Rapid discrimination of multiple myeloma patients by artificial neural networks coupled with mass spectrometry of peripheral blood plasma.

Multiple myeloma (MM) is a highly heterogeneous disease of malignant plasma cells. Diagnosis and monitoring of MM patients is based on bone marrow biopsies and detection of abnormal immunoglobulin in serum and/or urine. However, biopsies have a single-site bias; thus, new diagnostic tests and early detection strategies are needed. Matrix-Assisted Laser Desorption/Ionization Time-of Flight Mass Spectrometry (MALDI-TOF MS) is a powerful method that found its applications in clinical diagnostics. Artificial intelligence approaches, such as Artificial Neural Networks (ANNs), can handle non-linear data and provide prediction and classification of variables in multidimensional datasets. In this study, we used MALDI-TOF MS to acquire low mass profiles of peripheral blood plasma obtained from MM patients and healthy donors. Informative patterns in mass spectra served as inputs for ANN that specifically predicted MM samples with high sensitivity (100%), specificity (95%) and accuracy (98%). Thus, mass spectrometry coupled with ANN can provide a minimally invasive approach for MM diagnostics.

Clusters of Monoisotopic Elements for Calibration in (TOF) Mass Spectrometry.

Precise calibration in TOF MS requires suitable and reliable standards, which are not always available for high masses. We evaluated inorganic clusters of the monoisotopic elements gold and phosphorus (Au n+/Au n- and P n+/P n-) as an alternative to peptides or proteins for the external and internal calibration of mass spectra in various experimental and instrumental scenarios. Monoisotopic gold or phosphorus clusters can be easily generated in situ from suitable precursors by laser desorption/ionization (LDI) or matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS). Their use offers numerous advantages, including simplicity of preparation, biological inertness, and exact mass determination even at lower mass resolution. We used citrate-stabilized gold nanoparticles to generate gold calibration clusters, and red phosphorus powder to generate phosphorus clusters. Both elements can be added to samples to perform internal calibration up to mass-to-charge (m/z) 10-15,000 without significantly interfering with the analyte. We demonstrated the use of the gold and phosphorous clusters in the MS analysis of complex biological samples, including microbial standards and total extracts of mouse embryonic fibroblasts. We believe that clusters of monoisotopic elements could be used as generally applicable calibrants for complex biological samples. Graphical Abstract ᅟ.

Use of flower-like gold nanoparticles in time-of-flight mass spectrometry.

RATIONALE: Many kinds of nanoparticles (NPs) have been used for mass spectrometry (MS) so far. Here we report the first use of flower-like gold nanoparticles (AuNPs) as a mediator to enhance ionization in MS of peptides and proteins. METHODS: Flower-like AuNPs were characterized using transmission and scanning electron microscopy, UV-VIS spectrophotometry, and laser desorption/ionization (LDI)-MS and compared with polyhedral AuNPs. Mass spectra were obtained in positive ion mode using a time-of-flight (TOF) analyzer coupled with either matrix-assisted laser desorption/ionization (MALDI) or surface-assisted laser desorption/ionization (SALDI) methods. RESULTS: The intensities of peptide peaks (m/z 500-3500) were up to 7.5× and up to 7× higher using flower-like AuNPs and flower-like AuNPs-enriched α-cyano-4-hydroxycinnamic acid (CHCA) matrix respectively, than the classical CHCA matrix. The signals of higher mass peptide/protein peaks (m/z 3600-17000) were up to 2× higher with using flower-like AuNPs-enriched CHCA matrix than conventional CHCA matrix. The signal of profile peaks generated by intact cell MALDI-TOFMS of fibroblast suspension (m/z 4000-20000) was 2× higher with using flower-like AuNPs combined with sinapinic acid (SA) compared to SA matrix alone. The use of flower-like AuNPs as internal calibration standard for the calibration of MS spectra of peptides was performed. CONCLUSIONS: Flower-like AuNPs and flower-like AuNPs combined with CHCA or SA as combined matrices for MS measurement of peptides and proteins were used. Comparison of the conventional MALDI method and our method with flower-like AuNPs was carried out. In addition, gold clusters generated from flower-like AuNPs by SALDI provide a suitable internal calibration standard for MS analysis of peptides. Copyright © 2015 John Wiley & Sons, Ltd.

