A plasmonic MNAzyme signal amplification strategy for quantification of miRNA-4739 breast cancer biomarker.

A major challenge in the 21st century is the development of point-of-care diagnostic tools capable to detect and quantify disease biomarkers in a straightforward, affordable, sensitive, and specific manner. The remarkable plasmonic properties of gold nanoparticles (AuNPs) have promoted their use for development of simple methodologies for nucleic acid detection in combination with a variety of oligonucleotides amplification techniques. Here, assemblies of AuNPs with Multicomponent Nucleic Acid enzymes (MNAzymes) has been successfully used in the design of a highly sensitive and simple bioassay for rapid spectroscopic detection and quantification of miRNA-4739 in blood samples. The miRNA selected is a doxorubicin chemoresistant biomarker in breast cancer which overexpression promotes the proliferation, progression, and survival of cancer cells. In this work, two alternatives experimental designs, based on use of MNAzymes and AuNPs, have been optimized and applied for sensitive miRNA-4739 quantification: one based on a traditional direct measurement of wavelength shift and a second non-conventional simple approach based on isolation and measurement of free nanoparticles absorbance. Improvement in sensitivity and, higher measurement accuracy and precision were achieved with the second approach. The developed bioassay provides a detection limit as low as 7 pmolL-1 for miRNA-4739 quantification and performed satisfactory selectivity and well practical applicability by analysis of the miRNA-4739 in blood, demonstrating that the proposed strategy is a promising and suitable spectroscopic method for breast cancer diagnosis thought liquid biopsy of circulating tumoral cells.

Potential of GC-Combustion-MS as a Powerful and Versatile Nitrogen-Selective Detector in Gas Chromatography.

Here, we show the potential and applicability of the novel GC-combustion-MS approach as a nitrogen-selective GC detector. Operating requirements to achieve reproducible and compound-independent formation of volatile NO species as a selective N-signal during the combustion step are described. Specifically, high temperatures (≥1000 °C) and post-column O2 flows (0.4 mL min-1 of 0.3% O2 in He) turned out to be necessary when using a vertical oven without makeup flow (prototype #1). In contrast, the use of a horizontal oven with 1.7 mL min-1 He as an additional makeup flow (prototype #2) required milder conditions (850 °C and 0.2 mL min-1). A detection limit of 0.02 pg of N injected was achieved, which is by far the lowest ever reported for any GC detector. Equimolarity, linearity, and peak shape were also adequate. Validation of the approach was performed by the analysis of a certified reference material obtaining accurate (2% error) and precise (2% RSD) results. Robustness was tested with the analysis of two complex samples with different matrices (diesel and biomass pyrolysis oil) and N concentration levels. Total N determined after the integration of the whole chromatograms (524 ± 22 and 11,140 ± 330 µg N g-1, respectively) was in good agreement with the reference values (497 ± 10 and 11,000 ± 1200 µg N g-1, respectively). In contrast, GC-NCD results were lower for the diesel sample (394 ± 42 µg N g-1). Quantitative values for the individual and families of N species identified in the real samples by parallel GC-MS and additional GC × GC-MS analyses were also obtained using a single generic internal standard.

Certification of protein biomarker standards using element MS and generic standards: Application to human cytokines.

The availability of protein standards and methods for their characterization, quantification, and purity assessment are currently a bottleneck in absolute quantitative proteomics. In this work, we introduce an absolute quantitative analytical strategy based on ICP-MS sulfur detection that uses sulfate as generic standard to quantify and certify the mass purity of protein standards. The methodology combines capillary chromatographic separation with parallel detection with ICP-MS and ESI-MS to determine proteoforms concentration and identity, respectively. The workability of the methodology was demonstrated using recombinant human cytokine standards IP-10 and Flt3L (2 batches), which are relevant biomarkers for carcinoma or inflammatory diseases. Every key factor (transport efficiency, column recovery, signal stability and internal standard suitability) was taken into account and certified BSA standard was used as quality control for validation purposes. Protein quantification values and resulting mass purity certification of IP-10 and one batch of Flt3L were very high (100 and 86%, respectively). Lower mass purity obtained for another batch of Flt3L (<70%) concurred with the finding of significant proteoforms resulted from oxidation processes as observed by parallel ESI-MS.

