Friedreich's Ataxia and Cesarean Delivery: A Case Report of Epidural Anesthesia With Ropivacaine.

Friedreich's ataxia (FRDA), a rare inherited neurodegenerative disease, presents distinctive complexities in obstetrical anesthesia. Available research about FRDA in obstetrics is extremely limited. In this report, the anesthetic management of a 40-year-old primigravida with FRDA undergoing cesarean delivery is presented. An uneventful cesarean delivery with effective epidural anesthesia with ropivacaine at the L2-L3 intervertebral space was performed in our case. Neither hypotension nor bradycardia was observed, and vital signs remained stable, with no need for administration of vasoactive drugs. After discharge, the parturient reported no change in her neurologic symptoms. Conclusive recommendations are contingent upon more extensive studies. Overall management and the choice to proceed with neuraxial anesthesia in a woman with FRDA should be based on comprehensive consultations in both cardio-obstetrics and pre-anesthetic evaluations.

Tandem Mass Tag-10plex (TMT-10plex) phosphoproteomics dataset for comprehensive analysis of active compounds with tyrosine kinase inhibition activity.

Bioactive compounds derived from natural products demonstrate a wide range of beneficial properties in cancer treatment. One popular approach to inhibiting cancer cell growth is by stimulating apoptosis. Interestingly, our research has discovered that traditional mushroom and isolated compounds from traditional herbs can induce apoptosis in A549 cells while inhibiting tyrosine kinase activities. We have identified two extracts from traditional mushrooms, Phallus indusiatus and Fomes rimosus (Berk.) Cooke, which exhibit promising abilities to activate apoptotic events in cells. Additionally, isolated compounds such as Chamuangone, Cannabigerol (CBG), Cannabidiol (CBD), and NP1-cyclic peptide have also demonstrated significant apoptotic activation capabilities. To further our understanding, we analyzed phosphoprotein changes in A549 cells exposed to these extracts and compounds, both with and without epidermal growth factor (EGF) stimulation. Our positive controls were two known drugs, Afatinib and Osimertinib, which are tyrosine kinase inhibitors with apoptotic stimulation abilities. In order to enrich our understanding of the kinase pathway, we conducted phosphoprotein enrichment analysis and identified altered phosphoproteins using LC-MS/MS. Across these testing conditions, we found that 1228 phosphoproteins were altered, providing valuable insights into the biochemical mechanisms underlying cell apoptosis in A549 cells through post-translational modifications of proteins. Furthermore, our findings not only shed light on the mechanisms of cell apoptosis in A549 cells but also offer promising avenues for future research and therapeutic development.

A Handle on Mass Coincidence Errors in De Novo Sequencing of Antibodies by Bottom-up Proteomics.

Antibody sequences can be determined at 99% accuracy directly from the polypeptide product by using bottom-up proteomics techniques. Sequencing accuracy at the peptide level is limited by the isobaric residues leucine and isoleucine, incomplete fragmentation spectra in which the order of two or more residues remains ambiguous due to lacking fragment ions for the intermediate positions, and isobaric combinations of amino acids, of potentially different lengths, for example, GG = N and GA = Q. Here, we present several updates to Stitch (v1.5), which performs template-based assembly of de novo peptides to reconstruct antibody sequences. This version introduces a mass-based alignment algorithm that explicitly accounts for mass coincidence errors. In addition, it incorporates a postprocessing procedure to assign I/L residues based on secondary fragments (satellite ions, i.e., w-ions). Moreover, evidence for sequence assignments can now be directly evaluated with the addition of an integrated spectrum viewer. Lastly, input data from a wider selection of de novo peptide sequencing algorithms are allowed, now including Casanovo, PEAKS, Novor.Cloud, pNovo, and MaxNovo, in addition to flat text and FASTA. Combined, these changes make Stitch compatible with a larger range of data processing pipelines and improve its tolerance to peptide-level sequencing errors.

An efficient, amine-specific iTRAQ labeling method improves the peptide and protein identification rates.

