While real-time reverse transcriptase–polymerase chain reaction (RT-PCR) on nasopharyngeal swabs is the current standard for SARS-CoV-2 detection, saliva is an attractive alternative for diagnosis and screening due to ease of collection and minimal supply requirements. Studies on the sensitivity of saliva-based SARS-CoV-2 molecular testing have shown considerable variability.We conducted a prospective, longitudinal study to investigate the testing time frame that optimizes saliva sensitivity for SARS-CoV-2 detection.Methods
Between June 17, 2020, and February 15, 2021, a convenience sample of individuals exposed to a household member with RT-PCR–confirmed SARS-CoV-2 within 2 weeks were recruited from Children’s Hospital Los Angeles and nearby community testing sites into the Household Exposure and Respiratory Virus Transmission and Immunity Study (HEARTS). Paired nasopharyngeal and saliva samples were collected every 3 to 7 days for up to 4 weeks or until 2 negative nasopharyngeal test results. RT-PCR for SARS-CoV-2 N1 and N2 genes was performed; cycle threshold less than 40 defined a positive result. A nasopharyngeal N1 cycle threshold of 34 or less was defined as high viral load. Detailed specimen collection and RT-PCR methods are reported in the eMethods in the Supplement.
Saliva sensitivity was calculated using nasopharyngeal-positive RT-PCR as the reference standard. COVID-19 onset was defined as the earlier date between first symptom (collected by questionnaire daily) or first RT-PCR positivity. Pre- and postsymptomatic were defined as asymptomatic time points before and after a symptomatic interval, respectively.
Saliva sensitivity by week of collection and between symptomatic and asymptomatic individuals were compared using the χ2 test or the Fisher exact test. Generalized estimating equations were used to determine clinical characteristics (Table) associated with saliva sensitivity in nasopharyngeal-positive pairs while accounting for repeated samples from the same individuals. Analyses were performed using SPSS version 27.0 (IBM Corp) with a 2-sided P < .05 considered significant. Written informed consent was obtained from participants. The study was approved by the institutional review board of Children’s Hospital Los Angeles.Results
We tested 889 paired nasopharyngeal swab-saliva samples from 404 participants, of which SARS-CoV-2 was detected in 524 nasopharyngeal (58.9%) and 318 saliva (35.7%) specimens. SARS-CoV-2 was detected in both specimens in 258 pairs (29.0%). Of the 256 nasopharyngeal SARS-CoV-2–positive participants (63.4%), the mean age was 28.2 years (range, 3.0-84.5 years); 108 (42.2%) were male. Participants returned for a median of 3 visits (interquartile range, 2-4). Ninety-three participants (36.3%) were asymptomatic throughout their infection; 126 (77.3%) of 163 symptomatic individuals reported mild severity.
Saliva sensitivity was highest in samples collected during the first week of infection at 71.2% (95% CI, 62.6%-78.8%) but decreased each subsequent week . Participants who presented with COVID-19–associated symptoms on the specimen collection day during week 1 of infection had significantly higher saliva sensitivity compared with asymptomatic participants (88.2% [95% CI, 77.6%-95.1%] vs 58.2% [95% CI, 46.3%-69.5%]; P < .001).
Saliva sensitivity remained significantly higher in symptomatic participants in week 2 (83.0% [95% CI, 70.6%-91.8%] vs 52.6% [95% CI, 42.6%-62.5%]; P < .001). No difference was observed more than 2 weeks after COVID-19 onset . Sensitivities did not significantly differ for never-symptomatic (34.7% [95% CI, 27.3%-42.7%]), presymptomatic (57.1% [95% CI, 31.7%-80.2%]), and postsymptomatic (42.9% [95% CI, 36.8%-49.1%]) time points (P = .26).
For each day after COVID-19 onset, the odds ratio for saliva detection was 0.94 (95% CI, 0.91-0.96) compared with the previous day (P < .001) . Participants presenting with COVID-19–associated symptoms at the time of specimen collection or with high nasopharyngeal viral loads had 2.8 (95% CI, 1.6-5.1; P < .001) and 5.2 (95% CI, 2.9-9.3; P < .001) higher odds of having a saliva-positive RT-PCR result compared with those with asymptomatic presentation or low nasopharyngeal viral loads, respectively.