Signal amplification strategies for clinical biomarker quantification using elemental mass spectrometry.

The current trends in modern medicine towards early diagnosis, or even prognosis, of different diseases have brought about the need for the corresponding biomarker detection at ever lower levels in really complex matrices. To do so, it is necessary to use proper extremely sensitive detection techniques such as elemental mass spectrometry. However, target labelling with metals for subsequent sensitive ICP-MS detection falls short nowadays even if resorting to inorganic nanoparticles containing a high number of detectable elements. Thus, new amplification strategies are being proposed to face this analytical challenge that will be critically discussed in this paper. Fundamentals of different novel strategies developed to achieve signal amplification and sensitive elemental mass spectrometry detection are here discussed. Some representative examples of relevant clinical applications are highlighted, along with future prospects and challenges.

Assessment of the Potential and Limitations of Elemental Mass Spectrometry in Life Sciences for Absolute Quantification of Biomolecules Using Generic Standards.

Inductively coupled plasma-mass spectrometry (ICP-MS) has been widely used in Life Sciences for the absolute quantification of biomolecules without specific standards, assuming the same response for generic compounds including complex biomolecules. However, contradictory results have been published on this regard. We present the first critical statistical comparison of the ICP-MS response factors obtained for 14 different relevant S-containing biomolecules (three peptides, four proteins, one amino acid, two cofactors, three polyethylene glycol (PEG) derivatives, and sulfate standard), covering a wide range of hydrophobicities and molecular sizes. Two regular flow nebulizers and a total consumption nebulizer (TCN) were tested. ICP-MS response factors were determined though calibration curves, and isotope dilution analysis was used to normalize the results. No statistical differences have been found for low-molecular-weight biocompounds, PEGs, and nonhydrophobic peptides using any of the nebulizers tested. Interestingly, while statistical differences were still found negligible (96-104%) for the proteins and hydrophobic peptide using the TCN, significantly lower response factors (87-40%) were obtained using regular flow nebulizers. Such differential behavior seems to be related mostly to hydrophobicity and partially to the molecular weight. Findings were validated using IDA in intact and digested bovine serum albumin solutions using the TCN (98 and 100%, respectively) and the concentric nebulizer (73 and 97%, respectively). Additionally, in the case of a phosphoprotein, results were corroborated using the P trace in parallel to the S trace used along the manuscript. This work seems to suggest that ICP-MS operated with regular nebulizers can offer absolute quantification using generic standards for most biomolecules except proteins and hydrophobic peptides.

Quantitative multiplexed elemental (C, H, N and S) detection in complex mixtures using gas chromatography.

We present a novel and single detection approach that enables sensitive, accurate and compound-independent quantification of N, S and H in the individual compounds present in complex samples. Integration of the whole chromatographic profile gives the total content of the elements. Simultaneous universal detection is also achieved using the C profile.

Standardization approaches in absolute quantitative proteomics with mass spectrometry.

Mass spectrometry-based approaches have enabled important breakthroughs in quantitative proteomics in the last decades. This development is reflected in the better quantitative assessment of protein levels as well as to understand post-translational modifications and protein complexes and networks. Nowadays, the focus of quantitative proteomics shifted from the relative determination of proteins (ie, differential expression between two or more cellular states) to absolute quantity determination, required for a more-thorough characterization of biological models and comprehension of the proteome dynamism, as well as for the search and validation of novel protein biomarkers. However, the physico-chemical environment of the analyte species affects strongly the ionization efficiency in most mass spectrometry (MS) types, which thereby require the use of specially designed standardization approaches to provide absolute quantifications. Most common of such approaches nowadays include (i) the use of stable isotope-labeled peptide standards, isotopologues to the target proteotypic peptides expected after tryptic digestion of the target protein; (ii) use of stable isotope-labeled protein standards to compensate for sample preparation, sample loss, and proteolysis steps; (iii) isobaric reagents, which after fragmentation in the MS/MS analysis provide a final detectable mass shift, can be used to tag both analyte and standard samples; (iv) label-free approaches in which the absolute quantitative data are not obtained through the use of any kind of labeling, but from computational normalization of the raw data and adequate standards; (v) elemental mass spectrometry-based workflows able to provide directly absolute quantification of peptides/proteins that contain an ICP-detectable element. A critical insight from the Analytical Chemistry perspective of the different standardization approaches and their combinations used so far for absolute quantitative MS-based (molecular and elemental) proteomics is provided in this review.

