Identification of Mycobacterium abscessus using the peaks of ribosomal protein L29, L30 and hemophore-related protein by MALDI-MS proteotyping.

Mycobacteroides (Mycobacterium) abscessus, which causes a variety of infectious diseases in humans, is becoming detected more frequently in clinical specimens as cases are spreading worldwide. Taxonomically, M. abscessus is composed of three subspecies of M. abscessus subsp. abscessus, M. abscessus subsp. bolletii, and M. abscessus subsp. massiliense, with different susceptibilities to macrolides. In order to identify rapidly these three subspecies, we determined useful biomarker proteins, including ribosomal protein L29, L30, and hemophore-related protein, for distinguishing the subspecies of M. abscessus using the matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) profiles. Thirty-three clinical strains of M. abscessus were correctly identified at the subspecies-level by the three biomarker protein peaks. This study ultimately demonstrates the potential of routine MALDI-MS-based laboratory methods for early identification and treatment for M. abscessus infections.

A large-scale genomically predicted protein mass database enables rapid and broad-spectrum identification of bacterial and archaeal isolates by mass spectrometry.

MALDI-TOF MS-based microbial identification relies on reference spectral libraries, which limits the screening of diverse isolates, including uncultured lineages. We present a new strategy for broad-spectrum identification of bacterial and archaeal isolates by MALDI-TOF MS using a large-scale database of protein masses predicted from nearly 200,000 publicly available genomes. We verify the ability of the database to identify microorganisms at the species level and below, achieving correct identification for > 90% of measured spectra. We further demonstrate its utility by identifying uncultured strains from mouse feces with metagenomics, allowing the identification of new strains by customizing the database with metagenome-assembled genomes.

ADAMTS4 is involved in the production of the Alzheimer disease amyloid biomarker APP669-711.

Amyloid-ß (Aß) deposition in the brain parenchyma is one of the pathological hallmarks of Alzheimer disease (AD). We have previously identified amyloid precursor protein (APP)669-711 (a.k.a. Aß(-3)-40) in human plasma using immunoprecipitation combined with matrix-assisted laser desorption ionization time-of-flight mass spectrometry (IP-MALDI-MS). Furthermore, we found that the level of a composite biomarker, i.e., a combination of APP669-711/Aß1-42 ratio and Aß1-40/Aß1-42 ratio in human plasma, correlates with the amyloid PET status of AD patients. However, the production mechanism of APP669-711 has remained unclear. Using in vitro and in vivo assays, we identified A Disintegrin and Metalloproteinase with a Thrombospondin type 1 motif, type 4 (ADAMTS4) as a responsible enzyme for APP669-711 production. ADAMTS4 cleaves APP directly to generate the C-terminal stub c102, which is subsequently proteolyzed by γ-secretase to release APP669-711. Genetic knockout of ADAMTS4 reduced the production of endogenous APP669-711 by 30% to 40% in cultured cells as well as mouse plasma, irrespectively of Aß levels. Finally, we found that the endogenous murine APP669-711/Aß1-42 ratio was increased in aged AD model mice, which shows Aß deposition as observed in human patients. These data suggest that ADAMTS4 is involved in the production of APP669-711, and a plasma biomarker determined by IP-MALDI-MS can be used to estimate the level of Aß deposition in the brain of mouse models.

Hot Hydrogen Atom Irradiation of Protonated/Deprotonated Peptide in an Ion Trap Facilitates Fragmentation through Heated Radical Formation.

Tandem mass spectrometry with fragmentation involving the reaction with hydrogen atoms is expected to be useful for the analysis of peptides and proteins. In general, hydrogen atoms preferentially react with odd-electron radicals. The attachment of hydrogen atoms to even-electron peptide ions is barely observed because of their low reaction rate. To date, only the methodology developed by our group has successfully induced the fragmentation of even-electron peptide ions by reacting with hydrogen atoms. In the present study, we focused on the temperature of the peptide ions and hydrogen atoms in an ion trap mass spectrometer to understand the mechanism of the corresponding reaction. Because the reaction between even-electron peptide ions and hydrogen atoms has a significant transition state barrier, the use of hot hydrogen atoms is required to initiate the reaction. The reaction contributes to increase the internal energy of the resultant peptide radicals because the heat of reaction and kinetic energy of the hydrogen atom are converted to the internal energy of the product. The resultant oxygen- and carbon-centered peptide radicals undergo radical-induced fragmentation with sub-picosecond and sub-millisecond time scales, respectively.

