Protein phosphorylation mediated by protein kinases is one of the most significant posttranslational modifications in many biological events. The function and physiological substrates of specific protein kinases, which are highly associated with known signal transduction elements or therapeutic targets, have been extensively studied using various approaches; however, most protein kinases have not yet been characterized. In recent decades, many techniques have been developed for the identification of in vitro and physiological substrates of protein kinases. In this review, I summarize recent studies profiling the characteristics of kinases using mass spectrometry-based proteomics, focusing on the large-scale identification of in vitro substrates of the human kinome using a quantitative phosphoproteomics approach.
Temperature-resolved proton transfer reactions of multiply-protonated angiotensin I, disulfide-intact and -reduced lysozyme, and ubiquitin ions to primary, secondary and aromatic amines were examined in the gas phase. Absolute reaction rate constants for the proton transfer were determined from the intensities of the parent and product ions in mass spectra. Dramatic changes were observed in the distribution of product ions and the reaction rate constants. In particular, the rate constants for disulfide-intact lysozyme ions changed more drastically with the change in charge state and temperature compared to the corresponding values for disulfide-reduced ions. Proton transfer reactions were enhanced or suppressed as the result of the formation of complexes between the ions with gaseous molecules, which is related to changes in their conformation with changing.
In 2007, probe electrospray ionization/mass spectrometry (PESI/MS) was developed. In this technique, the needle is moved down along a vertical axis and the tip of the needle touched to the sample. After capturing the sample at the needle tip, the needle is then moved up and a high voltage is applied to the needle at the highest position to generate electrospray. Due to the discontinuous sampling followed by the generation of spontaneous electrospray, sequential and exhaustive electrospray takes place depending on the surface activity of the analytes. As modified versions of PESI, dipping PESI (dPESI), sheath-flow PESI (sfPESI) and adjustable sfPESI (ad-sfPESI) have been developed. These methods are complementary to each other and they can be applicable to surface and bulk analysis of various biological samples. In this article, the characteristics of these methods and their applications to real samples will be reviewed.
The rapid development of next-generation sequencing techniques has enabled single-cell genomic and transcriptomic analyses, which have revealed the importance of heterogeneity in biological systems. However, analytical methods to accurately identify and quantify comprehensive metabolites from single mammalian cells with a typical diameter of 10–20 μm are still in the process of development. The aim of this study was to develop a single-cell metabolomic analytical system based on highly sensitive nano-liquid chromatography tandem mass spectrometry (nano-LC-MS/MS) with multiple reaction monitoring. A packed nano-LC column (3-μm particle-size pentafluorophenylpropyl Discovery HSF5 of dimensions 100 μm i.d.×180 mm) was prepared using a slurry technique. The optimized nano-LC-MS/MS method showed 3–132-fold (average value, 26-fold) greater sensitivity than semimicro-LC-MS/MS, and the detection limits for several hydrophilic metabolites, including amino acids and nucleic acid related metabolites were in the sub-fmol range. By combining live single-cell sampling and nano-LC-MS/MS, we successfully detected 18 relatively abundant hydrophilic metabolites (16 amino acids and 2 nucleic acid related metabolites) from single HeLa cells (n=22). Based on single-cell metabolic profiles, the 22 HeLa cells were classified into three distinct subclasses, suggesting differences in metabolic function in cultured HeLa cell populations. Our single-cell metabolomic analytical system represents a potentially useful tool for in-depth studies focused on cell metabolism and heterogeneity.
Serotonin, an important neurotransmitter, is produced mainly in intestines, and serotonin levels in feces can be an indicator of the intestinal environment. Human feces, however, contain a large amount of contaminants, which vary widely owing to food contents and the intestinal environment, and these contaminants would be expected to interfere with the determination of serotonin levels in human feces. To remove these contaminants and determine serotonin levels, we developed a new method using solid phase extraction (SPE) and column-switching LC-MS/MS. Serotonin, labeled with a stable isotope, was added to human feces samples prior to SPE as an internal standard to correct for individual differences in matrix effects. The recovery rate for SPE was 55.9–81.0% (intraday) and 56.5–78.1% (interday) for feces from two subjects. We analyzed 220 fecal samples from 96 subjects including 76 pregnant and post-delivery women. The endogenous serotonin content per unit weight of dried feces was 0.09–14.13 ng/mg for pregnant and post-delivery women and 0.30–9.93 ng/mg for the remaining subjects.