Discussion
Saliva was sensitive for detecting SARS-CoV-2 in symptomatic individuals during initial weeks of infection, but sensitivity in asymptomatic SARS-CoV-2 carriers was less than 60% at all time points. As COVID-19 testing strategies in workplaces, schools, and other shared spaces are optimized, low saliva sensitivity in asymptomatic infections must be considered. This study suggests saliva-based RT-PCR should not be used for asymptomatic COVID-19 screening.
This study has limitations. Samples were collected following household exposure; therefore, pretest probability was high. Nasopharyngeal swab testing was the reference standard, but this is not a perfect test for SARS-CoV-2 infection, and a positive RT-PCR result from any sample past 10 days of infection may not be predictive of viral replication or infectivity.
Discussion
Our study is concordant with multiple published works supporting saliva as an alternative sample for COVID-19 screening and diagnosis, and one of a minority where saliva was shown to be more sensitive than the corresponding NP swab8,9,13, although the results by Leung et al. (53.7% saliva vs. 47.4% NP swab, 95 subjects) were not statistically different8. Several reasons may account for this difference in the studies, including enrichment from nasal and oropharyngeal secretions, where the viral load is potentially higher8,9, or a higher volume of samples collection, where approximately 10 mL of saliva was collected for testing13. Steps were taken to minimize biases and errors—NP swabs performed by trained healthcare staff, environmental testing of CAP-accredited laboratory (no evidence of contamination), conduction of tests for most of the samples in the same laboratory, and pre-processing of saliva samples with dithiothreitol before RNA extraction to resolve the issues of saliva specimen viscosity, which can lead to false negatives.
Panbio
Interestingly but perhaps unsurprisingly, the use of different RT-PCR kits in the present study resulted in different test-positive rates in saliva, suggesting that this can potentially be an important consideration for clinical laboratories, where more sensitive laboratory protocols should be deployed for clinical diagnosis as opposed to mass screening for low-prevalence populations. More validation would be required to confirm this finding.
SN swabs, however, appeared less sensitive compared to both saliva and NP swabs for the diagnosis of COVID-19. Although it was convenient, less time-consuming to perform relative to saliva collection, and caused less discomfort compared to NP swabs, the markedly lower sensitivity should preclude its use where other sample types can be collected.
In our study, NGS provided efficient whole-genome profiling of SARS-CoV-2 for phylogenetic analysis directly from the clinical samples without culture. NGS detection sensitivity was excellent with a threshold of 1.7% genome coverage or 5 amplicons targets, confirming all CDC-LDT positives tested. Other groups have reported highly sensitive performance for NGS with limits of detection ranging between a threshold of 5% genome coverage or 84 genome-equivalents per mL21, or at least 5 SARS-CoV-2 targets for detection22. The phylogeny results were consistent with the virus belonging to a viral type (Clade O, lineage B.6) known to be circulating in the geographical regions of Singapore and India.
There are several limitations to our work. Firstly, the study population was confined to young and middle-aged men who were either asymptomatic or had mild disease. The results cannot be extrapolated to other populations (e.g., paediatric), where there is a clear need for alternate sample types to NP swabs. Secondly, we did not extend the follow-up testing sufficiently to determine when saliva viral shedding stopped for the majority of subjects, although this has been explored in other studies. Thirdly, we did not test for the difference, if any, between saliva obtained from naso-oropharyngeal or the mouth alone, although it is biologically plausible that the latter would result in lower sensitivity for COVID-19 diagnosis.
In conclusion, our study adds to the body of evidence supporting saliva as a sensitive and less intrusive sample for COVID-19 diagnosis and further defines the role of naso-oropharyngeal secretions and the impact of different RT-PCR kits in increasing the sensitivity of testing. In our study, SN swabs were inferior to both saliva and NP swabs. Our study also provides evidence to support NGS in challenging samples for sensitive COVID-19 molecular diagnosis. Such an NGS workflow can also provide direct-from-sample phylogenetic analysis for public health decision-making, such as contact tracing.
