NEUTRAL LOSS ANALYSIS OF AMINO ACIDS BY DESORPTION ELECTROSPRAY IONIZATION USING AN UNMODIFIED TANDEM QUADRUPOLE MASS SPECTROMETER.

A new method to analyze free amino acids using desorption electrospray ionization (DESI) has been implemented. The method is based on the neutral loss mode determination of underivatized amino acids using a tandem quadrupole mass spectrometer equipped with an unmodified atmospheric interface. Qualitative and quantitative optimization of DESI parameters, including ESI voltage, solvent flow rate, angle of collection and incidence, gas flow and temperatures, was performed for amino acids detection. The parameters for DESI analysis were evaluated using a mixture of valine, leucine, methionine, phenylalanine and tyrosine standards. A few microliters of this mixture were deposited on a slide, dried and analyzed at a flow rate of 2mL/min. The optimal ionization response was obtained using laboratory glass slides and an equivalent solution of water/methanol doped with 2% of formic acid. The method specificity was evaluated by comparing product ion spectra and neutral loss analysis of amino acids obtained either by DESI or by electrospray ionization flow injection analysis (ESI-FIA). To evaluate the quantitative response on amino acids analyzed by DESI, calibration curves were performed on amino acid standard solutions spiked with a fixed amount of labelled amino acids. The method was also employed to analyze free amino acids from blood spots, after a rapid solvent extraction without other sample pretreatment, from positive and negative subjects. The method enables one to analyze biological samples and to discriminate healthy subjects from patients affected by inherited metabolic diseases. The intrinsic high-throughput analysis of DESI represents an opportunity, because of its potential application in clinical chemistry.....

High-resolution proteomics and machine-learning identify protein classifiers of honey made by Sicilian black honeybees (Apis mellifera ssp. sicula)

Apis mellifera ssp. sicula, also known as the Sicilian black honeybee, is a Slow Food Presidium that produces honey with outstanding nutraceutical properties, including high antioxidant capacity. In this study, we used high-resolution proteomics to profile the honey produced by sicula and identify protein classifiers that distinguish it from that made by the more common Italian honeybee (Apis mellifera ssp. ligustica). We profiled the honey proteome of genetically pure sicula and ligustica honeybees bred in the same geographical area, so that chemical differences in their honey only reflected the genetic background of the two subspecies, rather than botanical environment. Differentially abundant proteins were validated in sicula and ligustica honeys of different origin, by using the so-called "rectangular strategy", a proteomic approach commonly used for biomarker discovery in clinical proteomics. Then, machine learning was employed to identify which proteins were the most effective in distinguishing sicula and ligustica honeys. This strategy enabled the identification of two proteins, laccase-5 and venome serine protease 34 isoform X2, that were fully effective in predicting whether honey was made by sicula or ligustica honeybees. In conclusion, we profiled the proteome of sicula honey, identified two protein classifiers of sicula honey in respect to ligustica, and proved that the rectangular strategy can be applied to uncover biomarkers to ascertain food authenticity

Phenotypical and molecular assessment of the virulence potential of KPC-3-producing Klebsiella pneumoniae ST392 clinical isolates

Klebsiella pneumoniae is a Gram-negative bacterium of clinical importance, due to its resistance to several antibiotic classes. We have identified 4 clinical isolates of K. pneumoniae sequence type (ST) 392 KPC-3-producing strains from patients at the Istituto Mediterraneo per i Trapianti e Terapie ad Alta Specializzazione (IRCCS-ISMETT), a Southern Italian transplantation health facility, during a routine surveillance for carbapenemase-producing Enterobacterales from in-house clinical samples. Since those were among, to the best of our knowledge, the first KPC-producing K. pneumoniae ST392 isolated in Europe, we assessed their virulence potential, to understand if this particular ST can become an endemic clinical threat. ST392 isolates were investigated to assess their virulence potential, namely resistance to human sera, formation of abiotic biofilms, adhesion to biotic surfaces, exopolysaccharide production and in vivo pathogenesis in the wax moth Galleria mellonella animal model. ST392-belonging strains were highly resistant to human sera. These strains also have a high capacity to form abiotic biofilms and high levels of adhesion to the human epithelial colorectal adenocarcinoma HT-29 cell line. An increase of transcriptional levels of genes involved in serum resistance (aroE and traT) and adhesion (pgaA) was observed when compared with the Klebsiella quasipneumoniae subsp. similipneumoniae strain ATCC 700603 reference strain. Infection of G. mellonella larvae with ST392 clinical isolates showed that the latter were not highly pathogenic in this model. Together, our results indicate that ST392 isolates have the potential to become a strain of clinical relevance, especially in health settings where patients are immunosuppressed, e.g., transplant recipients

Evaluation of mobile phase, ion pairing, and temperature influence on an HILIC-MS/MS method for L-arginine and its dimethylated derivatives detection.

Asymmetric NG,–NG-dimethylarginine (ADMA) increases in diseases such as renal failure, diabetes mellitus, and hypercholesterolemia. The feasibility and utility of a hydrophilic interaction chromatography (HILIC) method for the separation of free Larginine (Arg), ADMA, and symmetric NG,–NG9-dimethylarginine (SDMA) on a typical silica column were explored and the impact of some experimental parameters on the chromatographic behavior of these analytes was investigated. The effect of water and TFA content in mobile phase and of column temperature was investigated during the development of a fast and simple HILIC-MS/MS method that might be suitable for the quantification of free Arg, ADMA, and SDMA in plasma for routine analysis. Our results show that a good compromise between efficiency and peak shape with acceptable retention and total chromatographic run time is achieved using an ACN/water (90:10) mobile phase with TFA% as additive ranging from 0.015 to 0.025% and column temperature ranging from 25 to....

Profiling of acylcarnitines and sterols from dried blood or plasma spot by atmospheric pressure thermal desorption chemical ionization (APTDCI) tandem mass spectrometry

Free carnitine and acylcarnitines play an important role in the metabolism of fatty acids. Sterols are structural lipids found in themembranes ofmany eukaryotic cells, and they also have functional roles such as the regulation of membrane permeability and fluidity, activity of membrane-bound enzymes and signals transduction. Abnormal profiles of these compounds in biological fluids may be useful markers of metabolic changes. In this review,we describe the subset of the lipidome represented by acylcarnitines and sterols, andwe summarize how these compounds have been analyzed in the past. Over the last 50 years, lipid mass spectrometry (MS) has evolved to become one of the most useful techniques for metabolic analysis. Today, the introduction of new ambient ionization techniques coupled to MS (AMS), which are characterized by the direct desorbing/ionizing of molecules from solid samples, is generating new possibilities for in situ analysis. Recently, we developed an AMS approach called APTDCI to desorb/ionize using a heated gas flow and an electrical discharge to directly analyze sterols and indirectly investigate acylcarnitines in dried blood or plasma spot samples. Here, we also describe the APTDCI method and some of its clinical applications, and we underline the common complications and issues that remain to be resolved. This article is part of a Special Issue entitled: Lipodomics and ImagingMass Spectrometry