Characterization and studies of proteome are challenging because biological samples are complex, with a wide dynamic range of abundance. At present the proteins are identified by digestion into peptides, with subsequent identification of the peptides by mass spectrometry (MS). MS is a powerful technique for the purpose, but it cannot identify every peptide in such complex mixtures simultaneously. For accurate analysis and quantification it is important to separate the peptides first by chromatographyinto fractions of a size that MS can handle. With these less complex fractions, the probability is increased of identifying peptides of low abundance that would otherwise experience ion suppression effects due to the presence of peptides of high abundance. Enrichment for peptides with certain post-translational modifications helps to increase their detection rates as well. Electrostatic repulsion-hydrophilic interaction chromatography (ERLIC) is a mixed-mode chromatographic technique which combines the use of electrostatic repulsion and hydrophilic interaction. This review provides an overview of ERLIC and its variousproteomics applications. ERLIC has been demonstrated to have good orthogonality to reverse phase liquid chromatography (RPLC), making it useful as a first dimension in multidimensional liquid chromatography (MDLC) and fractionation of digestsin general. Peptides elute in order of their isoelectric points and polarity. ERLIC has also been successfully utilized for the enrichment for phosphopeptides and glycopeptides, facilitating their identification. In addition, it is promising for the study ofpeptide deamidation. ERLIC performs comparably well or better than established methods for these various applications, and serves as a viable and efficient workflow alternative.
Nonmedical use of prescription stimulants such as methylphenidate (MPH) and amphetamine (AP) by normal persons has been increased to improve cognitive functions. Due to high potential for their abuse, reliable analytical methods were required to detect these prescription stimulants in biological samples. A direct injection liquid chromatography-tandem mass spectrometric (LC-MS/MS) method was developed and implemented for simultaneous determination of MPH, AP and their metabolites ritalinic acid (RA) and 4-hydroxyamphetamine (HAP) in human urine. Urine sample was centrifuged and the upper layer (100 μL) was mixed with 800 μL of distilled water and 100 μL of internal standards (0.2 μg/mL in methanol). The mixture was then directly injected into the LC-MS/MS system. The mobile phase was composed of 0.2% formic acid in distilled water (A) and acetonitrile (B). Chromatographic separation was performed by using a Capcell Pak MG-II C18 (150 mm × 2.0 mm i.d., 5 μm, Shiseido) column and all analytes were eluted within 5 min. Linear least-squares regression with a 1/x weighting factor was used to generate a calibration curve and the assay was linear from 20 to 1500 ng/mL (HAP), 40-3000 ng/mL (AP and RA) and 2-150 ng/mL (MPH). The intra- and inter-day precisions were within 16.4%. The intra- and inter-day accuracies ranged from -15.6% to 10.8%. The limits of detection for all the analytes were less than 4.7 ng/mL. The suitability of the method was examined by analyzing urine samples from drug abusers.
The method of direct mass spectrometry profiling is reliable and reproducible for the rapid identification of clinical isolates of bacteria and fungi. This is the first study evaluating the approach of MALDI-TOF mass spectrometry profiling for rapid identification of carbapenemase-resistant enterobacteriaceae (CRE). Proof of concept was achieved by the discrimination of CRE using MALDI Biotyper MS based on the protein. This profiling appears promising by the visual observation of consistent unique peaks, albeit low intensity, that could be picked up from the mean spectra (MSP) method. The Biotyper MSP creation and identification methods needed to be optimized to provide significantly improved differences in scores to allow for subspecies identification with and without carbapenemases. These spectra were subjected to visual peak picking and in all cases; there were pertinent differences in the presence or absence of potential biomarker peaks to differentiate isolates. We also evaluated this method for potential discrimination between different carbapenemases bacteria, utilizing the same strategy. Based on our data and pending further investigation in other CREs, MALDI-TOF MS has potential as a diagnostic tool for the rapid identification of even closely related carbapenemases but would require a paradigm shift in which Biotyper suppliers enable more flexible software control of mass spectral profiling methods.
The Brazil nut (Bertholletia excelsa - Lecythidaceae) is considered a product with high economic value, being a food widely appreciated for its nutritional qualities. Although previous studies have reported the biochemical composition of Brazil nut oil, the knowledge regarding the phospholipid composition exhibits a disagreement: the composition of fatty acids present in the structures of phospholipids is reported as being different from the composition of the free fatty acids present in the oil. In this work, solid phase extraction (SPE) was employed to provide a fast extraction of the phospholipids from Brazil nuts, in order to compare the phospholipid profile of the in nature nuts and their fatty acids precursor present in the oil. The major phospholipids were characterized by mass spectrometry approach. Their fragmentation pattern through direct infusion electrospray ionization ion-trap tandem mass spectrometry (ESI-IT-MS2) proved to be useful to unequivocal characterization of these substances. High resolution (HR) experiments through ESI using a quadruple time of flight mass spectrometry (QTOF) system were performed to reinforce the identifications.
The interference of water vapor on the chemical ionization (CI) of hydroxyl radicals (OH) by sulfur hexafluoride ion (SF6-) was investigated using a flow tube system coupled to a high-pressure CI mass spectrometer. Water vapor, which is required to study heterogeneous reactions of OH under real tropospheric conditions, transforms the reagent ion SF6- into SF4O-and F-(HF)n, resulting in a substantial loss in CI sensitivity. Therefore, under humid conditions, peaks corresponding to OH are drastically diminished, while those corresponding to OH-water complex ions ([OH(H2O)n]-) are enhanced. [OH(H2O)3]- was observed as the major OH species. The obsercation of [OH(H2O)n]- by isolating humid conditions to the CI region and preliminary ab initio calculations suggested that [OH(H2O)n]- ions were produced from reactions between OH ions (OH-) and water molecules. An additional helium buffer flow introduced into the CI region reduced loss of the reagent ion and resulted in a partial recovery of OH peak intensities under humid conditions.