Amyotrophic lateral sclerosis (ALS) is a disorder characterized by the death of both upper and lower motor neurons and by 3- to 5-yr median survival postdiagnosis. The only US Food and Drug Administration-approved drug for the treatment of ALS, Riluzole, has at best, moderate effect on patient survival and quality of life; therefore innovative approaches. …
RNA-Seq-enabled proteomics of a weakly electric fish
The brown ghost knifefish (Apteronotus leptorhynchus) is a weakly electric teleost fish of particular interest as a versatile model system for a variety of research areas in neuroscience and biology. The comprehensive information available on the neurophysiology and neuroanatomy of this organism has enabled significant advances in such areas as the study of the neural network.
Fine structure HDX-MS software
Background:
Hydrogen/deuterium exchange (HDX) coupled to mass spectrometry permits analysis of structure, dynamics, and molecular interactions of proteins. HDX mass spectrometry is confounded by deuterium exchange-associated peaks overlapping with peaks of heavy, natural abundance isotopes, such as carbon-13. Recent studies demonstrated that high-performance mass spectrometers could resolve isotopic fine structure and eliminate this peak overlap.
Anaerobic top-down
Bottom-up MS studies typically employ a reduction and alkylation step that eliminates a class of PTM, S-thiolation. Given that molecular oxygen can mediate S-thiolation from reduced thiols, which are abundant in the reducing intracellular milieu, we investigated the possibility that some S-thiolation modifications are artifacts of protein preparation. Cu/Zn-superoxide dismutase (SOD1), Gentaur Bvba, Kampenhout.
Differential neuropeptidomics
BACKGROUND: Neuropeptides are a diverse category of signaling molecules in the nervous system regulating a variety of processes including food intake, social behavior, circadian rhythms, learning, and memory. Both the identification and functional characterization of specific neuropeptides are ongoing fields of research. Matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS) analysis of nervous tissues.
Isotopic Fine Structure HDX MS
Hydrogen/deuterium exchange (HDX) mass spectrometry (MS) is used for analyzing protein dynamics, protein folding/unfolding, and molecular interactions. Until this study, HDX MS experiments employed mass spectral resolving powers that afforded only one peak per nominal mass in a given peptide’s isotope distribution, and HDX MS data analysis methods were developed accordingly.
Isotopic Fine Structure
Structural impact of Cys mod of SOD1
The metalloenzyme Cu/Zn-superoxide dismutase (SOD1) catalyzes the reduction of superoxide anions into molecular oxygen and hydrogen peroxide. Hydrogen peroxide can oxidize SOD1, resulting in aberrant protein conformational changes, disruption of SOD1 function, and DNA damage. Cells may have evolved mechanisms of regulation that prevent such oxidation. We observed that cysteinylation of cysteine 111 (Cys111).
Superoxide Dismutase
Cysteine modification blocks oxidative damage
Reactive oxygen species (ROS) are cytotoxic. To remove ROS, cells have developed ROS-specific defense mechanisms, including the enzyme Cu/Zn superoxide dismutase (SOD1), which catalyzes the disproportionation of superoxide anions into molecular oxygen and hydrogen peroxide. Although hydrogen peroxide is less reactive than superoxide, it is still capable of oxidizing, unfolding, and inactivating SOD1.
LC-MS performance evaluation
Selecting a suitable nano-liquid chromatography system (LC), ionization source and mass spectrometer for LC-tandem mass spectrometry (MS-MS) studies is complicated by numerous competing technologies. This study compares four popular nano-LC systems, four ionization sources and three MS facilities that use completely different LC-MS-MS systems. Statistically significant differences in LC performance.
X-linking MS analysis
The small quantities of protein required for mass spectrometry (MS) make it a powerful tool to detect binding (protein-protein, protein-small molecule, etc.) of proteins that are difficult to express in large quantities, as is the case for many intrinsically disordered proteins. Chemical cross-linking, proteolysis, and MS analysis, combined, are a powerful tool for the identification.