Gas-Phase Peptide Fragmentation Induced by Hydrogen Attachment, from Principle to Sequencing of Amide Nitrogen-Methylated Peptides.

Tandem mass spectrometry (MS/MS) with radical-based fragmentation was developed recently, which involves the reaction of hydrogen atoms and peptides in a process called hydrogen attachment/abstraction dissociation (HAD). HAD mainly produces [cn + 2H]+ and [zm + 2H]+ via hydrogen attachment to the carbonyl oxygen on the peptide backbone. In addition, HAD often generates [an + 2H]+ and [xm + 2H]+. To explain the formation of [an + 2H]+ and [xm + 2H]+, hydrogen attachment to the carbonyl carbon atom on the peptide backbone is proposed to initiate Cα-C bond cleavage. The resultant hydrogen-abundant oxygen-centered radical intermediate undergoes radical-induced dissociation to give [an + H]+• and [xm + 2H]+. Subsequently, [an + 2H]+ was produced by the reaction of [an + H]+• and a hydrogen atom. The fragment ions formed by the cleavage of N-Cα and Cα-C bonds are observed in the HAD-MS/MS spectra, and the mass differences of these fragment ions correspond to the mass of peptide bonds. Consequently, HAD-MS/MS allows the identification of post-translational modifications on the peptide backbone. In addition, HAD-MS/MS provides a consecutive series of [cn + 2H]+ and [an + 2H]+ as the N-terminal fragments, as well as [zm + 2H]+ and [xm + 2H]+, which enables the sequencing of peptides with post-translational modification, including the discrimination of modifications on the side chain and backbone.

Plasma Amyloid-ß Biomarker Associated with Cognitive Decline in Preclinical Alzheimer's Disease.

BACKGROUND: Using immunoprecipitation-mass spectrometry, we recently developed and validated a plasma composite biomarker for the assessment of amyloid-ß (Aß) levels. However, as yet, its relationship with clinical outcomes remains unclear. OBJECTIVE: We aimed to examine the relationship between this plasma Aß composite biomarker and cognitive function in cognitively normal older adults in two independent cohorts. METHODS: Participants enrolled in the Australian Imaging, Biomarkers and Lifestyle (AIBL) study and the National Centre for Geriatrics and Gerontology (NCGG) study had undergone Aß neuroimaging using positron emission tomography (PET), cognitive assessments and provided blood samples. We derived a high-performance plasma Aß composite biomarker by immunoprecipitation with mass-spectrometry. RESULTS: Both continuous and categorical measures of the plasma Aß composite biomarker were significantly related to decline in episodic memory and executive function. The magnitude of effects of the plasma Aß composite on episodic memory and executive function were comparable to that observed for the effects of PET Aß levels on these same outcome measures. CONCLUSION: Several plasma Aß biomarkers have been developed, but none have yet been applied to investigate their relationship with cognitive outcomes. Our results have important implications for the use of this biomarker in the detection of at-risk individuals.

Characterization of Polyethers Using Tandem Mass Spectrometry with Hydrogen Abstraction Dissociation and Thermal Activation.

