Nanoflow Liquid Chromatography-tandem Mass Spectrometry Research Articles

Improved Data Acquisition Settings on Q Exactive HF-X and Fusion Lumos Tribrid Orbitrap-Based Mass Spectrometers for Proteomic Analysis of Limited Samples.

Deep proteomic profiling of complex biological and medical samples available at low nanogram and subnanogram levels is still challenging. Thorough optimization of settings, parameters, and conditions in nanoflow liquid chromatography-tandem mass spectrometry (MS)-based proteomic profiling is crucial for generating informative data using amount-limited samples. This study demonstrates that by adjusting selected instrument parameters, e.g., ion injection time, automated gain control, and minimally altering the conditions for resuspending or storing the sample in solvents of different compositions, up to 15-fold more thorough proteomic profiling can be achieved compared to conventionally used settings. More specifically, the analysis of 1 ng of the HeLa protein digest standard by Q Exactive HF-X Hybrid Quadrupole-Orbitrap and Orbitrap Fusion Lumos Tribrid mass spectrometers yielded an increase from 1758 to 5477 (3-fold) and 281 to 4276 (15-fold) peptides, respectively, demonstrating that higher protein identification results can be obtained using the optimized methods. While the instruments applied in this study do not belong to the latest generation of mass spectrometers, they are broadly used worldwide, which makes the guidelines for improving performance desirable to a wide range of proteomics practitioners.

Spectral libraries from nucleobases and deoxyribonucleosides facilitate the identification of ribonucleosides by nano-flow liquid chromatography-tandem mass spectrometry.

The study addresses the challenge of identifying RNA post-transcriptional modifications when commercial standards are not available to generate reference spectral libraries. It proposes employing homologous nucleobases and deoxyribonucleosides as alternative reference spectral libraries to aid in identifying modified ribonucleosides and distinguishing them from their positional isomers when the standards are unavailable. Complete sets of ribonucleoside, deoxyribonucleoside and nucleobase standards were analyzed using high-performance nano-flow liquid chromatography coupled to an Orbitrap Eclipse Tribrid mass spectrometer. Spectral libraries were constructed from homologous nucleobases and deoxyribonucleosides using targeted MS2 and neutral-loss-triggered MS3 methods, and collision energies were optimized. The feasibility of using these libraries for identifying modified ribonucleosides and their positional isomers was assessed through comparison of spectral fragmentation patterns. Our analysis reveals that both MS2 and neutral-loss-triggered MS3 methods yielded rich spectra with similar fragmentation patterns across ribonucleosides, deoxyribonucleosides and nucleobases. Moreover, we demonstrate that spectra from nucleobases and deoxyribonucleosides, generated at optimized collision energies, exhibited sufficient similarity to those of modified ribonucleosides to enable their use as reference spectra for accurate identification of positional isomers within ribonucleoside families. The study demonstrates the efficacy of utilizing homologous nucleobases and deoxyribonucleosides as interchangeable reference spectral libraries for identifying modified ribonucleosides and their positional isomers. This approach offers a valuable solution for overcoming limitations posed by the unavailability of commercial standards, enhancing the analysis of RNA post-transcriptional modifications via mass spectrometry.

Serum Cathepsin D was Elevated in Patients with Limb Lymphedema.

Lymphedema (LE) is a chronic disease that can lead to disability. Currently, the pathogenesis of LE remains unclear, and there is a lack of serum proteins applicable for diagnosis in clinical practice. This study aimed to screen and identify the differentially expressed proteins in serum samples of limb lymphedema and normal subjects and to further explore their value in the diagnosis of LE. Nano-flow reverse phase liquid chromatography-tandem mass spectrometry (Nano RPLC-MS/MS) was used to establish the serum protein profiles of primary lymphedema (PLE), secondary lymphedema (SLE), and normal controls (NC). Differentially expressed serum proteins were screened and identified. Subsequently, enrichment analysis was performed for proteins that were upregulated in the LE group compared to the NC group. The target protein was validated by western blot (WB) and enzyme-linked immunosorbent assay (ELISA). Both the receiver operating characteristic (ROC) curve and Spearman's correlation test were employed to evaluate the diagnostic performance of the protein and its relationship with disease severity. A total of 362 serum proteins were identified, among which 241 proteins were differentially expressed among PLE, SLE, and NC subjects (p < 0.05, fold change > 1.2). The enriched pathway correlated with cornified envelope formation was selected for further analysis. Cathepsin D (CTSD), a target protein involved in the selected pathway, was found to be up-regulated in the serum of PLE and SLE patients compared to NC. The AUCs of CTSD were 0.849 and 0.880 for patients with PLE and SLE, respectively. There was a significant positive correlation between the levels of serum CTSD and disease severity in the PLE group. Proteomic analysis found that the levels of serum proteins related to cornified envelope formation were elevated in patients with limb lymphedema. Serum CTSD was highly expressed in patients with limb lymphedema and showed good diagnostic value.

