that poorly differentiated hepatocellular carcinoma (HCC) tissues on average showed 3.7 times lower concentration of bilirubins than the corresponding nontumor parts. The corresponding fluorescence lifetime measurements indicated that HCC tissues exhibited a longer lifetime (500 ps) than that of nontumor parts (300 ps). Similarly, oral cancer cell lines had longer lifetimes (>330 ps) than those of nontumor ones (250 ps). We anticipate the developed methods of bilirubin molecular imaging to be useful in diagnosing cancers or studying the dynamics of bilirubin metabolisms in live cells.

Monodispersity of magnetic immuno-nanoprobes enhances the detection sensitivity of low abundance biomarkers in one drop of serum.

To enhance the detection sensitivity of target clinical protein biomarkers, a simple and rapid nanoprobe-based immuno-affinity mass spectrometry assay employing biocompatible monodisperse magnetic nanoparticles (MNPs) is reported herein. The MNPs were synthesized via a streamlined protocol that includes (a) fabrication of core MNPs using the thermal decomposition method to minimize aggregation, (b) surface protection by gold coating (MNP@Au) and surfactant coating using MNP@IGEPAL to improve hydrophilicity, and lastly, (c) oriented functionalization of antibodies to maximize immuno-affinity. The enrichment performances of the monodisperse MNPs for the C-reactive protein (CRP) serum biomarker were then evaluated and compared with aggregated magnetic nanoparticles synthesized from the conventional co-precipitation method (MNP(CP)). The detection sensitivity for CRP at an extremely low amount of serum sample (1 µL) was enhanced ∼19- and ∼15-fold when monodisperse MNP@Au and MNP@IGEPAL, respectively, were used. Furthermore, the detection sensitivity of CRP by this approach (1 ng mL(-1), S/N = 3) provided a 1000-fold sensitivity enhancement to the clinical cut-off (1 µg mL(-1)) of CRP. We supposed that these observed improvements are due to the enhanced nanoparticle dispersibility and size uniformity which eliminated completely other non-specific binding of high-abundance serum proteins. Most interestingly, the enrichment efficiency correlates more closely with the MNP dispersibility than the ligand density. Our investigation revealed the critical role of MNP dispersibility, as well as provided mechanistic insight into its impact on immunoaffinity enrichment and detection of CRP in one drop of serum sample. This strategy offers an essential advantage over the other methods by providing a simple and facile biofunctionalization protocol while maintaining excellent solvent dispersibility of MNPs.

Interaction modes and approaches to glycopeptide and glycoprotein enrichment.

Protein glycosylation has received increased attention for its critical role in cell biology and diseases. Developing new methodologies to discern phenotype-dependent glycosylation will not only elucidate the mechanistic aspects of cell signaling cascades but also accelerate biomarker discovery for disease diagnosis or prognosis. In the analytical pipeline, enrichment at either the protein or peptide level is the most critical prerequisite for analyzing heterogeneous glycan composition, linkage, site occupancy and carrier proteins. Because the critical factor for choosing a suitable enrichment method is primarily a particular technique's selectivity and affinity towards target glycoproteins/glycopeptides, it is important to fully understand the working principles for the different approaches. For mechanistic insight into the enrichment protocol, we focused on the fundamental chemical and physical processes for the commonly used approaches based on: (a) glycan/peptide physicochemical properties (hydrophilic interactions, chelation/coordination chemistry) and (b) glycan-specific recognition (lectin-based affinity, covalent bond formation by hydrazide/boronic acid). Various interaction modes, such as hydrogen bonding, van der Waals interaction, multivalency, and metal- or water-mediated stabilization, are discussed in detail. In addition, we will review the design of and modifications to such methods, hyphenated approaches, and glycoproteomic applications. Finally, we will outline challenges to existing strategies and offer novel proposals for glycoproteome enrichment.

Iron oxide nanomatrix facilitating metal ionization in matrix-assisted laser desorption/ionization mass spectrometry.