Characterization of Ni(II) complexes of Schiff bases of amino acids and (S)-N-(2-benzoylphenyl)-1-benzylpyrrolidine-2-carboxamide using ion trap and QqTOF electrospray ionization tandem mass spectrometry.

This work demonstrates the application of electrospray ionization mass spectrometry (ESI-MS) using two different mass analyzers, ion trap and hybrid quadrupole time-of-flight (QqTOF) mass analyzer, for the structural characterization of Ni(II) complexes of Schiff bases of (S)-N-(2-benzoylphenyl)-1-benzylpyrrolidine-2-carboxamide with different amino acids. ESI enables the determination of molecular weight on the basis of rather simple positive-ion ESI mass spectra containing only protonated molecules and adducts with sodium or potassium ions. Fragmentation patterns are characterized by tandem mass spectrometric experiments, where both tandem mass analyzers provide complementary information. QqTOF data are used for the determination of elemental composition of individual ions due to mass accuracies always better than 3 ppm with the external calibration, while multistage tandem mass spectra obtained by the ion trap are suitable for studying the fragmentation paths. The novel aspect of our approach is the combination of mass accuracies and relative abundances of all isotopic peaks in isotopic clusters providing more powerful data for the structural characterization of organometallic compounds containing polyisotopic elements. The benefit of relative and absolute mean mass accuracies is demonstrated on the example of studied Ni(II) complexes.

Comparison of negative ion electrospray mass spectra measured by seven tandem mass analyzers towards library formation.

A library of negative ion electrospray ionization mass spectra and tandem mass spectra (MS/MS) of sulfonated dyes has been developed for fast identification purposes. The uniform protocol has been elaborated and applied to the measurements of more than 50 anionic dyes. Three collision energies are selected in our protocol which ensures that at least one of them provides a suitable ratio of product ions to the precursor ion. The robustness is investigated with altered values of tuning parameters (e.g. the pressure of the nebulizing gas, the temperature and the flow rate of drying gas, and the mobile phase composition). The results of the inter-laboratory comparison of product ion mass spectra recorded on seven different tandem mass spectrometers (three ion traps, two triple quadrupoles and two hybrid quadrupole time of flight instruments) are presented for four representative anionic dyes--azo dye Acid Red 118, anthraquinone dye Acid Violet 43, triphenylmethane dye Acid Blue 1 and Al(III) metal-complex azo dye. The fragmentation patterns are almost identical for all tandem mass analyzers, only the ratios of product ions differ somewhat which confirms the possibility of spectra transfer among different mass analyzers with the goal of library formation.

Electrospray ionization-multistage tandem mass spectrometry of complex multitin organometallic compounds.

The series of 14 complex organotin(IV) compounds containing many tin atoms and noncovalent bonds in the structure was characterized by electrospray ionization multistage tandem mass spectrometry (ESI-MS(n)). The mass spectra were measured in both polarity modes to obtain complementary structural information. The characteristic pattern of ten natural tin isotopes allowed the determination of the number of tin atoms in the molecular adducts and fragment ions by comparing theoretical and experimental isotopic distributions. Positive ion ESI spectra show unusual adduct formation depending on the type of organic solvent used for the direct infusion analysis owing to the ion-molecule reactions in the ion source. On the basis of the detailed spectral interpretation of organotin(IV) compounds, the fragmentation patterns of multitin organometallic compounds have been proposed. Noncovalent bonds in polymeric complexes are fragmented first, which is then followed by characteristic neutral losses in monomeric units.

Metabolic profiling of a potential antifungal drug, 3-(4-bromophenyl)-5-acetoxymethyl-2,5-dihydrofuran-2-one, in mouse urine using high-performance liquid chromatography with UV photodiode-array and mass spectrometric detection.