The recently developed hydrogen radical-mediated fragmentation technique using an ion trap involving hydrogen attachment/abstraction dissociation-tandem mass spectrometry (HAD-MS/MS) was applied to the analysis of polyethylene glycol (PEG) and its derivatives. HAD was found to be initiated by hydrogen abstraction from carbon atoms in the polyether. Subsequently, the produced carbon-centered radical intermediates underwent radical-induced cleavage of their C-O bonds, with this process being facilitated by heating of the ion trap. The bond cleavage resulted in the formation of b fragments containing double bonds between carbon atoms. A counterpart c• alkoxy radical was discovered to be a fragile radical species. Consequently, c• underwent further radical-induced dissociation to produce small fragments during HAD-MS/MS with thermal activation. As a result, HAD-MS/MS with thermal activation through ion trap heating preferentially provided b fragments, facilitating identification of repeating units and individual end groups of the polyether analytes.

Classification of Cutibacterium acnes at phylotype level by MALDI-MS proteotyping.

Cutibacterium acnes is a major commensal human skin bacteria. It is a producer of propionic acids that maintain skin acidic pH to inhibit the growth of pathogens. On the other hand, it is also associated with diseases such as acne vulgaris and sarcoidosis. C. acnes strains have been classified into six phylotypes using DNA-based approaches. Because several characteristic features of C. acnes vary according to the phylotype, the development of a practical method to identify these phylotypes is needed. For rapid identification of phylotypes for C. acnes strains, a matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) fingerprinting technique has been applied; however, some phylotypes have not been discriminated. We developed a high-throughput protein purification method to detect biomarker proteins by ultrafiltration. MALDI-MS proteotyping using profiling of identified biomarker peaks was applied for the classification of 24 strains of C. acnes, and these were successfully classified into the correct phylotypes. This is a promising method that allows the discrimination of C. acnes phylotypes independent of a DNA-based approach.

Improved MALDI-MS method for the highly sensitive and reproducible detection of biomarker peaks for the proteotyping of Salmonella serotypes.

The rapid identification and classification of pathogenic microorganisms, including Salmonella enterica, is important for the surveillance and prevention of foodborne diseases. Matrix-assisted laser desorption\ionization time-of-flight mass spectrometry (MALDI-TOFMS) has been shown to be an effective tool for the rapid identification of microorganisms. In a previous report, a mass database consisting of 12 biomarker proteins, S8, L15, L17, L21, L25, S7, superoxide dismutase (SodA), peptidylprolyl cis-trans isomerase C, Gns, YibT, YaiA, and YciF, was introduced for the serotyping of S. enterica via MALDI-MS (Applied Microbiology and Biotechnology, 2017, 101, 8557-8569). However, the reproducibility of peak detection of biomarkers such as SodA at m\z 23 000 was poor. We report here an optimized MALDI-MS method for detecting these biomarkers with high sensitivity and reproducibility. The issue was solved by controlling the bacterial concentration at 1 × 10 to 1 × 102 MFU (3 × 106 to 3 × 107 CFU\µL, as calculated from the MFU), using the colony suspension supernatant obtained by centrifugation, and using matrix additives such as methylenediphosphonic acid and N-decyl-ß-D-maltopyranoside. We propose that the method including the above steps is one of the best for detecting biomarkers with high sensitivity and reproducibility.

Hydrogen attachment dissociation of peptides containing disulfide bonds.

A combination of tandem mass spectrometry (MS/MS) and hydrogen attachment dissociation (HAD) is a useful method for peptide sequence analysis. In this study, gas-phase fragmentation induced by the attachment of hydrogen to peptides containing disulfide bonds was investigated. Hydrogen attachment induced the cleavage of either the disulfide or N-Cα bond, which competitively occurred during HAD. The disulfide bond cleavage proceeded through an intermediate, which contains a thiyl radical (-S˙) and a thiol group (-SH). In contrast, N-Cα bond cleavage produced an intermediate containing an enol-imine group and α-carbon radical. The intermediate α-carbon radical then attacked the disulfide bond, resulting in a cyclic [z]+ fragment. The counterpart, [c + H]+˙ with a thiyl radical underwent further hydrogen attachment, producing [c + 2H]+. Because both disulfide and N-Cα bonds were cleaved by a single hydrogen attachment event, HAD-MS/MS can provide sequence information for the backbone region in the disulfide loop.