Phospho-heavy-labeled-spiketide FAIMS stepped-CV DDA (pHASED) provides real-time phosphoproteomics data to aid in cancer drug selection

Global high-throughput phosphoproteomic profiling is increasingly being applied to cancer specimens to identify the oncogenic signaling cascades responsible for promoting disease initiation and disease progression; pathways that are often invisible to genomics analysis. Hence, phosphoproteomic profiling has enormous potential to inform and improve individualized anti-cancer treatment strategies. However, to achieve the adequate phosphoproteomic depth and coverage necessary to identify the activated, and hence, targetable kinases responsible for driving oncogenic signaling pathways, affinity phosphopeptide enrichment techniques are required and often coupled with offline high-pressure liquid chromatographic (HPLC) separation prior to nanoflow liquid chromatography–tandem mass spectrometry (nLC-MS/MS). These complex and time-consuming procedures, limit the utility of phosphoproteomics for the analysis of individual cancer patient specimens in real-time, and restrict phosphoproteomics to specialized laboratories often outside of the clinical setting. To address these limitations, here we have optimized a new protocol, phospho-heavy-labeled-spiketide FAIMS stepped-CV DDA (pHASED), that employs online phosphoproteome deconvolution using high-field asymmetric waveform ion mobility spectrometry (FAIMS) and internal phosphopeptide standards to provide accurate label-free quantitation (LFQ) data in real-time. Compared with traditional single-shot LFQ phosphoproteomics workflows, pHASED provided increased phosphoproteomic depth and coverage (phosphopeptides = 4617 pHASED, 2789 LFQ), whilst eliminating the variability associated with offline prefractionation. pHASED was optimized using tyrosine kinase inhibitor (sorafenib) resistant isogenic FLT3-mutant acute myeloid leukemia (AML) cell line models. Bioinformatic analysis identified differential activation of the serine/threonine protein kinase ataxia-telangiectasia mutated (ATM) pathway, responsible for sensing and repairing DNA damage in sorafenib-resistant AML cell line models, thereby uncovering a potential therapeutic opportunity. Herein, we have optimized a rapid, reproducible, and flexible protocol for the characterization of complex cancer phosphoproteomes in real-time, a step towards the implementation of phosphoproteomics in the clinic to aid in the selection of anti-cancer therapies for patients.

Gendered burial practices of early Bronze Age children align with peptide-based sex identification: A case study from Franzhausen I, Austria

Gendered burial practices that differentiate between men and women by the way the body was placed were used over large parts of Central Europe in the Late Neolithic and Early Bronze Age (c. 2900–1600 BC). Until recently, it was unknown if the sex-based differentiation of bodies was extended to children, and if the biological sex of the children matched the classification as men and women placed on the left/right side in the opposite orientation.We applied nanoflow liquid chromatography-tandem mass spectrometry (nanoLC-MS/MS) to identify sex-specific peptides in human tooth enamel in 75 children under 12 years at death buried at one of the largest Early Bronze Age cemeteries in Europe, Franzhausen I, Austria, 70 of which produced reliable results.The study confirmed that the sex of the children corresponds to the gendered body position in 98.4% of cases. For burials in which the gendered sidedness and orientation are not internally consistent with the male or female pattern, we found that the sidedness of the body corresponds to the sex of the children rather than the orientation.