Isotope tags for relative and absolute quantification (iTRAQ) are among the most widely used proteomics quantification techniques. These tags can be rapidly coupled to the primary amines of proteins/peptides through chemical reactions under mild conditions, making this technique universally applicable to any kind of sample. However, iTRAQ reagents also partially react with the hydroxyl groups of serine, threonine and tyrosine residues, particularly when these residues coexist with a histidine residue in the same peptide. This overlabeling of peptides causes systematic biases and significantly compromises protein/peptide identification rates. In this study, we report a novel iTRAQ labeling method that overcomes the detrimental overlabeling while providing high amine labeling efficiency. The impacts of reaction temperature, reactant concentrations, reaction time, buffer compositions, and pH on iTRAQ labeling performance were investigated in-depth. In a comparison experiment between our method and the standard labeling method provided by the iTRAQ manufacturer, our method reduced the number of overlabeled peptides by 55-fold while achieving comparable amine labeling efficiency. This improvement allowed our method to eliminates the systematic bias against histidyl- and hydroxyl-containing peptides, and more importantly, enabled the identification of 23.9% more peptides and 9.8% more proteins. SIGNIFICANCE: In addition to amines, the hydroxyl groups in serine, threonine, and tyrosine residues can also partially labeled by iTRAQ reagents, which leads to systematic biases and significantly compromises the analytical sensitivity. To address this issue, we developed a novel iTRAQ labeling method that overcomes the detrimental overlabeling while providing high labeling efficiency of amines. When benchmarking our method against the standard method provided by the reagent manufacturer, our method achieved comparable labeling efficiency but reduced the overlabeled species by 55-fold. This significant improvement eliminated the systematic biases, and more importantly, enabled the identification of 23.9% more peptides and 9.8% more proteins, demonstrating its superior performance and potential to enhance proteome quantification using iTRAQ labeling.

Fully resolved high-precision measurement of 36S for sulfur reference materials.

RATIONALE: Advances in sulfur isotope measurement techniques have led to increased analytical precision. However, accurate measurement of 36S remains a challenge. This difficulty arises primarily from unresolved isobaric interferences of 36SF5 + at m/z = 131 u, 186WF4 2+ and 12C3F5 +, which lead to scale compression. Theoretically, unresolved interference with 2% relative intensity could cause 1‰ underestimation in a sample with real δ36S = +60‰. METHODS: Our study develops an interference-free four-sulfur isotope measurement method by using the high-resolution mass spectrometer Panorama. The mass resolving power of Panorama allows the distinction of 186WF4 2+ and 12C3F5 + from 36SF5 +. RESULTS: The 186WF4 2+ relative intensity was initially 9.4% that of 36SF5 + but reduced to 1.5% through tuning, while 12C3F5 + relative intensity dropped from 74% to 40% after flushing with air. Three IAEA standards were analyzed with both Panorama and MAT 253. We obtained Δ36SIAEA-S-2 = 1.238 ± 0.040‰ and Δ36SIAEA-S-3 = -0.882 ± 0.030‰, relative to IAEA-S-1, from Panorama, and Δ36SIAEA-S-2 = 0.18 ± 0.02‰ and Δ36SIAEA-S-3 = 0.11 ± 0.14‰ from MAT 253, while δ34S values from the two instruments are consistent. CONCLUSION: The measurement discrepancies on 36S between Panorama and MAT 253 highlight the impact of scale compression due to unresolved isobaric interferences. Resolving this problem is crucial for accurate 36S analysis. We recommend replacing the filament material with rhenium, tuning the filament voltage, and avoiding carbon in instruments to eliminate or mitigate interferences. We propose future systematic efforts to calibrate the δ33S, δ34S, and δ36S of IAEA-S-1, IAEA-S-2, and IAEA-S-3 and advise bracketing all three reference materials in the measurement sequences, to enable calibration.

Serum proteins differentially expressed in gestational diabetes mellitus assessed using isobaric tag for relative and absolute quantitation proteomics.