Congenital disorders of glycosylation (CDG), an increasingly recognized group of diseases that affect glycosylation, comprise the largest known subgroup of approximately 100 responsible genes related to N-glycosylation. This subgroup presents various molecular abnormalities, of either the CDG-I or the CDG-II type, attributable to a lack of glycans or abnormal glycoform profiles, respectively. The most effective approach to identifying these N-glycosylation disorders is mass spectrometry (MS) using either released glycans, intact glycoproteins or proteolytic peptides as analytes. Among these, MS of tryptic peptides derived from transferrin can be used to reliably identify signature peptides that are characteristic of CDG-I and II. In the present study, matrix-assisted laser desorption/ionization (MALDI) MS was applied to various N-glycosylation disorders including ALG1-CDG, B4GALT1-CDG, SLC35A2-CDG, ATP6V0A2-CDG, TRAPPC11-CDG and MAN1B1-CDG. This method does not require the prior enrichment of glycopeptides or chromatographic separation, and thus serves as a practical alternative to liquid chromatography-electrospray ionization MS. The signature peptides are biomarkers of CDG.
We measured the Re/Os (185Re/188Os) and 187Os/188Os ratios from nanoparticles (NPs) using a multiple collector-inductively coupled plasma-mass spectrometer equipped with high-time resolution ion counters (HTR-MC-ICP-MS). Using the HTR-MC-ICP-MS system developed in this study, the simultaneous data acquisition of four isotopes was possible with a time resolution of up to 10 μs. This permits the quantitative analysis of four isotopes to be carried out from transient signals (e.g., <0.6 ms) emanating from the NPs. Iridium–Osmium NPs were produced from a naturally occurring Ir–Os alloy (ruthenosmiridium from Hokkaido, Japan; osmiridium from British Columbia, Canada; iridosmine from the Urals region of Russia) through a laser ablation technique, and the resulting nanoparticles were collected by bubbling water through a suspension. The 187Os/188Os ratios for individual NPs varied significantly, mainly due to the counting statistics of the 187Os and 188Os signals. Despite the large variation in the measured ratios, the resulting 187Os/188Os ratios for three Ir–Os bearing minerals, were 0.121±0.013 for Hokkaido, 0.110±0.012 for British Columbia, and 0.122±0.020 for the Urals, and these values were in agreement with the ratios obtained by the conventional laser ablation-MC-ICP-MS technique. The data obtained here provides a clear demonstration that the HTR-MC-ICP-MS technique can become a powerful tool for monitoring elemental and isotope ratios from NPs of multiple components.
Platinum, a transition metal that is widely used in anti-cancer agents, also results in the development of nephropathy due to severe adverse reactions caused by platinum-induced nephrotoxicity. Reports on imaging with metals other than platinum remain are limited, even in preclinical studies. Furthermore, most of these are case reports, and the relationship between the distribution of the metal and clinical observations in human samples is not well understood. Here we report on visualizing lanthanum (139La), a component of Fosrenol, which is usually used for the treatment of hyperphosphatemia. Gastric inflammation, also known as hemorrhagic gastritis, is the main adverse event caused by Fosrenol. To conduct this study, 139La was visualized in gastric biopsy samples obtained from a patient using quantitative laser ablation-inductively coupled plasma-mass spectrometry (LA-ICP-MS). We also compared the distribution of 139La in tissue and histochemical results. The areas where 139La accumulated corresponded to the macrophage-positive areas observed in immunohistochemistry studies using an anti-CD68 antibody. In contrast, we observed a debris-like crystal morphology in hematoxylin and eosin staining tissues. The debris was also associated with 139La accumulation. The abnormal accumulation of 139La crystals caused the observed inflammation. This phenomenon was previously characterized, but this is the first report in which 139La distribution and histochemical results are compared using LA-ICP-MS.
The compounds inside rice koji have been thoroughly investigated as an essential material in making many food-related products, including sake. However, these studies focused only on quantitative aspects, leaving features that can still be uncovered if seen from a new perspective. Visualization of the metabolites inside rice koji may as well be the new angle needed to retrieve more information regarding rice koji making. Here we utilized mass spectrometry imaging (MSI) to visualize the distribution of sugars, sugar alcohols, and amino acids inside rice koji. Imaging results revealed that several sugars alcohols and amino acids were shown to have characteristic distribution near the edges or surface of rice koji. Furthermore, the distribution appears to be correlated with the different structure of rice koji. This study is the first report of using MSI to visualize sugars, sugar alcohols, and amino acids in rice koji.
Post-translational modifications (PTMs) of histone N-terminal tails in nucleosome core particle (NCP), such as acetylation, play crucial roles in regulating gene expression. To unveil the regulation mechanism, atomic-level structural analysis of in-vitro modified NCP is effective with verifying the PTMs of histones. So far, identification of PTMs of NCP originating from living cells has mainly been performed using mass spectrometry (MS) techniques, such as bottom-up approach. The bottom-up approach is the most established method for protein characterization, but it does not always provide sufficient information on the acetylated sites of lysine residues in the histone tails if trypsin digestion is carried out. For histone proteins, which have many basic amino acids, trypsin generates too many short fragments that cannot be perfectly analyzed by tandem MS. In this study, we investigated the in vitro acetylation sites in the histone H3 tail using a top-down sequence analysis, matrix-assisted laser desorption/ionization in-source decay (MALDI-ISD) experiment, in combination with aminopeptidase digestion. Aminopeptidase can cleave peptide bonds one-by-one from the N-terminus of peptides or proteins, generating N-terminally truncated peptides and/or proteins. As a result, it was identified that this method enables sequence characterization of the entire region of the H3 tail. Also, application of this method to H3 in in-vitro acetylated NCP enabled assigning acetylation sites of H3. Thus, this method was found to be effective for obtaining information on in-vitro acetylation of NCP for structural biology study.