Comparative Glycomic Analysis of Sialyl Linkage Isomers by Sialic Acid Linkage-Specific Alkylamidation in Combination with Stable Isotope Labeling of α2,3-Linked Sialic Acid Residues.

Sialic acids form the terminal sugars in glycan chains on glycoproteins via α2,3, α2,6, or α2,8 linkages, and structural isomers of sialyl linkages play various functional roles in cell recognition and other physiological processes. We recently developed a novel procedure based on sialic acid linkage-specific alkylamidation via lactone ring opening (aminolysis-SALSA). Herein, we have investigated an isotope labeling of α2,3-linked sialic acid residues (iSALSA) using amine hydrochloride salts. One limitation of SALSA using amine hydrochloride salts may be solved by adding only tert-butylamine (t-BA) as an acid scavenger, and comparative and quantitative glycomic analyses can be performed using iSALSA. We also developed quantitative glycomic analysis using dual isotope-labeled glycans by derivatizing with aminooxy-functionalized tryptophanylarginine methyl ester (aoWR) and iSALSA at the reducing and nonreducing end, respectively. Furthermore, we demonstrate that the amount of α2,3-linked sialoglycans in serum are altered during liver fibrosis using matrix-assisted laser desorption ionization-time-of-flight mass spectrometry (MALDI-TOF MS) and liquid chromatography MS (LC/MS) analyses. We revealed that the ratio of A33,6,6 to A3F3,6,6 was gradually decreased along with liver fibrosis progression. Therefore, these glycan alterations are potential diagnostic markers of nonalcoholic steatohepatitis (NASH) fibrosis progression.

Quantification of a cell-mediated immune response against varicella zoster virus by assessing responder CD4high memory cell proliferation in activated whole blood cultures.

BACKGROUND: Herpes zoster (HZ) is caused by reactivation of a latent varicella zoster virus (VZV). The potential to develop HZ increases with age due to waning of memory cell-mediated immunity (CMI), mainly the CD4 response. Therefore, VZV-CD4-memory T cells (CD4-M) count in blood could serve as a barometer for HZ protection. However, direct quantification of these cells is known to be difficult because they are few in number in the blood. We thus developed a method to measure the proliferation level of CD4-M cells responding to VZV antigen in whole blood culture. METHODS: Blood samples were collected from 32 children (2-15 years old) with or without a history of varicella infection, 18 young adults (28-45 years old), and 80 elderly (50-86 years old) with a history of varicella infection. The elderly group was vaccinated, and blood samples were taken 2 months and 1 year after VZV vaccination. Then, 1 mL of blood was mixed with VZV, diluted 1/10 in medium, and cultured. CD4-M cells were identified and measured by flow cytometry. RESULTS: There was distinct proliferation of CD3+CD4highCD45RA-RO+ (CD4high-M) cells specific to VZV antigen at day 9. The majority of CD4high-M cells had the effector memory phenotype CCR7- and was granzyme B-positive. CD4high-M cells were detected in blood culture from varicella-immune but not varicella-non-immune children. Meanwhile, a higher level of CD4high-M proliferation was observed in young adults than in the elderly. The CD4high-M proliferation level was boosted 2 months after VZV vaccination and maintained for at least 1 year in the elderly. CONCLUSION: Quantifying VZV responder CD4high -M cell proliferation is a convenient way to measure VZV CMI using small blood volumes. Our method can be applied to measure VZV vaccine-induced CMI in the elderly. Clinical study registry numbers: (www.clinicaltrials.jp) 173532 and 183985.

Sequencing of Sulfopeptides Using Negative-Ion Tandem Mass Spectrometry with Hydrogen Attachment/Abstraction Dissociation.