BACKGROUND: As a well-known fact to the public, gestational diabetes mellitus (GDM) could bring serious risks for both pregnant women and infants. During this important investigation into the linkage between GDM patients and their altered expression in the serum, proteomics techniques were deployed to detect the differentially expressed proteins (DEPs) of in the serum of GDM patients to further explore its pathogenesis, and find out possible biomarkers to forecast GDM occurrence. AIM: To investigation serum proteins differentially expressed in GDM were assessed using isobaric tag for relative and absolute quantitation (iTRAQ) proteomics and bioinformatics analyses. METHODS: Subjects were divided into GDM and normal control groups according to the IADPSG diagnostic criteria. Serum samples were randomly selected from four cases in each group at 24-28 wk of gestation, and the blood samples were identified by applying iTRAQ technology combined with liquid chromatography-tandem mass spectrometry. Key proteins and signaling pathways associated with GDM were identified by bioinformatics analysis, and the expression of key proteins in serum from 12 wk to 16 wk of gestation was further verified using enzyme-linked immunosorbent assay (ELISA). RESULTS: Forty-seven proteins were significantly differentially expressed by analyzing the serum samples between the GDM gravidas as well as the healthy ones. Among them, 31 proteins were found to be upregulated notably and the rest 16 proteins were downregulated remarkably. Bioinformatic data report revealed abnormal expression of proteins associated with lipid metabolism, coagulation cascade activation, complement system and inflammatory response in the GDM group. ELISA results showed that the contents of RBP4, as well as ANGPTL8, increased in the serum of GDM gravidas compared with the healthy ones, and this change was found to initiate from 12 wk to 16 wk of gestation. CONCLUSION: GDM symptoms may involve abnormalities in lipid metabolism, coagulation cascade activation, complement system and inflammatory response. RBP4 and ANGPTL8 are expected to be early predictors of GDM.

Overcoming the detrimental O-acylation in TMTpro labeling improves the proteome depth and quantification precision.

BACKGROUND: With the advent of proline-based reporter isobaric Tandem Mass Tag (TMTpro) reagents, the sample multiplexing capacity of tandem mass tags (TMTs) has been expanded, and up to 18 samples can be quantified in a multiplexed manner. Like classic TMT reagents, TMTpro reagents contain a tertiary amine group, which markedly enhances their reactivity toward hydroxyl groups and results in O-acylation of serine, threonine and tyrosine residues. This overlabeling significantly compromises proteome analysis in terms of depth and precision. In particular, the reactivity of hydroxyl-containing residues can be dramatically enhanced when coexisting with a histidine in the same peptides, leading to a severe systematic bias against the analysis of these peptides. Although some protocols using a reduced molar excess of TMT under alkaline conditions can alleviate overlabeling of histidine-free peptides to some extent, they have a limited effect on histidyl- and hydroxyl-containing peptides. RESULTS: Here, we report a novel TMTpro labeling method that overcomes detrimental overlabeling while providing high labeling efficiency of amines. Additionally, our method is cost-effective, as it requires only half the amount of TMTpro reagents recommended by the reagent manufacturer. In a deep-scale analysis of a yeast/human two-proteome model sample, we compared our method with a typical alkaline labeling method using a reduced molar excess of TMTpro. Even at a depth of over 10,000 proteins, our method detected 23.7% more unique peptides and 8.7% more protein groups compared to the alkaline labeling method. Moreover, our method significantly improved the quantitative precision due to the reduced variability in labeling and increased protein sequence coverage. This substantially enhanced the statistical power of our method for detecting differentially abundant proteins, providing an average of 13% more yeast proteins that reached statistical significance. SIGNIFCANCE: We presented a novel TMTpro labeling method that overcomes the detrimental O-acylation and thus significantly improves the depth and quantitative precision for proteome analysis.

Charge distribution studies in the epi-cadmium neutron induced fission of 238Pu.