Stabilizing SOD1
Amyotrophic lateral sclerosis (ALS) is a disorder characterized by the death of both upper and lower motor neurons and by 3- to 5-yr median survival postdiagnosis. The only US Food and Drug Administration-approved drug for the treatment of ALS, Riluzole, has at best, moderate effect on patient survival and quality of life; therefore innovative approaches.
RNA-Seq-enabled proteomics of a weakly electric fish
The brown ghost knifefish (Apteronotus leptorhynchus) is a weakly electric teleost fish of particular interest as a versatile model system for a variety of research areas in neuroscience and biology. The comprehensive information available on the neurophysiology and neuroanatomy of this organism has enabled significant advances.
RNA-Seq-enabled proteomics of a weakly electric fishFine structure HDX-MS softwareAnaerobic top-downDifferential neuropeptidomicsIsotopic Fine Structure HDX MSStructural impact of Cys mod of SOD1Cysteine modification blocks oxidative damageLC-MS performance evaluationX-linking MS analysisStabilizing SOD1 Mar 20
The brown ghost knifefish (Apteronotus leptorhynchus) is a weakly electric teleost fish of particular interest as a versatile model system for a variety of research areas in neuroscience and biology. The comprehensive information available on the neurophysiology and neuroanatomy of this organism has enabled significant advances in such areas as the study of the neural basis of behavior, the development of adult-born neurons in the central nervous system and their involvement in the regeneration of nervous tissue, as well as brain aging and senescence. Despite substantial scientific interest in this species, no genomic resources are currently available. Here, we report de novo assembly and annotation of the A. leptorhynchus transcriptome. After evaluating several trimming and transcript reconstruction strategies, de novo assembly using Trinity uncovered 42,459 unique contigs containing at least a partial protein-coding sequence based on alignment to a reference set of known Actinopterygii sequences. As many as 11,847 of these contigs contained full or near-full length protein sequences, providing broad coverage of the proteome. A variety of non-coding RNA sequences were also identified and annotated, including conserved long intergenic non-coding RNA and other long non-coding RNA observed previously to be expressed in adult zebrafish (Danio rerio) brain, as well as a variety of miRNA, snRNA, and snoRNA. Shotgun proteomics confirmed translation of open reading frames from over 2,000 transcripts, including alternative splice variants. Assignment of tandem mass spectra was greatly improved by use of the assembly compared to databases of sequences from closely related organisms. The assembly and raw reads have been deposited at DDBJ/EMBL/GenBank under the accession number GBKR00000000. Tandem mass spectrometry data is available via ProteomeXchange with identifier PXD001285. Presented here is the first release of an annotated de novo transcriptome assembly from Apteronotus leptorhynchus, providing a broad overview of RNA expressed in central nervous system tissue. The assembly, which includes substantial coverage of a wide variety of both protein coding and non-coding transcripts, will allow the development of better tools to understand the mechanisms underlying unique characteristics of the knifefish model system, such as their tremendous regenerative capacity and negligible brain senescence.