3-(4-bromophenyl)-5-acetyloxymethyl-2,5-dihydrofuran-2-one (LNO-18-22) is a representative member of a novel group of potential antifungal drugs, derived from a natural 3,5-disubstituted butenolide, (-)incrustoporine, as a lead structure. This lipophilic compound is characterized by high in vitro antifungal activity and low acute toxicity. For the purpose of in vivo studies, a new bioanalytical high-performance liquid chromatographic method with UV photodiode-array and mass spectrometric detection (HPLC-PDA-MS), involving a direct injection of diluted mouse urine was developed and used in the evaluation of the metabolic profiling of this drug candidate. The separation of LNO-18-22 and its phase I metabolites was performed in 37 min on a 125 mmx4 mm chromatographic column with Purospher RP-18e using an acetonitrile-water gradient elution. Scan mode of UV detection (195-380 nm) was employed for the identification of the parent compound and its biotransformation products in the biomatrix. Finally, the identity of LNO-18-22 and its metabolites was confirmed using HPLC-MS analyses of the eluate. These experiments demonstrated the power of a comprehensive analytical approach based on the combination of xenobiochemical methods and the results from tandem HPLC-PDA-MS (chromatographic behaviour, UV and MS spectra of native metabolites versus synthetic standards). The chemical structures of five phase I LNO-18-22 metabolites and one phase II metabolite were elucidated in the mouse urine, with two of these metabolites having very unexpected structures.

Structural analysis of ionic organotin(IV) compounds using electrospray tandem mass spectrometry.

A novel electrospray ionization (ESI) mass spectrometric approach for the structure elucidation of ionic organotin(IV) compounds or complexes with weakly bonded ligands as for example monodentate carboxylates or sulfonates is proposed using both positive-ion and negative-ion ESI tandem mass spectra. The ionization mechanism of organotin(IV) compounds involves the cleavage of the most labile bond with an ionic character yielding two complementary ions, [Cat]+ and [An]-. Positively charged species containing tin atom, [Cat]+, are analyzed in the positive-ion mode and negatively charged species without the tin atom, [An]-, in the negative-ion mode. Fragmentation patterns of [C24H29N2Sn]+, [C21H22NSn]+, and [C17H30NSn]+ ions are proposed based on the detailed interpretation of MSn spectra, which is simplified by an easy recognition of characteristic tin isotopic clusters in particular fragment ions. Proposed fragmentation mechanisms are supported by comparison with MSn spectra of deuterium-labeled analogues. The applicability of this method is illustrated on two sets of organotin(IV) compounds, including seven [2,6-bis(dimethylaminomethyl)phenyl]diphenyltin(IV) derivatives with small inorganic counteranions X (Br, NO3, SCN, BF4, SeCN, CN, PF6), six organotin(IV) complexes containing two C,N-chelating ligands with azo dyes, and the identification of unknown hydrolysis products.

Two-dimensional liquid chromatography normal-phase and reversed-phase separation of (co)oligomers.

Many samples contain compounds with various numbers of two or more regular structural groups. Such "multidimensional" samples (according to the Giddings' notation) are best separated in orthogonal chromatographic systems with different selectivities for the individual repeat structural groups, described by separation factors. Correlations between the repeat group selectivities characterize the degree of orthogonality and suitability of chromatographic systems for two-dimensional (2D) separations of two-dimensional samples. The range of the structural units in that can be resolved in a given time can be predicted on the basis of a model describing the repeat group selectivity in the first- and second-dimension systems. Two-dimensional liquid chromatographic system combining reversed-phase (RP) mode in the first dimension and normal-phase (NP) mode in the second dimension were studied with respect to the possibilities of in-line fraction transfer between the two modes. Hydrophilic interaction liquid chromatography (HILIC) with an aminopropyl silica column (APS) is more resistant than classical non-aqueous NP systems against adsorbent desactivation with aqueous solvents transferred in the fractions from the first, RP dimension to the second dimension. Hence, HILIC is useful as a second-dimension separation system for comprehensive RP-NP LCxLC. A comprehensive 2D RP-NP HPLC method was developed for comprehensive 2D separation of ethylene oxide-propylene oxide (EO-PO) (co)oligomers. The first-dimension RP system employed a 120 min gradient of acetonitrile in water on a C18 microbore column at the flow-rate of 10 microL/min. In the second dimension, isocratic HILIC NP with ethanol-dichloromethane-water mobile phase on an aminopropyl silica column at 0.5 mL/min was used. Ten microliter fractions were transferred from the RP to the HILIC NP system at 1 min switching valve cycle frequency.

Phase system selectivity and two-dimensional separations in liquid column chromatography.