Studies on charge distribution have been carried out in the epi-cadmium neutron induced fission of 238Pu for the first time. Experimentally fractional cumulative yields (FCY) and independent yields (IY) of various fission products have been measured by using an off-line γ-ray spectrometric technique. From the FCY values, the charge distribution parameters such as the isobaric width parameter (σZ), most probable charge (ZP) and the charge polarization (ΔΖEXPT) as a function of fragment mass were obtained. On the other hand, from the measured IY values, isotopic width parameter (σA), the most probable mass (AP) and the elemental yields (YZ) of Sn, Sb, Te, I, Xe, Cs, Ba, La, Ce and Pr were determined by using a non-linear fit. From the YZ values, the proton even-odd effect (δp) was obtained for the first time. The isobaric and isotopic charge distribution parameters in the 238Pu(nf, f) reaction were compared with the similar data in the thermal neutron induced fission of 238Pu and other actinides to examine the role of excitation energy.

Quantification Quality Control Emerges as a Crucial Factor to Enhance Single-Cell Proteomics Data Analysis.

Mass spectrometry (MS)-based single-cell proteomics (SCP) provides us the opportunity to unbiasedly explore biological variability within cells without the limitation of antibody availability. This field is rapidly developed with the main focuses on instrument advancement, sample preparation refinement, and signal boosting methods; however, the optimal data processing and analysis are rarely investigated which holds an arduous challenge because of the high proportion of missing values and batch effect. Here, we introduced a quantification quality control to intensify the identification of differentially expressed proteins (DEPs) by considering both within and across SCP data. Combining quantification quality control with isobaric matching between runs (IMBR) and PSM-level normalization, an additional 12% and 19% of proteins and peptides, with more than 90% of proteins/peptides containing valid values, were quantified. Clearly, quantification quality control was able to reduce quantification variations and q-values with the more apparent cell type separations. In addition, we found that PSM-level normalization performed similar to other protein-level normalizations but kept the original data profiles without the additional requirement of data manipulation. In proof of concept of our refined pipeline, six uniquely identified DEPs exhibiting varied fold-changes and playing critical roles for melanoma and monocyte functionalities were selected for validation using immunoblotting. Five out of six validated DEPs showed an identical trend with the SCP dataset, emphasizing the feasibility of combining the IMBR, cell quality control, and PSM-level normalization in SCP analysis, which is beneficial for future SCP studies.

Inserting Pre-analytical Chromatographic Priming Runs Significantly Improves Targeted Pathway Proteomics with Sample Multiplexing.

GoDig, a platform for targeted pathway proteomics without the need for manual assay scheduling or synthetic standards, is a powerful, flexible, and easy-to-use method that uses tandem mass tags to increase sample throughput up to 18-fold relative to label-free methods. Though the protein-level success rates of GoDig are high, the peptide-level success rates are more limited, hampering assays of harder-to-quantify proteins and site-specific phenomena. To guide the optimization of GoDig assays as well as improvements to the GoDig platform, we created GoDigViewer, a new stand-alone software that provides detailed visualizations of GoDig runs. GoDigViewer guided the implementation of "priming runs," an acquisition mode with significantly higher success rates. In this mode, two or more chromatographic priming runs are automatically performed to improve the accuracy and precision of target elution orders, followed by analytical runs which quantify targets. Using priming runs, success rates exceeded 97% for a list of 400 peptide targets and 95% for a list of 200 targets that are usually not quantified using untargeted mass spectrometry. We used priming runs to establish a quantitative assay of 125 macroautophagy proteins that had a >95% success rate and revealed differences in macroautophagy expression profiles across four human cell lines.

An Efficient, Amine-Specific, and Cost-Effective Method for TMT 6/11-plex Labeling Improves the Proteome Coverage, Quantitative Accuracy and Precision.

Tandem mass tags (TMT) are widely used in proteomics to simultaneously quantify multiple samples in a single experiment. The tags can be easily added to the primary amines of peptides/proteins through chemical reactions. In addition to amines, TMT reagents also partially react with the hydroxyl groups of serine, threonine, and tyrosine residues under alkaline conditions, which significantly compromises the analytical sensitivity and precision. Under alkaline conditions, reducing the TMT molar excess can partially mitigate overlabeling of histidine-free peptides, but has a limited effect on peptides containing histidine and hydroxyl groups. Here, we present a method under acidic conditions to suppress overlabeling while efficiently labeling amines, using only one-fifth of the TMT amount recommended by the manufacturer. In a deep-scale analysis of a yeast/human two-proteome sample, we systematically evaluated our method against the manufacturer's method and a previously reported TMT-reduced method. Our method reduced overlabeled peptides by 9-fold and 6-fold, respectively, resulting in the substantial enhancement in peptide/protein identification rates. More importantly, the quantitative accuracy and precision were improved as overlabeling was reduced, endowing our method with greater statistical power to detect 42% and 12% more statistically significant yeast proteins compared to the standard and TMT-reduced methods, respectively. Mass spectrometric data have been deposited in the ProteomeXchange Consortium via the iProX partner repository with the data set identifier PXD047052.