The significance and epidemiological effects of metals to life necessitate the development of direct, efficient, and rapid method of analysis. Taking advantage of its simple, fast, and high-throughput features, we present a novel approach to metal ion detection by matrix-functionalized magnetic nanoparticle (matrix@MNP)-assisted MALDI-MS. Utilizing 21 biologically and environmentally relevant metal ion solutions, the performance of core and matrix@MNP against conventional matrixes in MALDI-MS and laser desorption ionization (LDI) MS were systemically tested to evaluate the versatility of matrix@MNP as ionization element. The matrix@MNPs provided 20- to >100-fold enhancement on detection sensitivity of metal ions and unambiguous identification through characteristic isotope patterns and accurate mass (<5 ppm), which may be attributed to its multifunctional role as metal chelator, preconcentrator, absorber, and reservoir of energy. Together with the comparison on the ionization behaviors of various metals having different ionization potentials (IP), we formulated a metal ionization mechanism model, alluding to the role of exciton pooling in matrix@MNP-assisted MALDI-MS. Moreover, the detection of Cu in spiked tap water demonstrated the practicability of this new approach as an efficient and direct alternative tool for fast, sensitive, and accurate determination of trace metal ions in real samples.

Biomarker identification of hepatocellular carcinoma using a methodical literature mining strategy.

Hepatocellular carcinoma (HCC), one of the most common causes of cancer-related deaths, carries a 5-year survival rate of 18%, underscoring the need for robust biomarkers. In spite of the increased availability of HCC related literatures, many of the promising biomarkers reported have not been validated for clinical use. To narrow down the wide range of possible biomarkers for further clinical validation, bioinformaticians need to sort them out using information provided in published works. Biomedical text mining is an automated way to obtain information of interest within the massive collection of biomedical knowledge, thus enabling extraction of data for biomarkers associated with certain diseases. This method can significantly reduce both the time and effort spent on studying important maladies such as liver diseases. Herein, we report a text mining-aided curation pipeline to identify potential biomarkers for liver cancer. The curation pipeline integrates PubMed E-Utilities to collect abstracts from PubMed and recognize several types of named entities by machine learning-based and pattern-based methods. Genes/proteins from evidential sentences were classified as candidate biomarkers using a convolutional neural network. Lastly, extracted biomarkers were ranked depending on several criteria, such as the frequency of keywords and articles and the journal impact factor, and then integrated into a meaningful list for bioinformaticians. Based on the developed pipeline, we constructed MarkerHub, which contains 2128 candidate biomarkers extracted from PubMed publications from 2008 to 2017. Database URL: http://markerhub.iis.sinica.edu.tw.

UV-activated multilayer nanomatrix provides one-step tunable carbohydrate structural characterization in MALDI-MS.

The structure-specific fragmentation of gas-phase ions in tandem mass spectrometry among other techniques provides an efficient analytical method for confirming unknown analytes or for elucidating chemical structures. Using concentration-dependent UV-absorbing matrix-functionalized magnetic nanoparticles and matrix-assisted laser desorption-ionization mass spectrometry (MALDI MS), we developed a single-step pseudo-MS/MS approach for tunable ionization and fragmentation to facilitate structure determination. Without chemical derivatization, we have demonstrated that this approach successfully distinguished isomeric sets of di-, tri- and tetrasaccharides. Low concentration of nanomatrix provided an enhanced signal for accurate mass determination of the intact molecular ions of analytes present in the sample. In contrast, high concentration of nanomatrix induced extensive and unique fragmentation, including high-energy facile bond breakage (A- and X-type cross-ring cleavages), which facilitated the linkage and sequence characterization of oligosaccharides without conventional tandem mass spectrometric instrumentation. The practicality of this approach for complex sample analysis was evaluated by an oligosaccharide mixture, wherein molecular ions are unambiguously observed and signature product ions are distinguishable enough for molecular identification and isomer differentiation by this simple tunable approach. By probing the roles of the multilayer nanomatrix components: matrix (energy absorption), silane-coating (energy pooling and dissipation) and core Fe3O4 (fragmentation), a plausible energy transfer mechanism was proposed based on a computational study and photoelectron experiments. The differentiation of tri- and tetra-oligosaccharide shown in this study not only demonstrated the first step toward glycan characterization by nanoparticle-assisted MALDI-MS, but also shed some insight on the nanoparticle-mediated energy transfer dynamics behind our approach.