Background: Hydrogen/deuterium exchange (HDX) coupled to mass spectrometry permits analysis of structure, dynamics, and molecular interactions of proteins. HDX mass spectrometry is confounded by deuterium exchange-associated peaks overlapping with peaks of heavy, natural abundance isotopes, such as carbon-13. Recent studies demonstrated that high-performance mass spectrometers could resolve isotopic fine structure and eliminate this peak overlap, allowing direct detection and quantification of deuterium incorporation. Results: Here, we present a graphical tool that allows for a rapid and automated estimation of deuterium incorporation from a spectrum with isotopic fine structure. Given a peptide sequence (or elemental formula) and charge state, the mass-to-charge ratios of deuterium-associated peaks of the specified ion is determined. Intensities of peaks in an experimental mass spectrum within bins corresponding to these values are used to determine the distribution of deuterium incorporated. A theoretical spectrum can then be calculated based on the estimated distribution of deuterium exchange to confirm interpretation of the spectrum. Deuterium incorporation can also be detected for ion signals without a priori specification of an elemental formula, permitting detection of exchange in complex samples of unidentified material such as natural organic matter. A tool is also incorporated into QUDeX-MS to help in assigning ion signals from peptides arising from enzymatic digestion of proteins. MATLAB-deployable and standalone versions are available for academic use at qudex-ms.sourceforge.net and agarlabs.com. Conclusion: Isotopic fine structure HDX-MS offers the potential to increase sequence coverage of proteins being analyzed through mass accuracy and deconvolution of overlapping ion signals. As previously demonstrated, however, the data analysis workflow for HDX-MS data with resolved isotopic fine structure is distinct. QUDeX-MS we hope will aid in the adoption of isotopic fine structure HDX-MS by providing an intuitive workflow and interface for data analysis. Click here for full text. This post has no tag
Anaerobic top-down
by agarlab
Bottom-up MS studies typically employ a reduction and alkylation step that eliminates a class of PTM, S-thiolation. Given that molecular oxygen can mediate S-thiolation from reduced thiols, which are abundant in the reducing intracellular milieu, we investigated the possibility that some S-thiolation modifications are artifacts of protein preparation. Cu/Zn-superoxide dismutase (SOD1) was chosen for this case study as it has a reactive surface cysteine residue, which is readily cysteinylated in vitro. The ability of oxygen to generate S-thiolation artifacts was tested by comparing purification of SOD1 from postmortem human cerebral cortex under aerobic and anaerobic conditions. S-thiolation was ∼50% higher in aerobically processed preparations, consistent with oxygen-dependent artifactual S-thiolation. The ability of endogenous small molecule disulfides (e.g. cystine) to participate in artifactual S-thiolation was tested by blocking reactive protein cysteine residues during anaerobic homogenization. A 50-fold reduction in S-thiolation occurred indicating that the majority of S-thiolation observed aerobically was artifact. Tissue-specific artifacts were explored by comparing brain- and blood-derived protein, with remarkably more artifacts observed in brain-derived SOD1. Given the potential for such artifacts, rules of thumb for sample preparation are provided. This study demonstrates that without taking extraordinary precaution, artifactual S-thiolation of highly reactive, surface-exposed, cysteine residues can result.
Read the full paper: Artifacts to avoid while taking advantage of top-down mass spectrometry based detection of protein S-thiolation Tags: amyotrophic lateral sclerosis, anaerobic, cysteinylation, Fourier transform mass spectrometry, oxidative damage, PTMs, SOD1, top-down proteomics
Institute Core Mass Spectrometry Facility
Tags: electrospray ionization, Fourier transform mass spectrometry, isotopic fine structure, LC-MS, LC-MS-MS, nanoESI-LC-MS, nanoLC, performance, QqTOF
by agarlab
BACKGROUND: Neuropeptides are a diverse category of signaling molecules in the nervous system regulating a variety of processes including food intake, social behavior, circadian rhythms, learning, and memory. Both the identification and functional characterization of specific neuropeptides are ongoing fields of research. Matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS) analysis of nervous tissues from a variety of organisms allows direct detection and identification of neuropeptides. Here, we demonstrate an analysis workflow that allows for the detection of differences in specific neuropeptides amongst a variety of neuropeptides being simultaneously measured. For sample preparation, we describe a straight-forward and rapid (minutes) method where individual adult Drosophila melanogaster brains are analyzed. Using a MATLAB-based data analysis workflow, also compatible with MALDI-TOF mass spectra obtained from other sample preparations and instrumentation, we demonstrate how changes in neuropeptides levels can be detected with this method.
RESULTS: Over fifty isotopically resolved ion signals in the peptide mass range are reproducibly observed across experiments. MALDI-TOF MS profile spectra were used to statistically identify distinct relative differences in organ-wide endogenous levels of detected neuropeptides between biological conditions. In particular, three distinct levels of a particular neuropeptide, pigment dispersing factor, were detected by comparing groups of preprocessed spectra obtained from individual brains across three different D. melanogaster strains, each of which express different amounts of this neuropeptide. Using the same sample preparation, MALDI-TOF/TOF tandem mass spectrometry confirmed that at least 14 ion signals observed across experiments are indeed neuropeptides. Among the identified neuropeptides were three products of the neuropeptide-like precursor 1 gene previously not identified in the literature.