Removal of isobaric interference using pseudo-multiple reaction monitoring and energy-resolved mass spectrometry for the isotope dilution quantification of a tryptic peptide.

Energy-resolved mass spectrometry (ERMS) and an isotopically labelled internal standard were successfully combined to accurately quantify a tryptic peptide despite the presence of an isobaric interference. For this purpose, electrospray ionisation tandem mass spectrometry (ESI-MS/MS) experiments were conducted into an ion trap instrument using an unconventional 8 m/z broadband isolation window, which encompassed both the tryptic peptide and its internal standard. Interference removal was assessed by determining an excitation voltage that was high enough to maintain a constant value for the analyte/internal standard peaks intensity ratio, thus ensuring accurate quantification even in the presence of isobaric contamination. Pseudo-multiple reaction monitoring (MRM) was employed above this excitation voltage to quantify the trypic peptide. The internal standard calibration model showed no lack of fit and exhibited a linear dynamic range from 0.5 µM up to 2.5 µM. The detection limit was 0.08 µM. The accuracy of the method was evaluated by quantifying the tryptic peptide of three reference samples intentionally contaminated with the isobaric interference. All the reference samples were accurately quantified with ∼1% deviation despite the isobaric contamination. Furthermore, we have demonstrated that this methodology can also be applied to quantify the isobaric peptide by standard additions down to 0.2 µM. Finally, liquid chromatography ERMS (LC ERMS) experiments yielded similar results, suggesting the potential of the proposed methodology for analysing complex samples.

Quantitative Peptidomics: General Considerations.

Peptidomics is the detection and identification of the peptides present in a sample, and quantitative peptidomics provides additional information about the amounts of these peptides. It is possible to perform absolute quantitation of peptide levels in which the biological sample is compared to synthetic standards of each peptide. More commonly, relative quantitation is performed to compare peptide levels between two or more samples. Relative quantitation can measure differences between all peptides that are detectable, which can exceed 1000 peptides in a complex sample. In this chapter, various techniques used for quantitative peptidomics are described along with discussion of the advantages and disadvantages of each approach. A guide to selecting the optimal quantitative approach is provided, based on the goals of the experiment and the resources that are available.

An Updated Guide to the Identification, Quantitation, and Imaging of the Crustacean Neuropeptidome.

Crustaceans serve as a useful, simplified model for studying peptides and neuromodulation, as they contain numerous neuropeptide homologs to mammals and enable electrophysiological studies at the single-cell and neural circuit levels. Crustaceans contain well-defined neural networks, including the stomatogastric ganglion, oesophageal ganglion, commissural ganglia, and several neuropeptide-rich organs such as the brain, pericardial organs, and sinus glands. As existing mass spectrometry (MS) methods are not readily amenable to neuropeptide studies, there is a great need for optimized sample preparation, data acquisition, and data analysis methods. Herein, we present a general workflow and detailed methods for MS-based neuropeptidomic analysis of crustacean tissue samples and circulating fluids. In conjunction with profiling, quantitation can also be performed with isotopic or isobaric labeling. Information regarding the localization patterns and changes of peptides can be studied via mass spectrometry imaging. Combining these sample preparation strategies and MS analytical techniques allows for a multi-faceted approach to obtaining deep knowledge of crustacean peptidergic signaling pathways.

Stabilizing the Proteomes of Acute Myeloid Leukemia Cells: Implications for Cancer Proteomics.