Tandem mass spectrometry (MS/MS) with radical-based fragmentation involving the attachment or abstraction of hydrogen to peptides, in a process called hydrogen attachment/abstraction dissociation (HAD), has been recently developed. HAD-MS/MS is considered a useful method for the analysis of proteins with post-translational modification (PTM) because of its ability to determine the PTM site on proteins. In the present investigation, we analyzed highly acidic sulfopeptides and sulfoprotein digests using negative-ion HAD-MS/MS combined with matrix-assisted laser desorption/ionization (MALDI) and electrospray ionization (ESI). In general, MALDI and ESI produced singly and multiply charged peptides, respectively. HAD of singly deprotonated sulfopeptides preferentially produced fragment ions with sulfonation, whereas both sulfonated and nonsulfonated fragment ions were observed in the HAD-MS/MS spectrum of multiply deprotonated sulfopeptides. A comparison of the MALDI and ESI HAD-MS/MS spectra allows the discrimination of sulfonated and nonsulfonated fragments, which would be helpful in performing de novo sequencing of sulfopeptides. In addition, the combination of ESI-based HAD-MS/MS and liquid chromatography (LC) allows the analysis of sulfopeptides present in protein digests. LC-ESI-MS/MS with HAD is a potentially useful method for sulfoproteomic application.

Hydrogen atom attachment to histidine and tryptophan containing peptides in the gas phase.

In this study, we use a combination of tandem mass spectrometry and hydrogen radical-mediated fragmentation techniques to analyze the sequence of peptides. We focus on fragmentation induced by the attachment of hydrogen atoms to the histidine and tryptophan residue side-chains in the peptide that occurs in the gas-phase. The hydrogen atom attached to the imidazole and indole rings in the histidine and tryptophan residues, respectively, and the resulting intermediate experienced Cα-Cß bond cleavage. The detailed fragmentation mechanism is investigated by computational analysis using density functional theory. According to the results, hydrogen attachment occurs at the C-5 position in histidine and at the C-2 position in the tryptophan, which has a lower activation energy compared with the other positions and the resulting intermediate radicals yielded fragments due to Cα-Cß bond cleavage. For the peptides that contain the histidine and tryptophan residues, cleavages in the Cα-Cß and N-Cα bonds occurred independently. Therefore, the method presented in this study is applicable when analyzing peptides that contain histidine and tryptophan residues.

Identifying Double Bond Positions in Phospholipids Using Liquid Chromatography-Triple Quadrupole Tandem Mass Spectrometry Based on Oxygen Attachment Dissociation.

Lipids, a class of biomolecules, play a significant role in the physiological system. In this study, gas-phase hydroxyl radicals (OH·) and atomic oxygens (O) were introduced into the collision cell of a triple quadruple mass spectrometer (TQ-MS) to determine the positions of the double bond in unsaturated phospholipids. A microwave-driven compact plasma generator was used as the OH·/O source. The reaction between OH·/O and the precursor ions passing through the collision cell generates product ions that correspond to the double bond positions in the fatty acyl chain. This double bond position specific fragmentation process initiated by the attachment of OH·/O to the double bond of a fatty acyl chain is a characteristic of oxygen attachment dissociation (OAD). A TQ-MS incorporating OAD, in combination with liquid chromatography, permitted a high throughput analysis of the double bond positions in complex biomolecules. It is important to know the precise position of double bonds in lipids, since these molecules can have widely different functionalities based on the position of the double bonds. The assignment of double bond positions in a mixture of eight standard samples of phosphatidylcholines (phospholipids with choline head groups) with multiple saturated fatty acyl chains attached was successfully demonstrated.

Sialic Acid Linkage Specific Derivatization of Glycosphingolipid Glycans by Ring-Opening Aminolysis of Lactones.

Sialic acids occur widely as glycoconjugates at the nonreducing ends of glycans. Glycosphingolipids (GSLs) include a large number of sialyl-linked glycan isomers with α2,3-, α2,6-, and α2,8-linked polysialic acids. Thus, it is difficult to distinguish structural isomers with the same mass by mass spectrometry. The sialic acid linkage specific alkylamidation (SALSA) method has been developed for discriminating between α2,3- and α2,6-linked isomers, but sequential amidation of linkage-specific sialic acids is generally complicated and time-consuming. Moreover, analysis of GSL-glycans containing α2,8-linked polysialic acids using solid-phase SALSA has not been reported. Herein, we report a novel SALSA method focused on ring-opening aminolysis (aminolysis-SALSA), which shortens the reaction time and simplifies the experimental procedures. We demonstrate that aminolysis-SALSA can successfully distinguish serum GSL-glycan isomers by mass spectrometry. In addition, ring-opening aminolysis can easily be applied to amine and hydrazine derivatives.