Capping of Mn-Doped ZnS Quantum Dots with DHLA for Their Stabilization in Aqueous Media: Determination of the Nanoparticle Number Concentration and Surface Ligand Density.

Colloidal Mn2+-doped ZnS quantum dots (QDs) were synthesized, surface modified, and thoroughly characterized using a pool of complementary techniques. Cap exchange of the native l-cysteine coating of the QDs with dihydrolipoic acid (DHLA) ligands is proposed as a strategy to produce nanocrystals with a strong phosphorescent-type emission and improved aqueous stability. Moreover, such a stable DHLA coating can facilitate further bioconjugation of these QDs to biomolecules using established reagents such as cross-linker molecules. First, a structural and morphological characterization of the l-cysteine QD core was performed by resorting to complementary techniques, including X-ray powder diffraction (XRD) and microscopy tools. XRD patterns provided information about the local structure of ions within the nanocrystal structure and the number of metal atoms constituting the core of a QD. The judicious combination of the data obtained from these complementary characterization tools with the analysis of the QDs using inductively coupled plasma-mass spectrometry (ICP-MS) allowed us to assess the number concentration of nanoparticles in an aqueous sample, a key parameter when such materials are going to be used in bioanalytical or toxicological studies. Asymmetric flow field-flow fractionation (AF4) coupled online to ICP-MS detection proved to be an invaluable tool to compute the number of DHLA molecules attached to the surface of a single QD, a key feature that is difficult to estimate in nanoparticles and that critically affects the behavior of nanoparticles when entering the biological media (e.g., cellular uptake, biodistribution, or protein corona formation). This hybrid technique also allowed us to demonstrate that the elemental composition of the nanoparticle core remains unaffected after the ligand exchange process. Finally, the photostability and robustness of the DHLA-capped QDs, critical parameters for bioanalytical applications, were assessed by molecular luminescence spectroscopy.

Instrumental Setup for Simultaneous Total and Speciation Analysis of Volatile Arsenic Compounds in Gas and Liquefied Gas Samples.

Although analysis of metals and metalloids, such as arsenic, is widely spread in many different fields, their analysis in gas and liquefied gas samples is still a challenge. A new GC-ICP-MS set up has been developed for their simultaneous total and speciation analysis in gas and liquefied gas samples without the need of a preconcentration step. An arsine in nitrogen standard was used for optimization and evaluation of the system. Good linearity and detection limits in the very low ppt level for both total and speciation analyses were found. Liquefied butane pressurized under nitrogen and doped with arsine and a propylene real sample from a cracker plant were analyzed using both external calibration and standard additions methods. The good match between both quantifying approaches demonstrated almost negligible matrix effects, even for the total analysis. Application of the approach to check repartition of volatile elements or species between gas and liquid phases was performed in the real propylene sample. Finally, its potential applicability for the simultaneous total and speciation analysis of other elements, such as Hg, was also proved.

Protein-species quantitative venomics: looking through a crystal ball

Abstract In this paper we discuss recent significant developments in the field of venom research, specifically the emergence of top-down proteomic applications that allow achieving compositional resolution at the level of the protein species present in the venom, and the absolute quantification of the venom proteins (the term protein species is used here to refer to all the different molecular forms in which a protein can be found. Please consult the special issue of Jornal of Proteomics Towards deciphering proteomes via the proteoform, protein speciation, moonlighting and protein code concepts published in 2016, vol. 134, pages 1-202). Challenges remain to be solved in order to achieve a compact and automated platform with which to routinely carry out comprehensive quantitative analysis of all toxins present in a venom. This short essay reflects the authors view of the immediate future in this direction for the proteomic analysis of venoms, particularly of snakes.