A novel ribosome-dimerization protein found in the hyperthermophilic archaeon Pyrococcus furiosus using ribosome-associated proteomics

Ribosome dimerization is one of the bacterial events that suppresses protein synthesis in the stationary phase. Protein factors responsible for ribosome dimerization in bacteria are well characterized, whereas no information is available for the corresponding factors in archaeal and eukaryotic cells. Here we describe a protein found among the ribosome-associated proteins which dimerizes the 30S ribosomal subunit of the archaeon Pyrococcus furiosus. The ribosome-associated proteins were prepared by high-salt wash of crude ribosomes, and analyzed by nanoflow liquid chromatography-tandem mass spectrometry (nano LC-MS/MS). Of the detected proteins we focused on a protein (PF0560) whose Protein Score was the highest of all of the function-unknown proteins. PF0560 protein had a pronounced effect on the sedimentation pattern of the 30S ribosomal subunit; addition of this protein to isolated 30S subunit reduced the 30S fraction and increased the amount of the 50S fraction. This increase presumably corresponds to the dimer of the 30S subunit. The PF0560-dependent 30S-dimerization, was also observed by gel electrophoretic analysis. This effect was not observed in EDTA-treated 30S subunit, with protein-free 16S rRNA or with bacterial/eukaryotic ribosomal small subunits. Furthermore, PF0560 protein suppressed the formation of functional 70S ribosomes. These results suggest that PF0560 is a novel 30S dimerization factor, which might participate in regulation of archaeal translation.

Identification of Decrease in TRiC Proteins as Novel Targets of Alpha-Amanitin-Derived Hepatotoxicity by Comparative Proteomic Analysis In Vitro

Alpha-amanitin (α-AMA) is a cyclic peptide and one of the most lethal mushroom amatoxins found in Amanita phalloides. α-AMA is known to cause hepatotoxicity through RNA polymerase II inhibition, which acts in RNA and DNA translocation. To investigate the toxic signature of α-AMA beyond known mechanisms, we used quantitative nanoflow liquid chromatography–tandem mass spectrometry analysis coupled with tandem mass tag labeling to examine proteome dynamics in Huh-7 human hepatoma cells treated with toxic concentrations of α-AMA. Among the 1828 proteins identified, we quantified 1563 proteins, which revealed that four subunits in the T-complex protein 1-ring complex protein decreased depending on the α-AMA concentration. We conducted bioinformatics analyses of the quantified proteins to characterize the toxic signature of α-AMA in hepatoma cells. This is the first report of global changes in proteome abundance with variations in α-AMA concentration, and our findings suggest a novel molecular regulation mechanism for hepatotoxicity.

Quantitative proteomic profiling of Cervicovaginal fluid from pregnant women with term and preterm birth

BackgroundPreterm birth (PTB) is one of major causes of perinatal mortality and neonatal morbidity, but knowledge of its complex etiology is still limited. Here we present cervicovaginal fluid (CVF) protein profiles of pregnant women who subsequently delivered at spontaneous preterm or term, aiming to identify differentially expressed CVF proteins in PTB and term birth.MethodsThe CVF proteome of women who sequentially delivered at preterm and term was analyzed using isobaric tags for relative and absolute quantitation (iTRAQ) coupled with two-dimensional nanoflow liquid chromatography-tandem mass spectrometry (2D-nLC-MS/MS). We compared the CVF proteome of PTB (n = 5) and control subjects (term birth, n = 7) using pooled control CVF (term birth, n = 20) as spike-in standard.ResultsWe identified 1294 CVF proteins, of which 605 were newly identified proteins. Of 990 proteins quantified in both PTB and term birth, 52 proteins were significantly up/down-regulated in PTB compared to term birth. The differentially expressed proteins were functionally associated to immune response, endopeptidase inhibitors and structural constituent of cytoskeleton. Finally, we confirm the down-regulation of SERPINB7 (a serine-type protease inhibitor) in PTB compared to control by Western blot.ConclusionsTaken together, our study provide quantitative CVF proteome profiles of pregnant women who ultimately delivered at preterm and term. These promising results could help to improve the understanding of PTB etiology and to discover biomarkers for asymptomatic PTB.