Correlations between the separation selectivity in aqueous and non-aqueous reversed-phase systems and in normal-phase LC systems were investigated for samples containing different numbers of two repeat structural elements. Such samples are best separated in "orthogonal" two-dimensional chromatographic systems, showing selectivity for one type of the repeat structural element only in the first dimension and for the other structural element only in the second dimension. The number of resolved compounds improves as the degree of orthogonality of the separation systems increases with decreasing correlation between the selectivities for the sample structural distribution in the two dimensions. Orthogonal systems with non-correlated selectivities for each repeat structural element provide the highest number of separated peaks and regular arrangement of the peaks over the two-dimensional retention space according to the individual structural element distribution and the best use of the available peak capacity. Fully orthogonal systems are difficult to find in practice. Partially orthogonal system with correlated selectivities for one structural type distribution, but with one system non-distinguishing the distribution for the other structural element are still useful for the two-dimensional separations. The correlations between the selectivities for repeat regular structural increments were employed to evaluate the suitability of phase systems for two-dimensional HPLC separations. The selectivity correlation in various reversed-phase and normal-phase systems was evaluated for two sample types: (1) Various RP columns show significantly inversely correlated selectivities for acyl lengths and numbers of double bonds distribution, but the differences in the double bond selectivity can be used for practical separations of triacylglycerols with the same equivalent carbon numbers. (2) Synthetic EO-PO block (co)oligomers with two-dimensional distribution of oxyethylene and oxypropylene monomer units were separated according to the two distribution types using on-line two-dimensional reversed-phase-normal-phase LC with a C18 column in the first dimension and an aminopropyl silica column in the second dimension.

Structural analysis of 2,6-[bis(alkyloxy)methyl]-phenyltin derivatives using electrospray ionization mass spectrometry.

Electrospray ionization (ESI) mass spectrometry was applied to the structural analysis of 23 2,6-[bis(alkyloxy)methyl]phenyltin(IV) derivatives. The mass spectra were measured in both polarity modes and multistage tandem mass spectrometric (MS(n)) measurements were performed on the ion trap analyser for positively charged tin-containing ions. The sum of complementary ions observed in the positive-ion mode (i.e. [M-R(3)](+) ion) and in the negative-ion mode (i.e. [R(3)](-) ion) permits molecular mass determination in spite of the fact that the molecular adducts were often missing even in the first-order mass spectra. The subsequent fragmentation of [M-R(3)](+) ions studied by MS(n) and the correlation of observed fragment ions with the expected structures of synthesized organotin(IV) compounds allowed us to understand the fragmentation behaviour and the mechanism of the ion formation for studied compounds. The typical neutral losses are alkenes, alcohols and aldehydes. The fragmentation pattern of one selected compound was supported by MS(n) measurements of an isotopically labelled analogue to confirm unusual ion-molecule reactions of some fragment ions with water in the ion trap.

Respiratory viruses and severe lower respiratory tract complications in hospitalized patients.

BACKGROUND: Acute respiratory viral infections are generally self-limited in healthy subjects but can lead to severe complications in immunocompromised hosts. We report the clinical impact of acute lower respiratory tract viral infections in hospitalized patients. MATERIALS AND METHODS: Of 1,001 fiberoptic bronchoscopies performed during a period of 5 years, 33 BAL samples were positive for respiratory viruses by cell culture. The main diagnosis, length of hospitalization, response to initial treatment, and the mortality rate at 30 days were analyzed. Spirometry performed before and after infection was compared in lung transplant recipients. RESULTS: The following respiratory viruses were identified in 33 cases: influenza A or B (n = 13), parainfluenza virus 1-3 (n = 7), rhinovirus (n = 5), respiratory syncytial virus (n = 4), and adenovirus (n = 4). All cases were immunocompromised patients who acquired new respiratory symptoms and/or radiologic abnormalities suggesting a pulmonary infection. Twenty-five patients (74%) did not respond to initial broad-spectrum antibiotics, and 11 patients (33%) required intensive care for respiratory failure. The overall mortality rate at 1 month was 24%. In patients with a sole viral pathogen identified in their BAL, the mortality rate was 39%. In lung transplant recipients (n = 10), the mean FEV(1) decreased from 2.2 to 1.9 L/s before and during the infection episode, respectively (p < 0.01); 3 months later, 60% of the patients had still not completely recovered to baseline values. CONCLUSION: Respiratory viruses recovered in BAL samples of immunocompromised patients are associated with severe lower respiratory complications. In lung transplant recipients, we observed a persisting impairment of pulmonary function.