Fundamental study of hydrogen-attachment-induced peptide fragmentation occurring in the gas phase and during the matrix-assisted laser desorption/ionization process.

Mass spectrometry with hydrogen-radical-mediated fragmentation techniques has been used for the sequencing of proteins/peptides. The two methods, matrix-assisted laser desorption/ionization in-source decay (MALDI-ISD) and hydrogen attachment/abstraction dissociation (HAD) are known as hydrogen-radical-mediated fragmentation techniques. MALDI-ISD occurs during laser induced desorption processes, whereas HAD utilizes the association of hydrogen with peptide ions in the gas phase. In this study, the general mechanisms of MALDI-ISD and HAD of peptides were investigated. We demonstrated the fragmentation of four model peptides and investigated the fragment formation pathways using density functional theory (DFT) calculations. The current experimental and computational joint study indicated that MALDI-ISD and HAD produce aminoketyl radical intermediates, which immediately undergo radical-induced cleavage at the N-Cα bond located on the C-terminal side of the radical site, leading to the c'/z˙ fragment pair. In the case of MALDI-ISD, the z˙ fragments undergo a subsequent reaction with the matrix to give z' and matrix adducts of the z fragments. In contrast, the c' and z˙ fragments react with hydrogen atoms during the HAD processes, and various fragment species, such as c˙, c', z˙ and z', were observed in the HAD-MS/MS mass spectra.

Structural Analysis of Phospholipid Using Hydrogen Abstraction Dissociation and Oxygen Attachment Dissociation in Tandem Mass Spectrometry.

Gas-phase hydrogen radicals were introduced into a quadrupole ion trap containing singly charged phospholipids to obtain structural fragmentation patterns in tandem mass spectrometry (MS/MS). Saturated and unsaturated phosphatidylcholines were used as a model phospholipid, whose chain-length ranges between 16 and 24. The MS/MS spectrum yielded a continuous series of fragment ions with a mass difference of 14 Da, representing the saturated fatty acyl chains. The fragment ions corresponding to the double-bond position within a single fatty acyl chain showed a characteristic mass difference of 12 Da. The detection of these diagnostic product ions enabled the structural analysis of double-bond isomers of phospholipids. To further investigate the potential of radical-induced dissociation for the isomeric analysis of phospholipids, gas-phase hydroxyl radicals, and triplet oxygen atoms were employed in tandem mass spectrometry. The methylene bridges adjacent to the double-bond positions were selectively dissociated, accompanied by oxidation of the double bonds. Tandem mass spectrometry incorporating multiple radical species facilitates the structural analysis of isomeric phospholipids.

Tandem Mass Spectrometry of Peptide Ions by Microwave Excited Hydrogen and Water Plasmas.

A thermal cracking cell that served as the atomic hydrogen source for hydrogen attachment/abstraction dissociation (HAD) analysis has an intrinsic problem to produce a beam of atoms reactive against heated tungsten capillary. A plasma excited by 2.45 GHz microwave discharge can deliver reactive species to a quadrupole ion trap confining analyte ions without excessive heating of the radical source components. The radical (H•) production performance of the developed source was evaluated by optical emission spectroscopy and H• attachment reaction to fullerene ions. The source exhibited the H• attachment rate as high as a thermal cracking source forming H• in the high temperature tungsten capillary to induce fragmentation processes preserving post-translational modifications. Water vapor was introduced to the source to confirm the stability to generate oxygen containing radicals, which were found present in the water vapor plasma together with atomic hydrogen. Injection of radicals from a water vapor plasma successfully dissociated peptide ions to c-/z- and a-/x-type ions as the case of HAD induced by a thermal cracking cell.