The oral microbiome and salivary proteins influence caries in children aged 6 to 8 years

BackgroundOral microbiome and salivary proteins play a critical role in the occurrence and development of caries. In this study, we used metagenomic and metaproteomic analyses to explore the microbiological and proteinic biomarkers and investigate the etiology of caries in 6–8 years old children. Our study aims to offer a better comprehension of these factors and the relationship with caries, and these findings might facilitate caries risk assessment and provide a basis for future prevention strategies.MethodsChildren 6 to 8 years old living in rural isolated areas including 40 caries-active subjects and 40 caries-free subjects were recruited. Supragingival plaque and unstimulated saliva were collected for 16S rDNA pyrosequencing and isobaric tags for relative and absolute quantitation (iTRAQ) technique coupled with quantitative nano-flow liquid chromatography-tandem mass spectrometry (LC-MS/MS), respectively.ResultsWe found 6 phyla and 13 genera predominant in all the samples, and differences in relative abundances can be observed. The Alpha diversity analysis demonstrated that the richness and diversity of the bacterial communities were similar between children with caries-free and caries-active groups; LEfSe detected differences in the bacterial community including Dialister, Selenomonas, Actinomyces, and Mogibacterium in the caries-active group (P < 0.05) and Capnocytophaga, Fusobacterium, Desulfuromonadales, Haemophilus, and Porphyromonas in the caries-free group(P < 0.05). The core microbiome was defined as 18 predominant genera in children with caries. The results of the salivary proteome identified 9135 unique peptides and 1662 proteins group from 20 salivary samples. Two hundred fifty-eight proteins were differentially expressed between the caries-free and caries-active groups.ConclusionsThe diversity of the microbial community has little effect on caries but some bacteria with different relative abundance between the caries-active and caries-free group could be considered as potential biomarkers for children with caries. In addition, as a critical host factor of caries, the salivary proteins are different in caries-free and caries-active groups.

Child murder in the Early Bronze Age: proteomic sex identification of a cold case from Schleinbach, Austria

The identification of sex-specific peptides in human tooth enamel by nanoflow liquid chromatography-tandem mass spectrometry (nanoLC-MS/MS) represents a quantum leap for the study of childhood and social relations more generally. Determining sex-related differences in prehistoric child rearing and mortality has been hampered by the insufficient accuracy in determining the biological sex of juveniles. We conducted mass spectrometric analysis to identify sex-specific peptides in the dental enamel of a child from a settlement pit of the Early Bronze Age settlement of Schleinbach, Austria (c. 1950–1850 bc). Four perimortal impression fractures on the skull of a 5–6-year-old child indicate an intentional killing, with a co-buried loom weight as possible murder weapon. Proteomic analysis, conducted for the first time on prehistoric teeth in Austria, determined the child’s sex as male. While we cannot conclusively determine whether the child was the victim of conflicts between village groups or was slain by members of his own community, we suggest that contextual evidence points to the latter. A possible trigger of violence was the follow-on effects of an uncontrolled middle ear infection revealed by an osteological analysis. The boy from Schleinbach highlights the potential for further investigation of gender-biased violence, infanticide and child murder based on the recently developed method of proteomic sex identification.

Protein Composition of the Subretinal Fluid Suggests Selective Diffusion of Vitreous Proteins in Retinal Detachment.

PurposeTo study the proteome of the subretinal fluid (SRF) from rhegmatogenous retinal detachment (RRD) in search for novel markers for improved diagnosis and prognosis of RRD.MethodsHuman undiluted SRF obtained during vitrectomy for primary RRD using a 41-gauge needle (n = 24) was analyzed and compared to vitreous humor from 2-day postmortem eyes (n = 20). Sample preparation underwent nanoflow liquid chromatography–tandem mass spectrometry. Label-free quantification (LFQ) using MaxQuant was used to determine differentially expressed proteins between SRF and vitreous humor. The intensity-based absolute quantification (iBAQ) was used to rank proteins according to their molar fractions within groups. Identification of proteins beyond the quantitative level was performed using the Mascot search engine.ResultsThe protein concentration of the control vitreous humor was lower and more consistent (1.2 ± 0.4 mg) than that of the SRF (17.9 ± 22 mg). The iBAQ analysis showed high resemblance between SRF and vitreous humor, except for crystallins solely identified in vitreous humor. The LFQ analysis found 38 protein misregulations between SRF and vitreous humor of which the blood coagulation pathway was found to be enriched using the PANTHER Classification System. Combined, the iBAQ, LFQ, and Mascot analysis found an overlap only in chitinase-3-like protein 1 and galectin-3-binding protein unique to the SRF.ConclusionsThe proteome of the SRF was highly represented by proteins involved in proteolysis. Such proteins can possibly serve as targets in modulating the effects of SRF in RD.Translational RelevanceTo identify potential novel biomarkers for therapeutic targeting in RD.