CONCLUSIONS: Using MALDI-TOF MS and preprocessing/statistical analysis, changes in relative levels of a particular neuropeptide in D. melanogaster tissue can be statistically detected amongst a variety of neuropeptides. While the data analysis methods should be compatible with other sample preparations, the presented sample preparation method was sufficient to identify previously unconfirmed D. melanogaster neuropeptides.
Read the full paper: A rapid MALDI-TOF mass spectrometry workflow for Drosophila melanogaster differential neuropeptidomics Tags: drosophila melanogaster, isotopic resolution, MALDI-TOF, mass spectrometry, neuropeptidomics, neuroscience, NPLP1
Isotopic Fine Structure HDX MS
by agarlab
Hydrogen/deuterium exchange (HDX) mass spectrometry (MS) is used for analyzing protein dynamics, protein folding/unfolding, and molecular interactions. Until this study, HDX MS experiments employed mass spectral resolving powers that afforded only one peak per nominal mass in a given peptide’s isotope distribution, and HDX MS data analysis methods were developed accordingly. A level of complexity that is inherent to HDX MS remained unaddressed, namely, various combinations of natural abundance heavy isotopes and exchanged deuterium shared the same nominal mass and overlapped at previous resolving powers. For example, an A + 2 peak is comprised of (among other isotopomers) a two-2H-exchanged/zero-13C isotopomer, a one-2H-exchanged/one-13C isotopomer, and a zero-2H-exchanged/two-13C isotopomer. Notably, such isotopomers differ slightly in mass as a result of the ~3 mDa mass defect between 2H and 13C atoms. Previous HDX MS methods did not resolve these isotopomers, requiring a natural-abundance-only (before HDX or “time zero”) spectrum and data processing to remove its contribution. It is demonstrated here that high-resolution mass spectrometry can be used to detect isotopic fine structure, such as in the A + 2 profile example above, deconvolving the isotopomer species resulting from deuterium incorporation. Resolving isotopic fine structure during HDX MS therefore permits direct monitoring of HDX, which can be calculated as the sum of the fractional peak magnitudes of the deuterium-exchanged isotopomers. This obviates both the need for a time zero spectrum as well as data processing to account for natural abundance heavy isotopes, saving instrument and analysis time.
Read the full paper: Resolving Isotopic Fine Structure to Detect and Quantify Natural Abundance- and Hydrogen/Deuterium Exchange-Derived Isotopomers
Tags: Fourier transform mass spectrometry, hydrogen-deuterium exchange, isotopic fine structure, isotopic resolution
Structural impact of Cys mod of SOD1
by agarlab
The metalloenzyme Cu/Zn-superoxide dismutase (SOD1) catalyzes the reduction of superoxide anions into molecular oxygen and hydrogen peroxide. Hydrogen peroxide can oxidize SOD1, resulting in aberrant protein conformational changes, disruption of SOD1 function, and DNA damage. Cells may have evolved mechanisms of regulation that prevent such oxidation. We observed that cysteinylation of cysteine 111 (Cys111) of SOD1 prevents oxidation by peroxide (DOI 10.1021/bi4006122 ). In this article, we characterize cysteinylated SOD1 using differential scanning fluorometry and X-ray crystallography. The stoichiometry of binding was one cysteine per SOD1 dimer, and there does not appear to be free volume for a second cysteine without disrupting the dimer interface. Much of the three-dimensional structure of SOD1 is unaffected by cysteinylation. However, local conformational changes are observed in the cysteinylated monomer that include changes in conformation of the electrostatic loop (loop VII; residues 133-144) and the dimer interface (loop VI; residues 102-115). In addition, our data shows how cysteinylation precludes oxidation of cysteine 111 and suggests possible cross-talk between the dimer interface and the electrostatic loop.