Improved monitoring of cytomegalovirus infection after allogeneic hematopoietic stem cell transplantation by an ultrasensitive plasma DNA PCR assay.

Cytomegalovirus (CMV) DNA amplification assays in plasma have shown limited sensitivity compared to the detection of pp65 antigen in leukocytes. Our goal was to increase the sensitivity of a commercial CMV DNA PCR quantitative assay. After modification, the new assay was able to reproducibly detect 20 CMV DNA copies/ml of plasma. We compared this new ultrasensitive PCR assay with the standard PCR and the pp65 test for CMV detection and quantification in 22 consecutive allogeneic hematopoietic stem cell recipients. CMV infection or reactivation was detected in 84 of 319 (26%) samples by the ultrasensitive PCR assay compared to 38 of 319 (12%) samples by the pp65 assay (P < 0.01). All samples positive by the pp65 assay were positive by the ultrasensitive PCR, and CMV episodes were detected on average 4 days earlier and 7 days later than the first and the last pp65-positive test, respectively. In addition, during CMV episodes, the ultrasensitive assay identified positive samples that were inconsistently detected by the pp65 assay. The ultrasensitive assay was also much more sensitive than the standard PCR, with 26 versus 12% of CMV DNA-positive samples (P < 0.01). This assay improved the monitoring of CMV infection or reactivation in hematopoietic allogeneic stem cell recipients.

Secular trends of candidemia over 12 years in adult patients at a tertiary care hospital.

The incidence of fungal infections has been increasing for the last 3 decades, especially among neutropenic, cancer, and critically ill patients. These infections are associated with high mortality rates. We retrospectively reviewed medical charts of adult patients with fungemia from 1989 to 2000 at our institution. The characteristics of the population groups served by the hospital were described. Of 328 patients with fungemia, we reviewed 315 (96%) medical records, and focused on those with candidemia (n = 294). The species distribution in patients with candidemia showed that the most commonly identified species were Candida albicans (66%), followed by C. glabrata (17%), and C. parapsilosis (6%). The incidence of candidemia ranged from 0.2 to 0.46 per 10,000 patient-days with the highest incidence in 1993 and the lowest in 1997. Although most studies show an increased incidence of candidemia, we observed a reduction over the study period. Furthermore, we observed no shift from C. albicans to non-albicans Candida species despite a significant increase in the use of fluconazole. The overall mortality among patients with candidemia was 44%, with the highest rate in patients over 65 years (52%). Factors independently associated with higher mortality were patient age greater than 65 years, intensive care unit admission, and underlying cancer.

Exon 1 polymorphism of the B2BKR gene does not influence the clinical status of patients with hereditary angioedema.

Polymorphic variants of B2 receptors for bradykinin (B2BKR) have been postulated to influence a clinical manifestation of hereditary angioedema. In this study, exon 1 nonanucleotide deletion polymorphism in the B2BKR gene was examined in 37 patients with hereditary angioedema. The patients were grouped according to disease severity or the age of the first clinical manifestation of disease. No significant differences in allelic frequencies were found between particular subgroups of patients. Therefore, we concluded that this polymorphism does not seem to have any significant effect on the course and severity of hereditary angioedema in Caucasians.

Five novel mutations in the C1 inhibitor gene (C1NH) leading to a premature stop codon in patients with type I hereditary angioedema.

Hereditary angioedema (HAE) is a disorder characterised by recurrent attacks of localized subcutaneous or submucosal edema. It is inherited in an autosomal dominant fashion and caused by a deficiency of C1 inhibitor (C1 inh, or C1NH). Most patients with HAE have an absolute deficiency of C1 inh (type I HAE) while the rest (15% of kindreds) synthetize a dysfunctional C1 inh protein (type II HAE). In this report a novel use of denaturing gradient gel electrophoresis (DGGE) followed by direct sequencing of the C1 inhibitor gene is presented. Five novel mutations, one nonsense (p.S48X) and four small deletions resulting in frameshifts (g.2264-2265delAG, g.2304delC, g.8493-8494delCC and g.16676-16677delTG) have been identified in the C1 inhibitor gene in five families with type I HAE. All of these mutations lead to premature termination of translation and thus can be considered causative of the C1 inh deficiency. Moreover, two previously described mutations in the reactive center of C1 inh, p.R444C and p.R444H, have been detected in four unrelated patients with type II HAE.