Comparative Leaf Proteomics of Brassica napus Genotypes with Distinctive Levels of Early Cold Acclimation

Rapeseed/canola (Brassica napus L.) is considered amongst the “big four” oilseed crops with varying degrees of cold tolerance. Differential protein expression of cold acclimated young leaves of two canola genotypes, Sari Gul (spring type cold-sensitive) and Zarfam (winter type cold-tolerant), were determined via two dimensional polyacrylamide gel electrophoresis. Seedling leaves were cold acclimated by temperature regime and collected before and after cold treatment at 4 and − 4 °C after 24 h, proteins were isolated and separated on 2-DE. Gel analysis of 651 protein spots revealed that 19 protein spots were significantly different between the two conditions in response to early cold acclimation. Following nanoflow liquid chromatography–tandem mass spectrometry, 19 protein sequences were identified in gel spots, which were reported for the first time in response to early cold acclimation in B. napus and nearly half of them were associated with various aspects of chloroplast physiology; suggesting that the cold stress response of B. napus is achieved, at least partly, by regulation of chloroplast function. Our work has provided novel insights into the plant response to cold stress and should pave the way for future studies towards functional analysis of candidate genes in cold response.

Associations of smoking and air pollution with peripheral blood RNA N6-methyladenosine in the Beijing truck driver air pollution study

BackgroundPost-transcriptional modifications of RNA constitute fundamental mechanisms of gene regulation. N6-methyladenosine (m6A) is critical for health and disease and is modulated by cellular stressors. However, associations between environmental exposures and m6A have not been studied in humans. We aimed to examine associations between tobacco smoking and particulate air pollution with m6A and mRNA expression levels of its reader, writer and eraser (RWE) genes in blood. MethodsUsing the Beijing Truck Driver Air Pollution Study, we investigated global m6A in RNA from peripheral blood collected from 106 human subjects in Beijing, China, in 2008. We measured m6A with nano-flow liquid chromatography-tandem mass spectrometry and investigated gene expression of six m6A RWEs with real-time-quantitative PCR. Using linear models, we examined associations with smoking status, pack-years, and smoking on day of visit in men, and with environmental tobacco smoke in nonsmokers. We also examined associations with ambient PM10 (particulate matter ≤ 10 µm in diameter), and personal black carbon (BC) and PM2.5 measured with a portable monitor. ResultsSmoking in men was significantly associated with a relative 10.7% decrease in global m6A levels in comparison to nonsmokers (p = 0.02). In men, smoking greater than 3.8 pack-years was associated with a 14.9% lower m6A than in nonsmokers. BC exposure trended towards positive associations with m6A (5.95% per 10 μg/m3 increase in BC; 95% CI: −0.96, 13.3). Global m6A levels were not correlated with RWE gene expression levels. No associations were detected between smoking or air pollutants and m6A RWE gene expression. Discussionm6A was negatively associated with long-term smoking, yet positively associated with short-term BC exposure. These results indicate variable m6A responses to environmental stressors, providing early evidence into the impacts of toxicants on RNA modifications and suggesting potential for m6A as a biomarker or mechanism in environmental health research.

Analysis of the nonobese diabetic mouse islet MHC-II peptidome reveals posttranslationally modified autoantigens