Protein-species quantitative venomics: looking through a crystal ball

In this paper we discuss recent significant developments in the field of venom research, specifically the emergence of top-down proteomic applications that allow achieving compositional resolution at the level of the protein species present in the venom, and the absolute quantification of the venom proteins (the term "protein species" is used here to refer to all the different molecular forms in which a protein can be found. Please consult the special issue of Jornal of Proteomics "Towards deciphering proteomes via the proteoform, protein speciation, moonlighting and protein code concepts" published in 2016, vol. 134, pages 1-202). Challenges remain to be solved in order to achieve a compact and automated platform with which to routinely carry out comprehensive quantitative analysis of all toxins present in a venom. This short essay reflects the authors' view of the immediate future in this direction for the proteomic analysis of venoms, particularly of snakes.(AU)

Comparison of gas chromatography-combustion-mass spectrometry and gas chromatography-flame ionization detector for the determination of fatty acid methyl esters in biodiesel without specific standards.

GC-FID has been effectively used as a universal quantification technique for volatile organic compounds for a long time. In most cases, the use of the ECN allows for quantification by GC-FID without external calibration using only the response of a single internal standard. In this paper we compare the performance characteristics of GC-FID with those of post-column (13)C Isotope Dilution GC-Combustion-MS for the absolute quantification of organic compounds without the need for individual standards. For this comparison we have selected the quantification of FAMEs in biodiesel. The selection of the right internal standard was critical for GC-FID even when ECN were considered. On the other hand, the nature of the internal standard was not relevant when GC-Combustion-MS was employed. The proposed method was validated with the analysis of the certified reference material SRM 2772 and comparative data was obtained on real biodiesel samples. The analysis of the SRM 2772 biodiesel provided recoveries in the range 100.6-103.5% and 96.4-103.6% for GC-combustion-MS and GC-FID, respectively. The detection limit for GC-combustion-MS was found to be 4.2ng compound/g of injected sample. In conclusion, the quantitative performance of GC-Combustion-MS compared satisfactorily with that of GC-FID constituting a viable alternative for the quantification of organic compounds without the need for individual standards.

Highly sensitive nanoparticle-based immunoassays with elemental detection: Application to Prostate-Specific Antigen quantification.

One of the major challenges in developing novel assay methods for the detection of biomolecules is achieving high sensitivity, because of the ultralow concentrations typically in clinical samples. Here, a Mn-doped ZnS quantum dots-based immunoassay platform is presented for highly sensitive detection of cancer biomarkers. Ultrahigh sensitivity is achieved through gold deposition on the surface of the nanoparticle tags acting as catalytic seeds, thus effectively amplifying the size of the metallic nanoparticles after the immunoassay and before the tag detection. Elemental mass spectrometry measurement of the gold content allowed detection of Prostate-Specific Antigen (PSA) at the low attog mL(-1) level. Moreover, the developed method showed not only an extremely high sensitivity for PSA detection but also a broad dynamic range, higher than 8 orders of magnitude, particularly useful for clinical studies involving quantitative detection of diverse biomarkers at their very different relevant concentration levels. Its applicability to discriminate small differences in PSA concentrations at low levels (few pgmL(-1)) in real serum samples was successfully evaluated.

Elemental and molecular detection for Quantum Dots-based immunoassays: a critical appraisal.