Combined therapy using photodynamic therapy (PDT) and chemotherapy has been proposed for anticancer-drug-resistant cancer cells. To evaluate the efficacy of such a combined therapy, the uptakes of an anticancer drug and a photosensitizer in cancer cells must be assessed. Mass spectrometry using matrix-assisted laser desorption/ionization can detect multiple drugs simultaneously. Human prostate cancer cells PC-3 or docetaxel-resistant cancer cells PC-3-DR were incubated in a serum-free medium containing a photosensitizer, protoporphyrin IX (PpIX), and an anticancer drug, docetaxel. A zeolite matrix was created by mixing 6-aza-2-thiothymine and NaY5.6 zeolite, and dissolving in water with 50% acetone. Ions were obtained with a time-of-flight mass spectrometer using a Nd:YAG laser at a wavelength of 355 nm. The cell morphology was preserved by washing the cells with ammonium acetate and drying in a vacuum after drug administration. Protonated PpIX (m/z 563.3) and the sodium adduct ion of docetaxel (m/z 829.9) were obtained from PC-3 cells simultaneously using the zeolite matrix. On the other hand, PpIX was detected but ions originating from docetaxel were not detected from PC-3-DR cells. The result indicated the efficacy of PDT for docetaxel-resistant cancer cells.
The Kendrick analysis is used for the processing and visualization of mass spectra obtained from polymers containing C, H, O and/or Si with simple isotopic patterns (monoisotope=lightest isotope=most intense isotope for short chains). In the case of heteroatoms with complex isotopic patterns, the impact of the chosen isotope on point alignments in Kendrick plots has not been examined extensively. Rich isotopic patterns also make the evaluation of the mass and nature of the repeating unit and end-groups more difficult from the mass spectrum in the case of unknown samples due to the number of peaks and the absence of a monoisotopic peak. Using a polybrominated polycarbonate as running example, we report that horizontal point alignments can be obtained in a Kendrick plot using the mass of the most abundant isotope instead of the monoisotopic mass as is usually done. Rotating the plot (“reverse Kendrick analysis”) helps to accurately evaluate the mass of the most abundant isotope of the repeating unit, as well as the nature of the brominated neutral expelled upon gentle heating (debromination or dehydrobromination). The whole procedure is then applied to the characterization of an unknown polybrominated flame retardant in an industrial formulation before and after heating.
Peptides larger than 3–4 kDa, such as neuropeptide Y (NPY), orexin-B, and alpha-MSH, have practical issues that arise when conducting direct and sensitive quantitative liquid chromatography (LC) orbitrap-FT mass spectrometry (MS) due to their adsorption and low ionization efficiency, especially in standard solutions. A mixing solvent consisting of 0.5% trifluoroacetic acid (TFA) and 35–50% aq. acetonitrile was developed as the standard NPY for creating calibration curves, as well as a matrix to block the experimental tube surface to minimize adsorption. The mixture matrix effectively blocked non-specific adsorption of the standard peptides with tryptic digested bovine serum albumin (BSA) (small fragment peptides) and orexin-B (a large chain peptide). A sample containing 1 : 100 peptide:water was detected in the developed sample solution. Finally, 2 to 1,000 fmol/μL NPY could be analyzed quantitatively and reproducibly using conventional LC-MS. Parameters of the calibration curves, such as X-intercept, Bias (%), and relative standard deviation (RSD), were adjusted to optimize the sample solutions and the sensitive and quantitative LC-MS analyses.
Using a multi-turn time-of-flight (TOF) mass spectrometer, we have extracted a single xenon isotope ion, 129Xe+, from its orbit at given a lap number without disturbing the rest of isotopes. After detecting the 129Xe+ at 20 laps, the rest of the xenon isotope spectrum was obtained at 30 laps, which generated a TOF spectrum where the TOF difference between 129Xe+ and 130Xe+ was 87.4 μs while 130Xe+ and 131Xe+ were 1.03 μs. The time distance between 129Xe+ and other isotopes can be set by any lap difference that is a factor of 8.7 μs, which depends on the acceleration voltage and the mass of the ion. Method accuracy was verified by comparing the isotopic abundance ratio of the xenon sample after withdrawing one of the ions from the isotope cluster to the abundance ratio obtained from the conventional method. The TOF stability was also evaluated at various lap numbers between 10 to 230.