Abstract Autoimmune diabetes results from the destruction of insulin-producing beta cells, a process initiated by CD4 T cells recognizing MHC-II bound beta cell-derived peptides. We identified MHC-II bound peptides in the islets and throughout the peripheral lymphatic system in order to better understand diabetes initiation and progression. Toward this end, we performed unbiased nanoflow liquid chromatography-tandem mass spectrometry (nLC-MS/MS) analysis to obtain sequence information for MHC-II peptides isolated from islets, pancreatic lymph nodes and spleens of nonobese diabetic (NOD) mice. Peptides derived from beta cell proteins were further examined for their relative I-Ag7 binding strength and CD4 T cell autoreactivity. For a discussion of the major immunogenic peptides derived from the B chain of insulin and from C peptide, see the abstract of Wan et al. Herein we discuss the finding of a deamidated C peptide fragment and our search for fused peptides. We identified the deamidated insulin-1 C peptide fragment Ins1C:51–61, LQTLALEVARE. Evidence in the literature supports this C-terminal deamidation as being spontaneous rather than enzyme-mediated. Deamidation improved I-Ag7 binding compared to the wild type peptide and increased its immunogenicity, demonstrating the biological importance of this modification. The homologous peptide from insulin-2 was not identified, indicating a possible sequence bias for spontaneous deamidation. We identified only one fused peptide bound to I-Ag7, an insulin C peptide-islet amyloid polypeptide hybrid insulin peptide (HIP) that matches the previously reported HIP6.9. We also identified several free fused peptides in crinosomes and posit this as the probable site of their formation.

System-Wide Analysis of Protein Acetylation and Ubiquitination Reveals a Diversified Regulation in Human Cancer Cells.

Post-translational modifications are known to be widely involved in the regulation of various biological processes, through the extensive diversification of each protein function at the cellular network level. In order to unveil the system-wide function of the protein lysine modification in cancer cell signaling, we performed global acetylation and ubiquitination proteome analyses of human cancer cells, based on high-resolution nanoflow liquid chromatography–tandem mass spectrometry, in combination with the efficient biochemical enrichment of target modified peptides. Our large-scale proteomic analysis enabled us to identify more than 5000 kinds of ubiquitinated sites and 1600 kinds of acetylated sites, from representative human cancer cell lines, leading to the identification of approximately 900 novel lysine modification sites in total. Very interestingly, 236 lysine residues derived from 141 proteins were found to be modified with both ubiquitination and acetylation. As a consequence of the subsequent motif extraction analyses, glutamic acid (E) was found to be highly enriched at the position (−1) for the lysine acetylation sites, whereas the same amino acid was relatively dispersed along the neighboring residues of the lysine ubiquitination sites. Our pathway analysis also indicated that the protein translational control pathways, such as the eukaryotic initiation factor 2 (EIF2) and the ubiquitin signaling pathways, were highly enriched in both of the acetylation and ubiquitination proteome data at the network level. This report provides the first integrative description of the protein acetylation and ubiquitination-oriented systematic regulation in human cancer cells.

Proteomic analysis of decedent scalp hair segments exhibiting a postmortem hair root band

A postmortem root band (PMRB) is an opaque microscopic band that can be observed near the root area of hairs from a decomposing body. Although PMRB is a recognized phenomenon in the forensic trace evidence community, protein signatures that are associated with PMRB formation are not well understood. To characterize the specific hair proteins affected by PMRB formation, comparative protein profiles of banded and non-banded hair sections were performed via site specific enzymatic digestion followed by nano-flow liquid chromatography tandem-mass spectrometry (nLC-MS/MS). The sequence coverage in banded sections of hair was ~35% less than in non-banded sections of hair, which reduces the confidence in protein identification and indicates that the abundance or integrity of the proteins is compromised. These findings suggest that proteolysis may play a role in the formation of PMRB.

Label-Free Proteomic Approach to Study the Non-lethal Effects of Silver Nanoparticles on a Gut Bacterium.

Among all the food-related nanoparticles consumed every day, silver nanoparticles (AgNPs) have become one of the most commonly utilized because of their antimicrobial properties. Despite their common use, the effects of sublethal concentrations of AgNPs, especially on gut biofilms, have been poorly investigated. To address this issue, we investigated in vitro the proteomic response of a monospecies Escherichia coli gut biofilm to chronic and acute exposures in sublethal concentrations of AgNPs. We used a new gel- and label-free proteomic approach based on shotgun nanoflow liquid chromatography–tandem mass spectrometry. This approach allows a quantification of the whole proteome at a dynamic range that is higher than the traditional proteomic investigation. To assess all different possible exposure scenarios, we compared the biofilm proteome of four treatments: (i) untreated cells for the control treatment, (ii) cells treated with 1 μg/ml AgNPs for 24 h for the acute treatment, (iii) cells grown with 1 μg/ml AgNPs for 96 h for the chronic treatment, and (iv) cells grown in the presence of 1 μg/ml AgNPs for 72 h and then further treated for 24 h with 10 μg/ml AgNPs for the chronic + acute treatment. Among the 1,917 proteins identified, 212 were significantly differentially expressed proteins. Several pathways were altered including biofilm formation, bacterial adhesion, stress response to reactive oxygen species, and glucose utilization.