A critical comparison between Elemental Mass Spectrometry (ICP-MS) and molecular fluorescence, as detection techniques for CdSe/ZnS Quantum Dots (QDs)-based immunoassays is presented here. Using a QDs-based progesterone immunoassay as "model" analytical system the features of both detection modes has been investigated. Minimal changes, compared to the previously developed fluorescent approach, were necessary to build the corresponding inhibition curve for the progesterone immunoassay using ICP-MS detection of cadmium (contained in the QDs core). Adequate agreement between results obtained using both elemental and molecular techniques for the determination of progesterone in cow milk has been obtained. Moreover, results from the comparison showed that fluorescence detection of the QDs is simpler, less time consuming and less expensive, but ICP-MS detection affords alternative and useful information unattainable using luminescence detection. First of all, ICP-MS allowed mass balances to be carried out (all along the sample preparation) providing an internal validation of the immunoassay procedure. Secondly, matrix-independent quantification as provided by ICP-MS enabled a direct determination of progesterone in raw milk without any further sample preparation (dilution) step. As a matter of fact, ICP-MS results showed that the quenching matrix effect suffered on bioconjugated QDs fluorescence emission (e.g. when the immunoassay was carried out directly in whole milk without any dilution) could be unequivocally attributed to nonspecific interactions between the matrix of the whole milk and the QDs surface. Finally, better sensitivity could be obtained with ICP-MS detection, IC(10)=0.028 ng/mL, versus 0.11 ng/mL using conventional fluorimetric detection, just by using lower reagents concentrations.

The emerging role of ICP-MS in proteomic analysis.

Quantitative proteomics and absolute determination of proteins are topics of fast growing interest, since only the quantity of proteins or changes in their abundance reflect the status and extent of changes of a given biological system. Quantification of the desired proteins has been carried out by molecule specific MS techniques, but relative quantifications are commonplace so far even resorting to stable isotope labelling techniques such as ICAT and SILAC. In the last decade the idea of using element-selective mass spectrometric detection (e.g. ICP-MS instruments) to achieve absolute quantification has been realised and ICP-MS stands now as a new tool in the field of quantitative proteomics. In this review the emerging role of ICP-MS in protein and proteomic analysis is highlighted. The potential of ICP-MS methods and strategies for screening multiple heteroatoms (e.g. S, P, Se, metals) in proteins and their mixtures and extraordinary capabilities to tackle the problem of absolute protein quantifications, via heteroatom determinations, are discussed and illustrated. New avenues are also open derived from the use of ICP-MS for precise isotope abundance measurements in polyisotopic heteroatoms. The "heteroatom (isotope)-tagged proteomics" concept is focused on the use of naturally present element tags and also extended to any protein by resorting to bioconjugation reactions (i.e. labelling sought proteins and peptides with ICP-MS detectable heteroatoms). A major point of this review is displaying the possibilities of using a "hard" ion source, the ICP, to complement well-established "soft" ion sources for mass spectrometry to tackle present proteomic analysis.

Selenium bioaccessibility assessment in selenized yeast after "in vitro" gastrointestinal digestion using two-dimensional chromatography and mass spectrometry.

In order to investigate the potentially bioavailable selenium-containing compounds in the selenized yeast candidate reference material SEAS 6, a two-dimensional (size exclusion-reversed phase) chromatography approach has been worked out. Electrospray tandem mass spectrometry (ESI Q-TOF MS) was then used for off-line identification of low molecular weigh selenocompounds generated during the gastrointestinal digestion. Selenomethionine (SeMet) was the major compound identified in the gastrointestinal extract while SeMet selenoxide was its main degradation product formed after medium and long-term sample storage, respectively. Total Se and SeMet were quantified in both the soluble extracts and the residue. Results showed that 89+/-3% of total Se was extracted after gastrointestinal digestion, but only 34+/-1% was surprisingly quantified as free SeMet. The rest of Se was present as many other low, medium and high molecular weight Se-species, which could be detected and further characterized by using the two-dimensional chromatography approach proposed here. Interestingly, most of Se-species seemed to be Se-peptides unspecifically produced by the gastrointestinal juice. These results show for the first time that while the efficiency of human gastrointestinal digestion to dissolve Se-containing proteins present in yeast may be high, its efficiency to convert them into free SeMet is much lower. Se-species present in the insoluble residue (not assimilated by the organism), accounting for 11+/-1% of the total Se in selenized yeast, were also studied. After treatment with SDS (denaturing agent) only 13+/-2% of this "insoluble" Se was solubilized, indicating that it was mainly non-protein bound and likely associated to other insoluble matrix components.