Read the full paper: Structural Consequences of Cysteinylation of Cu/Zn-Superoxide Dismutase
Tags: crystallography, cysteinylation, dimer stability, protein structure, PTMs, SOD1
Cysteine modification blocks oxidative damage
by agarlab
Reactive oxygen species (ROS) are cytotoxic. To remove ROS, cells have developed ROS-specific defense mechanisms, including the enzyme Cu/Zn superoxide dismutase (SOD1), which catalyzes the disproportionation of superoxide anions into molecular oxygen and hydrogen peroxide. Although hydrogen peroxide is less reactive than superoxide, it is still capable of oxidizing, unfolding, and inactivating SOD1, at least in vitro. To explore the relevance of post-translational modification (PTM) of SOD1, including peroxide-related modifications, SOD1 was purified from postmortem human nervous tissue. As much as half of all purified SOD1 protein contained non-native post-translational modifications (PTMs), the most prevalent modifications being cysteinylation and peroxide-related oxidations. Many PTMs targeted a single reactive SOD1 cysteine, Cys111. An intriguing observation was that unlike native SOD1, cysteinylated SOD1 was not oxidized. To further characterize how cysteinylation may protect SOD1 from oxidation, cysteine-modified SOD1 was prepared in vitro and exposed to peroxide. Cysteinylation conferred nearly complete protection from peroxide-induced oxidation of SOD1. Moreover, SOD1 that has been cysteinylated and peroxide oxidized in vitro comprised a set of PTMs that bear a striking resemblance to the myriad of PTMs observed in SOD1 purified from human tissue.
Read the full paper: Post-Translational Modification by Cysteine Protects Cu/Zn-Superoxide Dismutase from Oxidative Damage
Tags: amyotrophic lateral sclerosis, cysteinylation, Fourier transform mass spectrometry, mass spectrometry, oxidative damage, protein stability, PTMs
LC-MS performance evaluation
by agarlab
Selecting a suitable nano-liquid chromatography system (LC), ionization source and mass spectrometer for LC-tandem mass spectrometry (MS-MS) studies is complicated by numerous competing technologies. This study compares four popular nano-LC systems, four ionization sources and three MS facilities that use completely different LC-MS-MS systems. Statistically significant differences in LC performance were identified with similarly performing Proxeon, Waters and Eksigent nanoLC-Ultra systems [retention time routinely at 0.7-0.9% relative standard deviation (RSD)], and all outperformed the Eksigent nanoLC-2D (RSD ∼2%). In addition, compatibility issues were identified between the Bruker HCT ion trap mass spectrometer and both the Eksigent nanoLC-2D and the Bruker nanoelectrospray source. The electrospray source itself had an unexpected and striking effect on chromatographic reproducibility on the Bruker HCT ion trap. The New Objective nanospray source significantly outperformed the Bruker nanospray source in retention time RSD (1% RSD versus 14% RSD, respectively); and the Bruker nebulized nanospray source outperformed both of these traditional, non-nebulized sources (0.5% RSD in retention time). Finally, to provide useful benchmarks for overall proteomics sensitivity, different LC-MS-MS platforms were compared by analyzing a range of concentrations of tryptic digests of bovine serum albumin at three MS facilities. The results indicate that similar sensitivity can be realized with a Bruker HCT-Ultra ion trap, a Thermo LTQ-Velos Linear ion trap and a Thermo LTQ-Orbitrap XL-ETD.
Read the full paper: Performance Comparisons of Nano-LC Systems, Electrospray Sources and LC–MS-MS Platforms
Tags: electrospray ionization, LC-MS, LC-MS-MS, mass spectrometry, nanoESI-LC-MS, nanoLC, performance
X-linking MS analysis
by agarlab
The small quantities of protein required for mass spectrometry (MS) make it a powerful tool to detect binding (protein-protein, protein-small molecule, etc.) of proteins that are difficult to express in large quantities, as is the case for many intrinsically disordered proteins. Chemical cross-linking, proteolysis, and MS analysis, combined, are a powerful tool for the identification of binding domains. Here, we present a traditional approach to determine protein-protein interaction binding sites using heavy water ((18)O) as a label. This technique is relatively inexpensive and can be performed on any mass spectrometer without specialized software.