Uniformly 15N-Labeled Recombinant Ricin A-Chain as an Internal Retention Time Standard for Increased Confidence in Forensic Identification of Ricin by Untargeted Nanoflow Liquid Chromatography-Tandem Mass Spectrometry.

Ricin, a toxic protein from the castor plant, is of forensic and biosecurity interest because of its high toxicity and common occurrence in crimes and attempted crimes. Qualitative methods to detect ricin are therefore needed. Untargeted liquid chromatography-tandem mass spectrometry (LC-MS/MS) proteomics methods are well suited because of their high specificity. Specificity in LC-MS/MS comes from both the LC and MS components. However, modern untargeted proteomics methods often use nanoflow LC, which has less reproducible retention times than standard-flow LC, making it challenging to use retention time as a point of identification in a forensic assay. We address this challenge by using retention times relative to a standard, namely, the uniformly 15N-labeled ricin A-chain produced recombinantly in a bacterial expression system. This material, added as an internal standard prior to trypsin digestion, produces a stable-isotope-labeled standard for every ricin tryptic peptide in the sample. We show that the MS signals for 15N and natural isotopic abundance ricin peptides are distinct, with mass shifts that correspond to the numbers of nitrogen atoms in each peptide or fragment. We also show that, as expected, labeled and unlabeled peptides coelute, with relative retention time differences of less than 0.2%.

In Situ Peroxidase Labeling and Mass Spectrometry of Alpha-Synuclein in Rat Cortical Neurons.

In this chapter, we describe a novel ascorbate peroxidase (APEX)-based labeling method that in combination with mass spectrometry identifies proteins in the immediate vicinity of αSyn in living rat cortical neurons. To isolate these interactions, we transduced primary cortical neurons with a lentivirus encoding APEX2 tagged to the C-terminus of alpha-synuclein (αSyn) and under the control of a synapsin promoter. Neural protein lysates were then incubated with streptavidin magnetic beads, washed, eluted from the beads, and digested overnight. The desalted peptides were then labeled with iTRAQ (4-plex) reagents and analyzed by nanoflow liquid chromatography-tandem mass spectrometry (LC-MS/MS). Collected data were analyzed using Spectrum Mill software, ultimately shedding light on αSyn physiological function and abnormal behavior during pathology.

PON-1 carbamylation is enhanced in HDL of uremia patients

High-density lipoprotein (HDL) carbamylation has been known in uremia patients. Paraoxonase-1 (PON-1) is an important HDL protein responsible for HDL anti-oxidant, arylesterase and lactonase activities. PON-1 carbamylation in uremic HDL has never been explored. We isolated HDL from uremia patients and control healthy subjects for study. Sandwich ELISA was used to estimate carbamylated PON-1 protein expression in HDL, and nanoflow liquid chromatography-tandem mass spectrometry (nanoLC-MS/MS) was applied to identify the amino acid in PON-1 carbamylated. PON-1 enzyme activities were estimated by substrates conversion method. HDL anti-oxidant activity was gauged by fluorescence changes of indicator dye in the presence of H2O2. Our study results proved that the degree of PON-1 carbamylation was higher in uremic HDL than in control HDL. Sandwich ELISA study showed that carbamylated PON-1 concentration in uremic HDL was 1.49 ± 0.08 fold higher than that in HDL from controls (p < 0.05). The nanoLC-MS/MS showed that the carbamylation of lysine 290 (K290) of PON-1, a residue adjacent to PON-1 activity determining site, was detected in uremic HDL but not detected in control HDL. K290 carbamylation leads to local conformation changes that reduce accessible solvent accessibility. The HDL paraoxonase, arylesterase, and lactonase activities were all significantly lower in uremia patients than in control subjects. Additionally, HDL anti-antioxidant ability was also lower in uremia patients. Carbamylation of PON-1 in uremia patients could be one of the factors in impairing PON-1 enzyme activities and HDL anti-oxidation function.