Previous work has shown that inhibition of abundant myeloid azurophil granule-associated serine proteases (ELANE [neutrophil elastase], PRTN3 [protease 3], and CTSG [Cathepsin G]) is required to stabilize some proteins in myeloid cells. We therefore hypothesized that effective inhibition of these proteases may be necessary for quantitative proteomic analysis of samples containing myeloid cells. To test this hypothesis, we thawed viably preserved acute myeloid leukemia cells from cryovials in the presence or the absence of diisopropyl fluorophosphate (DFP), a cell-permeable and irreversible serine protease inhibitor. Global proteomic analysis was performed, using label-free and isobaric peptide-labeling quantitation. The presence of DFP resulted in an increase of tryptic peptides (14-57%) and proteins (9-31%). In the absence of DFP, 11 to 31% of peptide intensity came from nontryptic peptides; 52 to 75% had cleavage specificity consistent with activities of ELANE-PRTN3. Treatment with DFP reduced the intensity of nontryptic peptides to 4-8% of the total. ELANE inhibition was 95%, based on diisopropyl phosphate modification of active site serine residue. Overall, the relative abundance of 20% of proteins was significantly altered by DFP treatment. These results suggest that active myeloid serine proteases, released during sample processing, can skew quantitative proteomic measurements. Finally, significant ELANE activity was also detected in Clinical Proteomics Tumor Analysis Consortium datasets of solid tumors (many of which have known myeloid infiltration). In the pancreatic cancer dataset, the median percentage of nontryptic intensity detected across patient samples was 34%, with many patient samples having more than half of their detected peptide intensity from nontryptic cleavage events consistent with ELANE-PRTN3 cleavage specificity. Our study suggests that in vitro cleavage of proteins by myeloid serine proteases may be relevant for proteomic studies of any tumor that contains infiltrating myeloid cells.

A Causal Model of Ion Interference Enables Assessment and Correction of Ratio Compression in Multiplex Proteomics.

Multiplex proteomics using isobaric labeling tags has emerged as a powerful tool for the simultaneous relative quantification of peptides and proteins across multiple experimental conditions. However, the quantitative accuracy of the approach is largely compromised by ion interference, a phenomenon that causes fold changes to appear compressed. The degree of compression is generally unknown, and the contributing factors are poorly understood. In this study, we thoroughly characterized ion interference at the MS2 level using a defined two-proteome experimental system with known ground-truth. We discovered remarkably poor agreement between the apparent precursor purity in the isolation window and the actual level of observed reporter ion interference in MS2 scans-a discrepancy that we found resolved by considering cofragmentation of peptide ions hidden within the spectral "noise" of the MS1 isolation window. To address this issue, we developed a regression modeling strategy to accurately predict reporter ion interference in any dataset. Finally, we demonstrate the utility of our procedure for improved fold change estimation and unbiased PTM site-to-protein normalization. All computational tools and code required to apply this method to any MS2 TMT dataset are documented and freely available.

Simple software solution for N2 diversion when measuring δ18O values of nitrogen-rich samples materials using a Thermo Scientific EA-IRMS System.

This method is a simple, cost-free, and reliable approach for the removal of N2 interference on a CO analyte when analysing nitrogen-rich (>0.5% w/w) samples by Elemental Analysis Isotope Ratio Mass Spectrometry. Specifically, the isobaric interference on m/z 30 is eliminated using only the open split of the Thermo Scientific ConFlo IV Universal Interface Device, improving the analytical workflow when using a static temperature Gas Chromatography (GC) column. It simplifies the N2 diversion methods described in recent decades. When applied, the method described here:•Provides sufficient baseline resolution between the N2 and CO analytes, to permit quantitative N2 diversion, using an extended length packed GC column;•Quantitatively eliminates all N2 analyte from the analytical gas stream ensuring that no N2 enters the ion source and therefore no isobaric interference is produced on m/z 30 ion trace of the CO analyte;•Allows reproducible measurement of δ18O values from nitrogen-rich sample materials without a N2 isobaric interference, where the CO analyte is measured on the analytical baseline that it was produced on in the reactor (i.e., no addition make-up helium or new baseline of pure helium for the CO analyte).

Recent progress in quantitative phosphoproteomics.

INTRODUCTION: Protein phosphorylation is a critical post-translational modification involved in the regulation of numerous cellular processes from signal transduction to modulation of enzyme activities. Knowledge of dynamic changes of phosphorylation levels during biological processes, under various treatments or between healthy and disease models is fundamental for understanding the role of each phosphorylation event. Thereby, LC-MS/MS based technologies in combination with quantitative proteomics strategies evolved as a powerful strategy to investigate the function of individual protein phosphorylation events. AREAS COVERED: State-of-the-art labeling techniques including stable isotope and isobaric labeling provide precise and accurate quantification of phosphorylation events. Here, we review the strengths and limitations of recent quantification methods and provide examples based on current studies, how quantitative phosphoproteomics can be further optimized for enhanced analytic depth, dynamic range, site localization, and data integrity. Specifically, reducing the input material demands is key to a broader implementation of quantitative phosphoproteomics, not least for clinical samples. EXPERT OPINION: Despite quantitative phosphoproteomics is one of the most thriving fields in the proteomics world, many challenges still have to be overcome to facilitate even deeper and more comprehensive analyses as required in the current research, especially at single cell levels and in clinical diagnostics.

Cathelicidin Antimicrobial Peptide Acts as a Tumor Suppressor in Hepatocellular Carcinoma.

Hepatocellular carcinoma (HCC) is associated with high rates of metastasis and recurrence, and is one of the most common causes of cancer-associated death worldwide. This study examined the protein changes within circulating exosomes in patients with HCC against those in healthy people using isobaric tags for a relative or absolute quantitation (iTRAQ)-based quantitative proteomics analysis. The protein levels of von Willebrand factor (VWF), cathelicidin antimicrobial peptide (CAMP), and proteasome subunit beta type-2 (PSMB2) were altered in HCC. The increased levels of VWF and PSMB2 but decreased CAMP levels in the serum of patients with HCC were validated by enzyme-linked immunosorbent assays. The level of CAMP (the only cathelicidin found in humans) also decreased in the circulating exosomes and buffy coat of the HCC patients. The serum with reduced levels of CAMP protein in the HCC patients increased the cell proliferation of Huh-7 cells; this effect was reduced following the addition of CAMP protein. The depletion of CAMP proteins in the serum of healthy people enhances the cell proliferation of Huh-7 cells. In addition, supplementation with synthetic CAMP reduces cell proliferation in a dose-dependent manner and significantly delays G1-S transition in Huh-7 cells. This implies that CAMP may act as a tumor suppressor in HCC.

Identifying Sex-Specific Serum Patterns of Alzheimer's Mice through Deep TMT Profiling and a Concentration-Dependent Concatenation Strategy.

Alzheimer's disease (AD) is the most prevalent form of dementia, disproportionately affecting women in disease prevalence and progression. Comprehensive analysis of the serum proteome in a common AD mouse model offers potential in identifying possible AD pathology- and gender-associated biomarkers. Here, we introduce a multiplexed, nondepleted mouse serum proteome profiling via tandem mass-tag (TMTpro) labeling. The labeled sample was separated into 475 fractions using basic reversed-phase liquid chromatography (RPLC), which were categorized into low-, medium-, and high-concentration fractions for concatenation. This concentration-dependent concatenation strategy resulted in 128 fractions for acidic RPLC-tandem mass spectrometry (MS/MS) analysis, collecting ∼5 million MS/MS scans and identifying 3972 unique proteins (3413 genes) that cover a dynamic range spanning at least 6 orders of magnitude. The differential expression analysis between wild type and the commonly used AD model (5xFAD) mice exhibited minimal significant protein alterations. However, we detected 60 statistically significant (FDR < 0.05), sex-specific proteins, including complement components, serpins, carboxylesterases, major urinary proteins, cysteine-rich secretory protein 1, pregnancy-associated murine protein 1, prolactin, amyloid P component, epidermal growth factor receptor, fibrinogen-like protein 1, and hepcidin. The results suggest that our platform possesses the sensitivity and reproducibility required to detect sex-specific differentially expressed proteins in mouse serum samples.